Alternative Toxicological Methods - H. Salem, S. katz (CRC, 2003) WW

ALTERNATIVETOXICOLOGICAL

METHODS

CRC PR ESSBoca Raton London New York Washington, D.C.

ALTERNATIVETOXICOLOGICAL

METHODS

Edited by

Harry SalemSidney A. Katz

This edition published in the Taylor & Francis e-Library, 2005.

“To purchase your own copy of this or any of Taylor & Francis or Routledge’scollection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”

ISBN 0-203-00879-0 Master e-book ISBN

Dedication

Preface

The Editors

Contributors

Contents

PART I

Progress in the Validation and RegulatoryAcceptance of Alternatives

2 ALTERNATIVE TOXICOLOGICAL METHODS

3

CHAPTER 1

Historical Developments in the HumaneCare and Use of Research Animals:

The First 4000 Years

CONTENTS

BIBLICAL ORIGINS


COLONIAL AMERICA

VICTORIAN ENGLAND

Table 1.1 Chronology for the Enactment of Animal Welfare Legislation

Year State Year State Year State

182818351838193818421845184818511851185218541856185718581859185918601861

New YorkMassachusettsConnneticutWisconsinNew HampshireMissouriVirginiaIowaMinnesotaKentuckyVermontTexasRhode IslandTennesseeKansasWashingtonPennsylvaniaNevada

18641864186718681868186918711871187118721873187318731875187918791880

IdahoOregonNew JerseyCaliforniaWest VirginiaIllinoisDistrict of ColumbiaMichiganMontanaColoradoDelawareIndianaNebraskaGeorgiaArkansasLouisianaMississippi

18801881188118831883188418871887188918901891189318951898191319131921

OhioNorth CarolinaSouth CarolinaAlabamaMaineHawaiiNew MexicoSouth DakotaFloridaMarylandNorth DakotaOklahomaWyomingUtahAlaskaArizonaVirgin Islands

THE FIRST 4000 YEARS 5


RUSSELL AND BURCH

THE THREE Rs

THE FIRST 4000 YEARS 7

REFERENCES

9

CHAPTER 2

A History of Interagency Approachesto Alternatives and Establishment of the

Interagency Regulatory Alternatives Group

CONTENTS

INTRODUCTION


ESTABLISHMENT OF IRAG

A HISTORY OF INTERAGENCY APPROACHES TO ALTERNATIVES 11

IRAG WORKSHOPS


EVOLUTION OF ICCVAM

A HISTORY OF INTERAGENCY APPROACHES TO ALTERNATIVES 13

REFERENCES

15

CHAPTER 3

The Interagency Coordinating Committeeon the Validation of Alternative

Methods (ICCVAM): Recent Progressin the Evaluation of Alternative

Toxicity Testing Methods

CONTENTS


INTRODUCTION

BACKGROUND AND HISTORY OF THE ICCVAM

ICCVAM EVALUATION OF ALTERNATIVE METHODS 17

Table 3.1 Member Agencies

Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)Consumer Product Safety CommissionDepartment of DefenseDepartment of EnergyDepartment of Health and Human Services

Agency for Toxic Substances and Disease RegistryFood and Drug AdministrationNational Institute for Occupational Safety and HealthNational Institutes of Health, Office of the DirectorNational Cancer InstituteNational Institute of Environmental Health SciencesNational Library of Medicine

Department of the InteriorDepartment of Labor

Occupational Safety and Health AdministrationDepartment of Transportation

Research and Special Programs AdministrationDepartment of AgricultureEnvironmental Protection Agency

Table 3.2 Test Method Validation and Acceptance Criteriaa

Validation Criteria

Clear statement of proposed useBiological basis/relationship to effect of interest providedFormal detailed protocolReliability assessedRelevance assessedLimitations describedAll data available for reviewData quality: Ideally good laboratory practices (GLPs)Independent scientific peer review

Acceptance Criteria

Fits into the regulatory testing structureAdequately predicts the toxic endpoint of interestGenerates data useful for risk assessmentAdequate data available for specified usesRobust and transferableTime and cost effectiveAdequate animal welfare consideration (3 Rs)

a These are shortened versions of the adopted criteria. For the full text see: National Instituteof Environmental Health Sciences (NIEHS), Validation and Regulatory Acceptance ofToxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committeeon the Validation of Alternative Methods (ICCVAM), NIH publication 97-3981, ResearchTriangle Park, NC, 1997.


Establishment of the ICCVAM

ICCVAM Authorization Act of 2000

Table 3.3 The Purposes of the ICCVAMa

Increase the efficiency and effectiveness of federal agency test method reviewEliminate unnecessary duplicative efforts and share experiences between federal regulatory agencies

Optimize use of scientific expertise outside the federal governmentEnsure that new and revised test methods are validated to meet the needs of federal agencies

Reduce, refine, or replace the use of animals in testing where feasible

a ICCVAM Authorization Act (U.S. Code, 2000).


THE NATIONAL TOXICOLOGY PROGRAM INTERAGENCY CENTER FOR THE EVALUATION OF ALTERNATIVE

TOXICOLOGICAL METHODS (NICEATM)

The ICCVAM Scientific Advisory Committee

Table 3.4 The Duties of the ICCVAMa

Consider petitions from the public for review and evaluation of new and revised test methods for which there is evidence of scientific validity

Coordinate the technical review and evaluation of new and revised test methods of interagency interest

Submit ICCVAM test recommendations to each appropriate federal agencyFacilitate and provide guidance on validation criteria and processesFacilitate:

Interagency and international harmonization of test protocols that encourage the reduction, refinement, and replacement of animal test methodsAcceptance of scientifically valid test methods and awareness of accepted methods

Make ICCVAM final test recommendations and agency responses available to the publicPrepare reports on the progress of this act and make these available to the public

a ICCVAM Authorization Act (U.S. Code, 2000).


THE ICCVAM TEST METHOD EVALUATION PROCESS

Test Method Validation

Test Method Submissions


ICCVAM Interagency Working Groups

Independent Scientific Peer-Review Panels

Figure 3.1 ICCVAM test method evaluation process.

NTP InteragencyNTP InteragencyCenter for the EvaluationCenter for the Evaluation

ofofAlternative ToxicologicalAlternative Toxicological

Methods (NICEATM)Methods (NICEATM)

Test SponsorTest SponsorSubmission of TestSubmission of Test

MethodMethod

Interagency CoordinatingInteragency CoordinatingCommittee on theCommittee on the

Validation of AlternativeValidation of AlternativeMethods (ICCVAM)Methods (ICCVAM)

Test RecommendationsTest Recommendationsto Agenciesto Agencies

Peer Review PanelsPeer Review PanelsExpert WorkshopsExpert Workshops

Advisory CommitteeAdvisory Committeeon Alternativeon AlternativeToxicologicalToxicological

MethodsMethods

AgencyAgencyDecisions/ActionsDecisions/Actions

ICCVAM InteragencyICCVAM InteragencyWorking GroupsWorking Groups

Report


Expert Panels and Workshops


ICCVAM Test Recommendations

REGULATORY AGENCY CONSIDERATION OF ICCVAM RECOMMENDATIONS

ICCVAM TEST METHOD EVALUATIONS

The Local Lymph Node Assay


Skin Corrosivity

Frog Embryo Teratogenesis Assay in Xenopus (FETAX)


Up-and-Down Procedure for Acute Oral Toxicity

In Vitro Methods for Assessing Acute Systemic Toxicity


SUMMARY

ACKNOWLEDGMENTS

REFERENCES





31

CHAPTER 4

Validation and Regulatory Acceptanceof Alternative Test Methods: Current

Situation in the European Union

CONTENTS

INTRODUCTION


VALIDATION AND REGULATORY ACCEPTANCE

ACCEPTANCE OF ALTERNATIVE TESTS IN THE EUROPEAN UNION 33


SKIN CORROSION



SKIN IRRITATION



DISCUSSION

ACKNOWLEDGMENTS

REFERENCES




43

CHAPTER 5

Integrated In Vitro Approachesfor Assessing Systemic Toxicity

CONTENTS

INTRODUCTION


THE STEPWISE APPROACH FOR INTEGRATED TESTING

THE ECITTS PROJECT

INTEGRATED IN VITRO APPROACHES FOR ASSESSING SYSTEMIC TOXICITY 45

Compounds and Test Battery

Figure 5.1 Building blocks of the ECITTS scheme.

Figure 5.2 (a) Native and (b) retinoic acid-differentiated SH-SY5Y cells.

1. Experimental 2. Modeling 3. Validation(literature data)

in vitro data on kinetics kinetic modeling kinetics in vivo

in vitro data on dynamics

(e.g., CNC, EC20, EC50)

prediction of target tissue

concentrations

(e.g., NOEL, LOEL)

prediction of dynamics

prediction of systemic

toxic doses

(e.g., NOED, LOED,

LD50)

in vivo systemic

toxic doses

(e.g., NOED, LOED,

LD50)

a b


Results

Table 5.1 Endpoints Studied in the in Vitro Neurotoxicity Test Battery for the ECITTS Project

Endpoint Assay Toxicity level

Cytotoxicity/Inhibition of cell growth Total cellular protein content Basal cytotoxicityNeurite degeneration (ND) Number of neurites per cell MorphologyProtein synthesis rate (PSR) [3H] leucine incorporation in

proteins during 2 hrPhysiology

Basal intracellular free Ca2+

concentration (basal Ca2+)Fura-2/Ca2+, fluorescence Physiology

Voltage operated Ca2+ channels(VOCC)

High potassium-induced Ca2+

flux, fluorescenceNeurochemistry

Phospholipase C-coupled acetylcholine receptor signal transduction (mAChR peak)

Carbachol-activated, immediate transient Ca2+ peak,fluorescence

Neurochemistry

Acetylcholine-induced capacitive Ca2+

entry (mAChR plateau)Carbachol-activated, secondary Ca2+ plateau, fluorescence

Neurochemistry


CONCLUSIONS AND FUTURE PERSPECTIVES

Refinement of the Neurotoxicity Test Battery

Table 5.2 Effects of the Test Compounds, Determined in Differentiated Human Neuroblastoma SH-SY5Y Cells, on in Vitro Endpoints as Presented in Table 5.1

Compound In vitro effectConcentration( M or M/hr)

Acrylamide 20% cytotoxicity 920Acrylamide CNC; neurite degeneration/time 11Caffeine CNC; inhibition of VOCC 10Diazepam CNC; inhibition of VOCC 49Lindane CNC; inhibition of VOCC 3.4Lindane 20% increased basal [Ca2+]i 35Lindane 50% cytotoxicity 150Parathion/paraoxon CNC; neurite degeneration/time 0.5Phenytoin CNC; inhibition of protein synthesis 87

CNC, critical cellular neurotoxic concentration; VOCC, voltage operated calcium channels.


Different Exposure Times

Figure 5.3 Estimated versus experimental doses after (a) acute and/or (s.c.) subchronicexposure. See Table 5.1 for endpoint definitions. The line represents the identity.

0.001

0.01

0.1

1

10

10.0

100.0

0.001 0.01 0.1 1 10 100 1000

Lindane (CNC: inhibition of VOCC vs. LOED: learning; s.c.)Lindane (EC20: increased basal Ca2+ vs. LOED: convulsions; s.c.)Lindane (50% cytotoxicity vs. lowest LD50; a.)Lindane (50% cytotoxicity vs. highest LD50; a.)Acrylamide (20% cytotoxicity vs. LOED: gait; a.)Acrylamide (CNC: ND vs. LOED: startle response; 10 days)Acrylamide (CNC: ND vs. LOED: startle response; 30 days)Acrylamide (CNC: ND vs. LOED: startle response; 90 days)Caffeine (CNC: inhibited VOCC vs. LOED: anti-nociception; a.)Diazepam (CNC: inhibited VOCC vs. LOED: time to emerge; a.)Phenytoin (CNC: inhibition of PSR vs. operant learning; a.)Parathion/paraoxon (CNC of paraoxon: ND vs. LOED: tail-pinch response of parathion; a.)

Experimental doses (mg/kg) or (mg/kg/day)

Est

imat

ed d

oses

(m

g/kg

) or

(m

g/kg

/day

)


ACKNOWLEDGMENTS

REFERENCES


51

CHAPTER 6

Summary of the OECD’s New GuidanceDocument on the Recognition,

Assessment, and Use of ClinicalSigns as Humane Endpoints for

Experimental Animals Usedin Safety Evaluation

CONTENTS

INTRODUCTION


DEVELOPMENT OF THE GUIDANCE DOCUMENT

SUMMARY OF THE OECD’S NEW GUIDANCE DOCUMENT 53

DEFINITIONS


GUIDING PRINCIPLES


INITIAL CONSIDERATIONS IN THE DESIGN OFANIMAL EXPERIMENTS


RECOGNITION AND ASSESSMENT OF PAIN, DISTRESS, AND SUFFERING AS AN APPROACH TO DETECTING CLINICAL

SIGNS AND ABNORMAL CONDITIONS


MAKING AN INFORMED DECISION TO HUMANELY KILL ANIMALS

Table 6.1 Common Conditions and Clinical Signs

Abdominal rigidityAbortionAgalactiaAnemiaAnalgesiaAnuriaApathyAtaxia/incoordinationBleedingBlepharospasmBlood in feces or urineBlood around nose, eyesBoarded abdomenBody temperature, abnormalBody weight loss or emaciation

Breathing difficulties (Dyspnea)

CachexiaChewing, persistentChromodachryorrheaCirclingComatoseCompulsive behaviorConstipationConvulsionsCorneal ulcerationCoughing/sneezingCyanosisDehydrationDiarrhea

Discharge, abnormalDyspnea (difficult breathing)Epistaxis (nasal bleeding)ExcitableEyelid closureEyes fixed/sunkenFractured boneGaspingGrooming—failure to doHunched/stiff postureHyperreflexiaImmobile/inactiveJaundice (icterus)Joints swollenKyphosisLateral positionLimping/lamenessLocomotory behaviorLordosisLoss of condition, body muscle

Mammary gland abnormalities

MoribundMotor excitationNot eating/drinkingOedemaPale mucous membranesParalysisParesisPiloerection

Pinna reflexProstratePruritisPupillary constriction/dilation

Rales, pulmonaryRectal prolapseRecumbency, prolongedRed eye(s)/noseReflexesRetention of fecesRighting reflexSalivation, excessive or abnormal

SeizuresSelf-mutilationSkin bruising/color/crepitusSpasmStaggeringSunken flanksSuppurationSwellingsTenesmusTetanyTremorUrine retentionVaginal prolapseVocalizationVomiting


SEVERE PAIN AND DISTRESS AS CRITERIA FOR HUMANE KILLING


GUIDANCE ON THE HUMANE CONDUCT OF SPECIFIC TYPES OF TOXICITY TESTING

REFERENCE

61

CHAPTER 7

Pain and Distress Management in AnimalResearch and Testing: The Humane

Society of the United States Painand Distress Initiative*

CONTENTS


INTRODUCTION

PUBLIC CONCERN ABOUT PAIN AND DISTRESS IN RESEARCH

PAIN AND DISTRESS MANAGEMENT IN ANIMAL RESEARCH AND TESTING 63

THE CHALLENGE


EXPERIMENTAL PROCEDURES THAT CAUSE PAIN AND DISTRESS IN RESEARCH

THE HSUS PAIN AND DISTRESS INITIATIVE

Outreach to IACUCs


Table 7.1 Models and Areas of Research and Specific Techniques That Cause Distress

Research Models or Areas

Non-Pain-Induced Distress

AggressionAnxiety (e.g., Vogel conflict-drinking model)Cancer (tumor burden, cachexia, carcinogenicity testing)Depression (e.g., learned helplessness, forced swimming, maternal

deprivation)DiabetesDrug addiction and withdrawalEnvironmental stress (e.g., hot, cold)FearImmunological research (e.g., vaccine potency testing)Infectious diseaseMotion sicknessNutrition research (e.g., nutrient deprivation)PanicPharmacology (some) (e.g., tumor necrosis factor, capsaicin research)Psychopathology (other than anxiety, depression, fear, etc., mentioned above)Radiation researchStress (psychological)Toxicology (induced effects)Transgenic research

Pain-Induced Distress

ArthritisBurn researchCancer research (tumor pain)Chronic pain studies1

Dental studiesInflammation studiesExperimental surgery (e.g., organ transplantation/rejection)Muricide (as a model of aggression, neophobia, etc.)Orthopedic studiesTrauma research

Specific Techniques

Non-Pain-Induced and Pain-Induced Distress

Anesthesia aftereffectsAntibody production (polyclonal and monoclonal)Aversive stimuli (e.g., electric shock)Bleeding techniques (including retro-orbital bleeding)Complete Freund’s AdjuvantControl group (animals denied experimental treatments)Deprivation (e.g., water, food, sleep, or social partners/experiences)Dosing techniques (e.g., gavage)Granuloma techniquesGut loop studies

(continued)


Regulatory Aspects

Table 7.1 (continued) Models and Areas of Research and Specific Techniques That Cause Distress

Knockout technologyRestraintSurgery sequelae

1 Acute pain should not be a problem if the guidelines of the International Association for theStudy of Pain (IASP, 1979) are followed.



Table 7.2 The USDA’s Current Pain and Distress Categories and a Proposed Modification, as Well as Related Features of the Two Systems

A. Current Scheme

USDA Category

Pain and/or Distress

Anesthesia/Analgesia

Full ACUC Review

AlternativeLiterature Search

C (63%)1 Little or None No Yes NoD (29%) Yes or No2 Yes Yes YesE (8%) Yes No Yes Yes

B. Proposed Scheme

CategoryPain and/or

DistressAnesthesia/Analgesia

Full IACUC Review

AlternativeLiterature Search

I Minor or None No No NoII Minor or None Yes Perhaps PerhapsIII Moderate Yes or No Yes YesIV Severe Yes or No Yes Yes

1 Numbers in parentheses are USDA figures for 2000.2 Animals listed in USDA category D were given pain- or distress-relieving drugs, but

these drugs may not have been sufficient to relieve all pain and distress throughout theexperiment.


Financial Support for Research on Pain and Distress

Table 7.3 State to State Variation in Reporting Animal Use in Column E (Unalleviated Pain or Distress) for States Using Greater Than 20,000 Animals

State

Percentage of Animals

in Column E State

Percentage of Animals

in Column E

Nationwide 8 Missouri 22 California 4 Nebraska 15Delaware 15 New Jersey 6Georgia 2 New York 11Illinois 3 North Carolina 6Indiana 18 Ohio 3Iowa 28 Pennsylvania 8Kansas 16 Texas 3Maryland 12 Virginia 0Massachusetts 1 Washington 20Michigan 15 Wisconsin 9Minnesota 3 Federal Agencies 7

Note: USDA data from 2000.

Table 7.4 States That Reported Less Than 1% of Animal Use in Column E between 1995 and 1997

Alaska (300) Mississippi (2,000) Tennessee (10,900)Arizona (5,000) Nevada (3,000) Utah (4,600)Hawaii (500) Oklahoma (4,300) Vermont (1,100)Kentucky (5,300) Oregon (4,700) Virginia (19,200)Louisiana (16,800) Rhode Island (2,100) West Virginia (1,700)Maine (800) S. Carolina (6,100) Wyoming (300)

Data from the USDA. Figures in parentheses indicate the average number of animalsused across all pain categories.


Development of a Technical Report on Animal Pain and Distress

BEST PRACTICES AND POLICIES


CONCLUSIONS

Table 7.5 Selected Elements of Institutional Policies on Monoclonal Antibody Production Available on the World Wide Web

Penn State Stanford U Iowa U Minnesota

Monitoring subj. with solid tumors

Not specified 3 /week Not specified 3 /week

Priming As low as 0.1 ml pristane

Not specified 0.2 ml max pristane

0.5 ml max pristane

Number of taps Max 3 taps, last terminal

Not specified 2 taps, last after euthanasia

Not specified

Monitoring postinoculation

Daily 3 /week for first week, then daily

Daily Daily

Replacementfluid after ascites harvest

Not specified Not specified 1–2 ml of saline subcutaneous

Not specified

Anesthesiaduring tap

Anesthesia can be used

Anesthesiaused for new personnel

Not specified Not specified


ACKNOWLEDGMENTS

References


PART II

Development of Predictive MethodsBased on Mechanisms of Eye Irritation

at the Ocular Surface: MeetingIndustry and Regulatory Needs

77

CHAPTER 8

Meeting Industry and Regulatory Needs forAlternative Test Methods to the Draize

Rabbit Eye Irritation Test

CONTENTS

INTRODUCTION


INDUSTRY PERSPECTIVE

INDUSTRY AND REGULATORY PERSPECTIVES 79


REGULATORY PERSPECTIVE




DISCUSSION AND CONCLUSIONS



REFERENCES



89

CHAPTER 9

The Ocular Surface: Barrier Functionand Mechanisms of Injury and Repair

CONTENTS

THE OCULAR SURFACE


ELECTROLYTES AND METABOLITES IN CORNEAL STROMA AND EPITHELIUM

Table 9.1 Stroma of rabbit cornea, mean ± SD, hydration = 3.2 ± 0.4

Mol/g H2O (n = 20) Mol/g net weight (n = 15)

Na 125 ± 42 Lactate 9.66 ± 1.2Cl 123 ± 25 Glucose 3.63 ± 0.8K 24 ± 4 ATP 0.20 ± 0.05S 105 ± 21 ADP 0.07 ± 0.03P 12 ± 4 ASC 4.72 ± 1.44

Note: ASC = ascorbate. Levels of some metabolites andelectrolytes in the corneal stroma. Adenosine triph-osphate (ATP) and adenosine diphosphate (ADP) arestrictly intracellular metabolites. Since the cornealstroma contains keratocytes only in about 10% of itsvolume, their levels are much lower than in the epi-thelium (see Table 9.2). Lactate is present in theextracellular as well as in the intracellular space andconsequently shows levels close to those of the epi-thelium (Reim et al., 1970, 1978; Fischern, 1996).

THE OCULAR SURFACE: BARRIER AND INJURY 91

Table 9.2 Metabolite levels of rabbit corneal epithelium Mol/g wet weight, m ± SEM

ATP 3.03 ± 0.10 (n = 14)ADP 0.26 ± 0.02 (n = 14)ATP/ADP 12.23 ± 0.89 (n = 14)AMP 0.93 ± 0.08 (n = 14)Glucose 2.02 ± 0.22 (n = 14)Lactate 9.89 ± 1.02 (n = 14)GSH 3.03 ± 0.43 (n = 21)GSSG 0.28 ± 0.05 (n = 21)ASC 11.55 ± 1.05 (n = 24)

Note: Levels of some metabolites in thecorneal epithelium, which may be sig-nificant to estimate vitality, to indicatemicrotrauma, and to show levels ofradical scavengers (Hennighausen etal., 1972; Reim et al., 1966, 1967,1970, 1976, 1982).


TRAUMA TO OCULAR SURFACE

CHEMICAL AND THERMAL INJURIES TO THE OCULAR SURFACE


Table 9.3 Grading of Eye Burns

I II III IV

Immediate signs

Erosion Large erosion Surface defect Epithelia destroyedHyperemia Ischemia 1/3 Ischemia >1/2 Deep ischemia >3/4

Chemosis Rose chemosis Dense corneal opacityCorneal opacity Conjunctival necroses

Sclera porcelain whiteDiscoloration and atrophy of irisFibrin exudate

Later signs

Regeneration Recirculation Persistent erosion ProliferationRegeneration Ulceration Large ulcerations

Vascularization Melting of cataractScars Glaucoma

Scarification

Note: Grading of eye burns according to clinical signs. The upper part lists immediatedamage visible, the lower one later secondary events. The classification of signswas developed from various authors and by clinical experiences (Hughes, 1946;Roper-Hall, 1965; Thoft, 1978; Reim and Kuckelkorn, 1995)

Figure 9.1 Eye with a mild alkali burn stage I (Table 9.3). The corneal epithelium was completelylost, but the stroma remained undamaged and clear. The conjunctiva showed hype-remia, but no swelling or ischemia. The damage healed within a few days.


Figure 9.2 Lime burn stage II (Table 9.3). The whole corneal and some conjunctival epitheliumwas destroyed. The corneal stroma exhibited little superficial turbidities. The lowerconjunctiva is demonstrated by upgaze. It was swollen (chemosis). Superficiallyin the conjunctiva, ischemia is recognized by the interrupted blood columns. Withthe lit lamp microscope, bloodstream could not be detected. Underneath theotherwise pale conjunctiva, intact sclera appeared with a faint rose background.

Figure 9.3 Clinical appearance of a severe chemical injury grade IV. The inner margin of theupper lid showed a white line of necrosis. The conjunctiva appeared flat and white,also from necrosis, which presumably included visible parts of the sclera. In theupper left region, some hemorrhages were deposited in necrotic conjunctiva.Ischemia was evident. The cornea was completely turbid. The outlines of iris andpupil could be hardly identified.


PATHOPHYSIOLOGY OF CHEMICAL EYE INJURIES

Ischemia and Necrosis

Inflammatory Response

Figure 9.4 Melting of the anterior eye segment, nine days after most severe burn from liquidmetal. There were extended necroses of all conjunctival, subconjunctival, andscleral tissues, appearing homogeneously white and slippery. Only in the rightupper region, some hemorrhages in necrotic tissues showed red color. The corneawas opaque in the upper marginal parts. The lower and central cornea was meltedaway and the iris and lens exposed. Since at that time (1977) corneal donormaterial was not available, the eye was lost and had to be removed. In the meltingtissues of the anterior eye segment, high activities of N-acetylglucose aminidase(NAcGA, E.C.3.2.1.50) and cathepsin-D (E.C.3.4.23.5) were found (Reim, 1982a).


Cytokines and Growth Factors in Cornea and Tears

Figure 9.5 Flow diagram of inflammatory cascade following chemical and thermal injuries ofthe eye. The inflammatory response is a quantitative process produced by theaffected tissues and the leucocytes involved (Ghattacherjee et al., 1979; Reim etal., 1980, 1993, 1997; Reim, 1982a; Rochels et al., 1982; Kulkarni and Srinivasan,1983; Becker et al., 1991, 1995; Reim and Leber, 1992; Reim and Becker, 1995).

Cornea and conjunctiva

PGE2α, Interleukins, LT 4, Subst-P, VIP, CGRP

Mild lesionweak response

Severe lesion, severe response

PMNs,macrophages

IL-1, IL-6IL-8, TNF

T-lymphocytesB-lymphocytes

Plasma cells

Cellular and humoral antibodies

O2

_

OH+ -radicalslysosomal enzymes

UlcerationInflammation Scars

PMNs

Restitution


Table 9.4 Cytokines in Tears

EGF regeneration of epitheliumTGFbeta 2 inhibits proliferationTNFalpha, in inflammationMany others, but presumably released from damaged surface epithelia

Note: Cytokines and growth factors in tears influencing the cornealepithelium (Mishima et al., 1991; Kruse and Tseng, 1994; Soto-zono and Kinshita, 1998).

Figure 9.6 Interleukin-1 (IL-1) and Interleukin-6 (IL-6) in human corneal buttons from kerato-plasty. Total number of cases: 127. The logarithmic ordinate shows the concen-trations found in pg/mg extractable protein. The symbols represent the median,squares stand for IL-1, rhombs for IL-6. The error bars demonstrate the 75%percentiles. In the abscissa, the diagnoses of the cases were indicated corre-sponding to the position of the symbols (Becker et al., 1995). Inflamed corneasrevealed very high levels of IL-1 and IL-6. The levels in the uninflamed, quietecorneas were lower by an order of magnitude.

1

10

100

1000

Keratitis DecompensationInflammat.Cone Dystrophy, Scars

Ulceration Keratoconus

Levels of IL-1 and IL-6 (n=127)pg/mg protein — human corneal buttons


Chemical Alteration of Extracellular Matrix

Stem Cell Insufficiency

Enzyme Activities and Metabolites on the Ocular Surface


Changes of the Contents of Na1+, K1+, Cl1–, and SO42–

Figure 9.7 Activity of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) in human tearscollected from nine human cases with eye burns stage I and II and an atopicpatient. Please note that the ordinate is in logarithmic scale! The enzyme activity( Mol/min/ml) increased considerably in surface diseases.

N-Acetylglucose aminidase in human tears

mol/min/ml

0.1

1

10

Normal 0.24 0.09 (9)

Range of 8 samplesfrom human burns

Atopic conjunctivitis


Calcification and Contamination

Scarring

Table 9.5 Stroma of Rabbit Cornea, Mol/g H2O, mean ± SD

EDXA Normal (n = 20)

Alkali Burn, Denuded, Rinsed for 16 Days, 4 daily

with 0.9% NaCl (n = 8)

Na 125 ± 42 90 ± 11Cl 123 ± 25 65 ± 15S 105 ± 21 24 ± 4P 12 ± 4 22 ± 22

Ca 3 ± 3 1 ± 3

Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn.The denuded stroma was rinsed with saline, four times daily for 16 days. Na, Cl,and especially S were decreased, P increased (Fischern et al., 1998).

Table 9.6 Stroma of Rabbit Cornea, Mol/g H2O, Mean ± SD

EDXA Normal (n = 20)

Alkali Burn, Denuded, Rinsed for 16 Days, 4 Daily with Phosphate Buffer (n = 8)

Na 125 ± 42 105 ± 22Cl 123 ± 25 88 ± 33S 105 ± 21 28 ± 4P 12 ± 4 623 ± 307

Ca 3 ± 3 435 ± 198

Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burnand rinsing with isotonic phosphate buffer, four times daily for 16 days. Na, Cl, andespecially S were decreased, but P and Ca were largely increased. Clinically,calcification of the cornea was observed (Schrage, 1997; Fischern et al., 1998;Haller, 2001).


Figure 9.8 Left eye of a 16-year-old boy six months after a most severe chemical injury. Inthis accident, a highly alkaline etching fluid used to work on electronic parts spilledinto both eyes of the patient. In this case, a severe inflammatory response haddeveloped and remained for years. The conjunctiva-like proliferation tissue sur-rounding the cornea was swollen and very hyperemic. The cornea was devoid ofepithelium. It showed extended ulceration especially in its marginal parts and wasgenerally thinned. The upper right cornea showed white calcification. To save theeye from melting, a keratoplasty was performed. The excised cornea was examinedwith electron dispersive x-ray analysis method (EDXA) (see Figures 9.9 and 9.10).

Figure 9.9 Scanning electron microscopy (SEM) on a cross section of the cornea seen inFigure 9.8. Magnification 200. The upper part shows calcification, the lower oneparallel corneal lamellae (Schrage et al., 1988, 1993, 1996).


ASPECTS OF REPAIR

Figure 9.10 Electron dispersive x-ray analysis (EDXA) of the calcified cornea as demonstratedin Figures 9.8 and 9.9. The spectra of the x-rays backscattered at scanningelectron microscopy (SEM) showed as expected high peaks for calcium (Ca) andphosphorus (P). But the most prominent peak from this sample was emitted fromsilicon (Si). Thus, EDXA revealed an unexpected high contamination of the corneaby silicone, which might have explained the severe and longstanding inflammatoryresponse in this case (Schrage et al., 1988, 1993, 1996).


Figure 9.11 Eye of a 42-year-old male 2 years after severe lime burn. Heavy scar formationcould not be prohibited. The cornea was covered with thick highly vascularizedproliferation tissue. The conjunctiva developed strong scars between the globeand the lids, reducing eye motility. The conjunctival scars also deformed the lidmargins. The hyperemic, red scar tissue showed that the inflammatory responsehad not subsided after 2 years. The eye was practically blind and had badprognoses for surgical rehabilitation.


References





109

CHAPTER 10

Evaluation and Refinement of the BovineCornea Opacity and Permeability Assay

CONTENTS

INTRODUCTION


METHODS

THE BCOP ASSAY 111

RESULTS

Figure 10.1 Cross-section of the new cornea holder. In contrast to the old holder, the newholder clamps onto sclera rather than cornea. Also, the shape of the chamber fitsthe normal curvature of the cornea in contrast to the flat chamber of the currentBCOP holder.

Quartz Window

Quartz Window

Stainless Steel Ring

Stainless Steel Ring

O-Ring

O - Ring

O-Ring

Bottom Holder

Top HolderEpithelialChamber

EndothelialChamber

Cornea

Vents

Sclera


Table 10.1 Absorbance at 570 nm (A570) of Bovine Corneas Exposed to Various Treatments and Incubated in MEM for 3 h

TreatmentExposure

None 10 min 1 min 30 sec

Intact, 35ºC, MEM 0.05 ± 0.03 — — —w/o Epi, 35ºC, MEM 0.11 ± 0.03 — — —w/o Epi/Endo, 4ºC, H2O 0.67 ± 0.13 — — —Isopropanol — 0.59 ± 0.08 0.23 ± 0.04 0.24 ± 0.07Acetone — 1.38 ± 0.22 1.07 ± 0.21 0.87 ± 0.2530% TCA — 1.43 ± 0.08 2.28 ± 0.20 1.96 ± 0.061% NaOH — 1.69 ± 0.22 1.28 ± 0.29 0.36 ± 0.1930% SLS — 0.095 ± 0.03 0.48 ± 0.28 0.21 ± 0.11

Note: Intact is untreated cornea incubated in MEM. w/o Epi is cornea with epitheliumremoved, not exposed to chemical, and incubated in MEM for 3 h. w/o Epi/Endo iscornea with both epithelium and endothelium removed, not exposed to chemical,and incubated in H2O at 4ºC. Values are mean ±SD, n = 5–10. (Data cited fromUbels et al. [2000]. With permission of Elsevier Science.)

Figure 10.2 Corneal hydration (mg H2O/mg cornea) following exposure to test substances for30 sec, 1 min, or 10 min and incubation in MEM for 3 h. Intact is untreated corneaincubated in MEM. w/o Epi is cornea with epithelium removed, not exposed tochemical, and incubated in MEM for 3 h. w/o Epi-Endo is cornea with bothepithelium and endothelium removed, not exposed to chemical, incubated in H2Oat 4 C. Mean ±SD, n = 5. Values for intact, without Epi, and without Epi-Endowere all significantly different from each other. 30-sec values and unmarked 1-min values are not different than intact value. # Significantly different than 30-secand 1-min values within group. * Significantly different than 30-sec and 10-minvalues within group. (ANOVA and Dunnett’s test, P 0.05). (Data cited from Ubelset al. [2000]. With permission of Elsevier Science.)

Aceto

neIP

A

1% N

aOH

30%

SLS

30%

TCA

mg

H20

/ m

g c

orn

ea

0

2

4

6

8

10

12

14

16

18

1 min

10 min

30 sec

## #* *

#

Inta

ct

w/o E

pi

w/o E

pi-Endo

THE BCOP ASSAY 113

DISCUSSION


Figure 10.3 Corneal endothelial cell layers stained with Alizarin Red S and trypan blue. Twentypercent of the endothelial layer is damaged after mounting in the old cornealholder (left), and none of the endothelial layer is damaged after mounting in thenew holder (right). The streaks of damaged cells exhibited after mounting in theold holder are characteristic of wrinkling caused by the holder. Magnification 35 .(Reproduced from Ubels et al. [2002]. With permission of Elsevier Science.)

a b

THE BCOP ASSAY 115

ACKNOWLEDGMENTS

REFERENCES

117

CHAPTER 11

Corneal Organ Culture forOcular Toxicity Test of Commercial

Hair Care Products

CONTENTS

INTRODUCTION


METHODS

Corneal Organ Culture

CORNEAL ORGAN CULTURE FOR OCULAR TOXICITY TEST 119

Culture Treatment

Surface Biotinylation-Tight Junction Permeability Assay

Electrophoretic Mobility Shift Assay (EMSA)

RESULTS AND DISCUSSION


Figure 11.1 Biotin surface labeling to visualize epithelial barrier. Cultured bovine cornea wasincubated with sulfo-NHS-LC-biotin for 30 min and then embedded in OCT, snap-frozen, and sectioned (6 m). Cryostat sections (8 m) were (A) stained directlywith hematoxylin to reveal corneal morphology (B) or incubated with rhodamine-avidin D to visualize the bound biotin. The rhodamine staining represents biotiny-lation of accessible surface of normal bovine cornea and linear staining at thecorneal surface indicates functional epithelial TJ barrier in cultured corneas. Ep,epithelium; BM, basement membrane; St, stroma that consists of fibroblasts. Aand B are mirror orientations of the same corneal sections.


Figure 11.2 Tight junction permeability assay of cultured bovine corneas in response to chal-lenge of three hair care products. Corneas in culture were treated with 100%,50% (not shown), and 25% chemicals, and TJ permeability of corneal epitheliumwas assessed by surface biotinylation as described in Figure 11.1. Inserts: cornealsections stained directly with hematoxylin to reveal corneal morphology. Note:extended biotinylation of the corneal surface caused by GA and GB exposure ina concentration dependent manner. However, no disruption of TJ was observedin GC treated cornea.


Figure 11.3 EMSA analysis of NF- B DNA-binding activity in bovine corneal epithelial cells inresponse to consumer product challenge. Panels showed cultured corneas weretreated with different concentrations of three hair care products for 5 min, untreatedcells were used as control (C). The corneas were then cultured for 10 min withoutthe presence of the chemicals. Cell extracts from corneal epithelial cells treatmentwere probed with 32P-labeled AP-1 (upper panel) or NF- B (lower panel) consen-sus oligonucleotide. EMSA experiments were repeated two times, and gels pre-sented in the figure are from a representative set.


REFERENCES


125

CHAPTER 12

Human Corneal Equivalentsfor In Vitro Testing

CONTENTS

INTRODUCTION


DEVELOPMENT OF CELL LINES AND CONSTRUCTION OF CORNEAS

CHANGES IN CORNEAL TRANSPARENCY IN RESPONSE TO CHEMICALS

HUMAN CORNEAL EQUIVALENTS FOR IN VITRO TESTING 127

CHANGES IN GENE EXPRESSION IN RESPONSE TO CHEMICAL EXPOSURE

DEVELOPMENT OF RELATED TISSUES


REFERENCES

Figure 12.1 Confocal image of a corneal equivalent with surrounding sclera containing chickembryonic dorsal root ganglion (not shown). Neurites, labeled with nerve-specificantineurofilament antibody are seen traveling through the sclera (S) parallel to thecorneal periphery and branching into the cornea (C). White dashed line, cornealperiphery; arrows, parallel neurites; arrowheads, branching neurites.

HUMAN CORNEAL EQUIVALENTS FOR IN VITRO TESTING 129

131

CHAPTER 13

The EpiOcular Prediction Model:A Reproducible In Vitro Means

of Assessing Ocular Irritancy

CONTENTS


INTRODUCTION

MATERIALS AND METHODS

EpiOcular (OCL-200) Tissue Source

THE EPIOCULAR PREDICTION MODEL 133

Histology

TISSUE VIABILITY ASSAY—MTT ET-50 METHOD

Preequilibration of Tissue

Preparation of Test Articles


Application of Test Article to EpiOcular Tissue

Exposure Times

Table 13.1 Choice of Additional Exposure Times Based on the Viability of the Initial 20-Min Exposure

Viability after 20-min exposure Additional exposure times (min)

90% 60, 240<90% but > 30% 5, 60

<30% 1, 5


Exposure Conditions

Removal of Test Article from EpiOcular Tissue

Tissue Viability: MTT Assay


Calculation of Effective Time 50 (ET-50)

Determination of Prediction Model

Testing of Prediction Model


Table 13.2 In Vitro and In Vivo Data Used to Generate the Prediction Model; In Vitro Data from EpiOcular ET-50 Determinations; In vivo Data from ECETOC Database or Commercial Sources

#Conc. tested

ET-50 (min)

Draize (MMAS)

1 Benzalkonium chloride (10%) 2.0% 1.07 108.0 2 Benzalkonium chloride (5%) 1.0% 1.0 83.8 3 Benzalkonium chloride (1%) 0.2% 5.9 45.3 4 Cetyl pyridinium bromide (10%) 2.0% 9.0 89.7 5 Cetyl pyridinium bromide (1%) 0.2% 30.1 36.0 6 Cetyl pyridinium bromide (0.1%) 0.02% 240.0 2.7 7 Glycerol 20.0% 240.0 1.7 8 Sodium hydroxide (10%) 2.0% 1.0 108.0 9 Sodium hydroxide (1%) 0.2% 2.3 25.8

10 Propylene glycol 20.0% 240.0 1.3 11 Sodium dodecyl sulfate (30%) 6.0% 2.1 60.5 12 Sodium dodecyl sulfate (15%) 3.0% 5.1 59.2 13 Sodium dodecyl sulfate (3%) 0.6% 9.0 16.0 14 Trichloro acetic acid (30%) 6.0% 1.0 106.0 15 Trichloro acetic acid (3%) 0.6% 155.1 6.7 16 Triton X-100 (10%) 2.0% 2.5 68.7 17 Triton X-100 (5%) 1.0% 5.3 33.1 18 Triton X-100 (1%) 0.2% 36.7 1.7 19 Tween 20 (100%) 20.0% 240.0 4.0 20 Body/hand wash 20% 9.1 3221 Body/hand wash 20% 14.8 3522 Eye gel/colorant 20% 240 023 Eye gel/colorant 20% 240 224 Face/body wash 20% 240 225 Face/body wash 20% 6.5 2526 Face/body wash 20% 10.2 4027 Hand/body lotion 20% 240 228 Hand/body lotion 20% 240 229 Hand/body lotion 20% 240 330 Shampoo—baby 20% 30.8 1031 Shampoo—baby 20% 25.7 1832 Conditioner 20% 240 233 Shampoo—regular 20% 6.0 3034 Shampoo—regular 20% 8.7 3535 Na2-ricinoleadmido MEA

sulfosuccinate20% 108.9 0

36 Sodium trideceth sulfate 20% 2.5 3337 Cetrimonium chloride 20% 116.9 6.6738 Stearalkonium chloride 20% 240 1439 Cocamide DEA 20% 240 040 Disodium cocoamphodipropionate 20% 11.2 15.341 Surfactant blend 20% 19.2 6.0 42 Surfactant blend 20% 40.4 2.6743 Final formulation shampoo 20% 26.1 4.044 Final formulation shampoo 20% 29.1 12.545 Final formulation shampoo 20% 4.2 32.746 Final formulation shampoo 20% 9.3 31.647 Final formulation shampoo 20% 9.0 34.4

(continued)


Quality Control Testing

Interlaboratory Testing

Table 13.2 (continued) In Vitro and In Vivo Data Used to Generate the Prediction Model; In Vitro Data from EpiOcular ET-50 Determinations; In vivo Datafrom ECETOC Database or Commercial Sources

#Conc. tested

ET-50 (min)

Draize (MMAS)

48 Final formulation shampoo 20% 31.0 3.949 Final formulation shampoo 20% 63.1 3.550 Final formulation shampoo 20% 47.1 8.351 Final formulation shampoo 20% 29.4 6.5752 Final formulation shampoo 20% 42.1 4.853 Hydro-alcohol (hair spray) solution 20% 84.1 6.0 54 10% fatty alcohol ethoxylate 20% 189 3.555 Eye makeup remover (surfactant sol.) 20% 240 056 Lactic acid (3% solution) 20% 240 057 Oleic acid 20% 240 258 Skin care emulsion 20% 240 059 Body spray 20% 240 0


Testing of Ultramild Materials

RESULTS

Histological Characterization of the EpiOcular Tissue Model

Figure 13.1 Hematoxylin and Eosin (H&E) stained histological cross sections of (A) EpiOculartissue model and (B) rabbit cornea epithelium and underlying stroma. Tissueswere fixed in 10% formalin, embedded in paraffin, and stained with H&E. Finalmagnification 360 .

A

B


Determination of Prediction Model

Testing of Prediction Model

Figure 13.2 Graphical depiction of in vivo and in vitro data used to derive the predictionequation. All materials tested that had specific gravity >0.95 were diluted to 20%in ultrapure water. If actual ET-50 exceeded 240 min, ET-50 was set equal to 240min. If ET was less than 1 min, ET-50 was set to 1 min.

Prediction Equation:Draize (MMAS) = –4.74 + 101.7/ (ET-50)

95% Prediction Intervals:Draize (MMAS) = –30.54 + 100.4/ (ET-50)Draize (MMAS) = 21.07 + 102.9/ (ET-50)

r = 0.9059 materials

150

125

100

75

50

25

01 10 100 300

OCL-200 Prediction Equation

Dra

ize

Scor

e (M

MA

S)

ET-50 (min)


Table 13.3 EpiOcular Prediction Model Testing: Comparison between In Vivo and InVitro Predicted Draize

Code ProductPredicted

DraizeActual Draize

(MMAS)

1 10599 A Body wash 14.6 16.72 10599 B Body wash 31.3 44.73 10599 C Dishwashing liquid 51.3 38.34 10599 D Hand soap liquid 28.0 24.75 10599 E Dishwashing liquid 25.2 39.36 10599 F Facial soap 15.6 9.37 10599 G Dishwashing liquid 50.8 39.08 10599 H Dishwashing liquid 60.5 50.39 10599 I Laundry detergent 37.3 37.3

10 10599 J Laundry detergent 1.8 0.711 10599 K Laundry detergent 33.4 37.712 10599 L Dishwashing liquid 96.9 37.713 10599 M Shampoo 6.5 4.014 10599 N Shampoo 32.9 41.715 10599 O Shampoo 8.4 3.316 10599 P Hand soap liquid 18.1 13.317 10599 Q Skin lotion 1.8 0.718 10599 R Shampoo 46.9 33.719 10599 S Skin lotion 1.8 0.020 10599 T Shampoo 39.3 37.721 10599 U Body wash 41.2 33.022 10599 V Laundry detergent 1.8 0.723 10599 W Laundry detergent 39.9 44.024 10599 X Skin lotion 1.8 0.7

Figure 13.3 Comparison of predicted and actual Draize scores for consumer products includ-ing: shampoos (5), face/body soap (6), dishwashing liquids (4), laundry detergents(5), and skin lotions (4). The predicted Draize scores were calculated based onthe ET-50 using the prediction equation shown in Figure 13.2.


Quality Control Results: 1996–2000

Interlaboratory Reproducibility

Testing of Ultramild Materials

Table 13.4 EpiOcular QC Testing Results for Positive (0.3% Triton X-100) and Negative (ultrapure water) Controls

Calendar Year Tissue LotsTriton ET-50

(min)Neg. Control

(OD)Avg. Lot CV

(%)

2000 60 23.1 6.0 1.441 5.51999 84 22.6 5.0 1.433 5.61998 85 25.2 5.6 1.354 5.51997 81 22.9 4.7 1.343 5.41996 47 24.9 6.3 1.274 5.2

Table 13.5 Results of Interlaboratory Testing

ET-50 (mins): Intralaboratory Reproducibility Avg. CV (%)Laboratory: BAK SDS Triton

P&G: 5.75 3.12 25.40 7.75 IIVS: 6.39 3.30 26.59 7.14 MatTek: 5.97 3.72 29.75 9.56 Average: 6.04 3.38 27.24 Std. Dev.: 0.32 0.31 2.25 CV: 5.36 9.19 8.26


Table 13.6 ET-50 for Ultramild Materials for Which the Draize Test Is Insensitive

In Vivo Concentration Draize (MMAS) ET-50 (min)

Benzalkonium chloride (BAK)

10.00% 108.0 1.15.00% 83.8 1.01.00% 45.3 5.90.30% 8.67 28.90.10% 0 212.70.03% 0 2053.0

Sodium dodecyl sulfate (SDS)

30.00% 60.5 2.115.00% 59.2 5.13.00% 16.0 9.01.00% 0.67 29.50.30% 0 740.10.10% 0 1938.3

Figure 13.4 Use of EpiOcular to differentiate between very mild materials that cannot bedistinguished by the in vivo Draize test (MMAS < 1.0).

�

�


DISCUSSION


ACKNOWLEDGMENTS


REFERENCES

147

CHAPTER 14

Three-Dimensional Construct of the HumanCorneal Epithelium for In Vitro Toxicology

CONTENTS

INTRODUCTION



Three-Dimensional Corneal Epithelial Cell Constructs

Histology

CONSTRUCT OF THE HUMAN CORNEAL EPITHELIUM FOR TOXICOLOGY 149

IN VITRO Toxicology Assay

Western Blot Analysis


RESULTS

Histologic Characteristics of the Three-Dimensional Corneal Epithelial Constructs

Figure 14.1 Light micrograph of a plastic section from a construct after 24 hr equilibration at37°C. The tissue organization reveals a stratified appearance with columnar basalcells, defined wing cells, and flattened superficial cells. Toluidine blue and basicfuchsin, original magnification 160 .


Figure 14.2 Transmission electron micrograph of the construct after 24 hr equilibration at 37°C.The superficial cells give rise to numerous villus processes. The nucleus is orientedparallel to the surface. Some small, spot-like junction arrangements are seenbetween cells at the surface. Desmosomes are present between adjacent cells.Original magnification 12,600 .

Figure 14.3 Transmission electron micrograph of basal cells of the three-dimensional con-struct. The nucleus of the basal cell is upright. Within the cell, there is an extensivecytoskeletal network. Mitochondria surround the nucleus and desmosomes joinadjacent cells. Original magnification 10,000 .


Biochemical Characteristics of the Three-Dimensional Corneal Epithelial Constructs

Figure 14.4 High-power transmission electron micrograph of the basal membrane of a basalcell revealing numerous mature hemidesmosomes. Also visible are the compo-nents of the hemidesmosomes: the intracellular membrane placode, filamentsradiating through the membrane, and the typical band in what would be the laminalucida. The amorphous band at the bottom is the polycarbonate support membraneof the construct. Original magnification 75,000 .


Response to Benzalkonium Chloride

Figure 14.5 Western blots of the acidic (AE1) and the basic (AE3) keratin family in the three-dimensional construct: A. The reactivity pattern for AE1 in the three-dimensionalconstruct (Lane 1) is similar to that of immortalized human corneal epithelial cells(IHCEC) cultured for 4 weeks (Lane 2). B. The reactivity pattern for AE3 (Lane1) is similar to that of cultured IHCEC at 1 week (Lane 2) and 2 weeks (Lane 3)and human donor corneal epithelial cells obtained within 24 hr after death (Lane4). The molecular weights of several cytokeratin isoforms recognized by therespective antibodies are indicated below under AE1 and AE3, respectively.

Figure 14.6 Western blot of the 64 kDa cornea-specific keratin AE5 in the three-dimensionalconstruct. Lanes show the construct on Day 0 (immediately upon arrival), Day 1(24 hr equilibration at 37°C), and Day 2 (48 hr at 37°C), as well as the freshhuman donor corneal epithelial cell positive control (+) and the rabbit lacrimalgland negative control (–).


Figure 14.7 Western blot of laminin in the three-dimensional construct. One band is seen at220 kDa and another at 440 kDa. Lane 1, three-dimensional construct; Lane 2,fresh human corneal donor epithelial cells from cadaver eyes; Lane 3, immortal-ized human corneal epithelial cells cultured for 4 weeks.

Figure 14.8


Figure 14.8 (continued)


DISCUSSION

Figure 14.9 Top: Western blot analysis for phosphorylated (active) p42/p44 mitogen-activatedprotein kinase (MAPK) after treatment with benzalkonium chloride. Positive control(Ctrl +), epidermal growth factor-stimulated A431 cells. Negative control (Ctrl –),unstimulated A431 cells. Bottom: The histogram shows the increase in intensitylevel (activity) of p42/p44 with increasing concentrations of benzalkonium chloride(BAK). The absence of activity with 0.1% benzalkonium chloride is likely due tocell death resulting from toxic insult.



ACKNOWLEDGMENTS

REFERENCES


161

CHAPTER 15

The Human Corneal Epithelial HCE-T TEPAssay for Eye Irritation: Scientific Relevanceand Summary of Prevalidation Study Results

CONTENTS

INTRODUCTION


SCIENTIFIC RELEVANCE

Background and Rationale for the Development of the HCE-T TEP Assay

THE HUMAN CORNEAL HCE-T TEP ASSAY 163


Figure 15.1 The three major steps in the execution of the HCE-T TEP assay (A–C), a typicaldose-response curve (D), and the prediction model (E).

A. B.

C.

D. Determination of In Vitro Endpoint, FR85 E. Prediction Model


Scientific Basis for the HCE-T TEP Assay



Tab

le 1

5.1

Sim

ilari

ties

an

d D

iffe

ren

ces

in t

he

En

dp

oin

t M

easu

red

in

th

e H

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EP

Ass

ay a

nd

in

th

e D

raiz

e E

ye I

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atio

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est

Test

Sp

ecie

sTe

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of T

issu

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mat

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to

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are

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l has

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netr

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to

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Figure 15.2 Cross-sectional diagram of the human corneal epithelium. A drawing illustratingthe multiple cell layers, the apical tight junctions, and the intercellular desmosomaljunctions of the corneal epithelium.

stroma

desmosomaljunction

tightjunction

tear film


Figure 15.3 Cross sections of stratified HCE-T cultures visualized by transmission electronmicroscopy following 5-min superficial exposures to (A) cell culture medium (KGM)(5000 ); and (B) 0.01% benzalkonium chloride (BAC) (6000 ). Ap = apical surfaceof the culture; M = collagen membrane. [Photos contributed by S.D. Dimitrijevich.]

KGM 0.01% BAC

Ap

M

A B

M

Ap


Figure 15.4 Cross sections of stratified HCE-T cultures, visualized by transmission electronmicroscopy, which were fixed in a special buffer to retain the mucin layer. Thedark-stained surface material is the mucin produced by the corneal epithelial cells.(A) A continuous layer of mucin is found on the surface of a differentiated culture(maintained at the air–liquid interface in serum-free medium containing 1.15 mMcalcium) in which the TER is high (tight junctions are intact); and (B) mucin isfound between the cells in this non-differentiated culture (maintained submergedin serum-free medium containing 0.15 mM calcium) in which the TER is low (tightjunctions not intact).

A B


Figure 15.5 Three assays were used to evaluate the dose-dependent effects of sodium dodecylsulfate (SDS) on HCE-T cultures on the day of treatment (day 1), and 48 hr later(day 3). TEP, transepithelial permeability to fluorescein; TER, transepithelial elec-trical resistance; MTT, cell viability assay using the MTT dye [3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyl tetrazolium bromide]. Each dose is represented by triplicatecultures, and the error bars are the standard deviations.

100

80

60

40

20

00.00 0.02 0.04 0.06 0.00 0.02 0.04 0.06

SDS(%) SDS(%)

% o

f Con

trol

Day 1 Day 3

TEPMTTTER


The Mechanism of Action of Fluorescein TEP in the Test System Compared to the Human Eye


Figure 15.6 Fluorescein staining data from rabbit eyes following surfactant-containing formu-lation exposure correlates well with Draize tissue score data (modified maximumaverage score, MMAS and modified maximum average corneal score, MMCS)from the same animals. The error bars are the standard deviations; n = 5–6 rabbitsevaluated per Draize test; and n = 2–4 rabbits evaluated for fluorescein stainingat 24 hr. [Data from Gettings et al., 1996.]

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

–20.000 0.000 20.000 40.000 60.000 80.000 100.000 120.000

24 Hr Fluorescein

MMAS R2 = 0.9435

MMCS R2 = 0.9711

Dra

ize

Dat

a


Figure 15.7 The relationship between the in vivo 24-hour fluorescein staining data and the invitro log FR85 values for the subset of the CTFA formulations that are representedin the HCE-T TEP assay Prediction Model. The error bars are the standarddeviations; n = 2 TEP assays; n = 5–6 rabbits per Draize test; and n = 2–4 rabbitsper fluorescein data.

-20

0

20

40

60

80

100

120

-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6

logFR85

Dra

ize

Dat

aMMAS R2 = 0.632

MMCS R2 = 0.7057

24 Hr R2 = 0.7361Fluorescein


PREVALIDATION STUDY RESULTS

Purpose for the HCE-T TEP Assay

Study Structure


Data Analysis


Tab

le 1

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Pre

valid

atio

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0.5

–/+

Sha

mpo

o 1

100%

sodi

um la

uryl

sul

fate

diso

dium

laur

eth

sulfo

succ

inat

ebu

tyle

ne g

ylco

lla

uram

ide

DE

A

25.0

15.0 5.0

0.5

anio

nic

anio

nic

noni

onic

noni

onic

MA

S 3

7.8

CS

20.

0C

OS

1.0

+

Sha

mpo

o 2

100%

amm

oniu

m la

uryl

sul

fate

laur

amid

e D

EA

etho

xydi

glyc

olhy

drox

ypro

pyl m

ethy

lcel

lulo

se

12.0 2.0

0.4

0.15

anio

nic

noni

onic

— —

MA

S 5

7.4

CS

38.

0C

OS

2.0

+

aC

once

ntra

tion

used

in D

raiz

e te

st,

and

initi

al c

once

ntra

tion

for

dilu

tion

in T

EP

ass

ay.

bM

AS

is t

he m

axim

um a

vera

ge s

core

; C

S is

the

cor

neal

sco

re;

CO

S is

the

cor

neal

opa

city

sco

re.

cF

eder

al H

azar

dous

Sub

stan

ces

Act

(F

HS

A)

clas

sific

atio

ns:

–, n

egat

ive;

+,

posi

tive;

–/+

rep

eat

test

(F

HS

A,

1979

).


Results

Conclusions


Figure 15.8 Overlay of the prevalidation study test results for five test materials in the MASPrediction Model (PM). The solid line represents the nonlinear regression curveof the MAS PM, and the dashed lines are the 95% confidence intervals. (A)Overlay of the 60 log FR85 values in the MAS PM. There are 12 values for eachof the five test materials on the plot, but, due to overlap in the data, all 12 pointsmay not be distinct. (B) Fit of the average log FR85 value for each of the fivetest materials in the MAS PM. The result for each test material is the average of12 assays.

B

A

7065605550454035302520151050–0.8 –0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2

7065605550454035302520151050–0.8 –0.4 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2

Dra

ize

MA

S

LogFR85

Dra

ize

MA

S

LogFR85


SUMMARY

Table 15.3 The Nonirritant/Irritant Classification of the Five Test Materials as Determined by the Draize Test and by the HCE-T TEP Assay

Test MaterialDraize Score

Classificationa

TEP Assay MAS PMb

TEP Assay CS PMb

TEP Assay COS PMb

Bubble bath 1 1 1 1Hair conditioner 1 1 1 1Cleansing gel 2 2 2 1Shampoo 1 2 2 2 2Shampoo 2 2 2 2 2

a Draize classification was the same across the MAS, CS, and COS scores, except for thecleansing gel which was an irritant by the MAS and CS, but a nonirritant by the COS.

b Draize scores for the classifications: nonirritant MAS 15; nonirritant CS < 5; nonirritantCOS < 0.667.

Note: Draize maximum average score, MAS; corneal score, CS; corneal opacity score, COS.

Table 15.4 Average HCE-T TEP Assay Results for Five Test Materials in Three Laboratories; The Predicted MAS and Class from the TEP assay Are Compared to the Draize MAS and Class for Each Test Result

Test Material Draize MASa

Draize Classb

PredictedMASa

PredictedClassb

Bubble bath 4.8 1 4.20 1Hair conditionerc 14.2 1 1.82 1Cleansing gel 22.0 2 16.15 2Shampoo 1 37.8 3 34.62 3Shampoo 2 57.4 4 41.18 3

a MAS, maximum average score.b The four classification cutoffs for MAS are based on the scheme proposed by Kay and

Calandra (1962): MAS 0–15, minimal (class 1); MAS 15.1–25, mild (class 2); MAS25.1–55, moderate (class 3); MAS > 55, severe (class 4).

c Hair conditioner was less water soluble than other test materials, which may account forits underprediction when it was diluted.


REFERENCES

Table 15.5 Variability in the Draize MAS Compared to the Predicted MAS for the HCE-T TEP Assay Prevalidation Study Data

Test MaterialIntralab CV (%) for

Draize MASa

Intralab CV (%) for Predicted MASb

Interlab CV (%) for Predicted MASc

Bubble bath 22.82 9.9410.06

4.94

6.03

Hair conditionerd 19.67 33.1915.8054.09

15.59

Cleansing gel 53.24 17.503.01

25.90

15.10

Shampoo 1 10.31 12.5421.2120.42

12.49

Shampoo 2 8.32 14.6014.6229.49

2.83

a Evaluated using five to six animals from one lab.b Evaluated using four replicates per lab for three labs.c Results from three testing labs.d Hair conditioner was less water soluble, which may account for its greater variability

when it was diluted.Notes: Intralab is the within lab variability (repeatability); interlab is the between lab

variability (reproducibility). Coefficient of variation, CV; maximum average score,MAS.






PART III

Dermal Testing Alternatives

189

CHAPTER 16

Alternative Methodsfor Dermal Toxicity Testing

CONTENTS

INTRODUCTION

SKIN CORROSION


SKIN IRRITATION

SKIN SENSITIZATION

ALTERNATIVE METHODS FOR DERMAL TOXICITY TESTING 191

SKIN PHOTOTOXICITY

PERCUTANEOUS ABSORPTION


REFERENCES

193

CHAPTER 17

Allergic Contact Hypersensitivity:Mechanisms and Methods*

CONTENTS


INTRODUCTION

PROCESS

ICCVAM and the LLNA Review

ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS 195

Figure 17.1 ICCVAM review process: the local lymph node assay. (Modified from Sailstad, 2000.)

Input/ guidance/ response

AGENCYLLNA

Recommendation

AdvisoryCommittee on

AlternativeToxicological

MethodsGuidance

LLNA SPONSORSData submission/ method support

ICCVAMImmunotoxicology

Working GroupFacilitation/ recommendation

NICEATMSupport

Peer Review Panel

Evaluation/ REPORT

REGULATORY ACTION

Agency Response


BACKGROUND

Contact Hypersensitivity and Allergic Contact Dermatitis Test Methods

Background: An Example of the Dilemma of GP Tests


Mechanisms: Contact Hypersensitivity/Allergic Contact Dermatitis

Induction Phase

Figure 17.2 Illustration of contact hypersensitivity (CH) mechanisms: induction and elicitationphase. During the induction phase of CH, immature dendritic cells of the skin,called “Langerhans cells” (LC), effectively uptake and process the allergen. Simul-taneously, epidermal keratinocytes and the LCs themselves release cytokinemediators which assist the LCs in the migration to the draining lymph node andmaturation into effective antigen-presenting cells. In the lymph node, LC to Tlymphocyte interactions occur, which is followed by lymphocyte proliferation. Theproliferation results in “primed” effector lymphocytes which maintain a memory forthe specific allergen. The elicitation phase of CH appears to involve two mech-anisms of action. Initially, allergens cause direct cellular action releasing a seriesof nonspecific inflammatory mediators. These mediators are responsible for someof the cellular influx into the site of allergen challenge. Additionally, the “primed”lymphocytes are called into the area in a very specific response. These responsesand the cytokines, costimulatory factors, and adhesion molecules act to producethe end results of erythema and edema. (Modified from Selgrade et al., 2001.)

CHEMICAL ALLERGEN

LANGERHANSCELL (LC)

EDEMA AND ERYTHEMA

CYTOKINES,COSTIMULATORY,

ADHESIONMOLECULESINCREASE

INDUCTION PHASE ELICITATION PHASE

LC ANDLYMPHOCYTEINTERACTION

“PRIMED”LYMPHOCYTES

CELLULARINFLUX

INITIAL NONSPECIFICINFLAMMATORY

MEDIATORS

MIGRATION TO LOCALLYMPH NODE

LYMPHOCYTEPROLIFERATION

SPECIFIC RESPONSE


Elicitation Phase


TEST METHODS

Traditional Contact Hypersensitivity Tests—Guinea Pig


The Local Lymph Node Assay (LLNA)—Murine

Figure 17.3 Guinea pig models. (Modified from Sailstad, 2002.)

Topical antigen application:closed patch

Days 0, 6–8, and 13–15

Day 27–28 topicalapplication (untreated flank

for 6 h)

21, 24, 48 h after removingpatch

Induction

Challenge

Endpoint Analysiserythema

20 animals/group

Guinea PigMaximization

Test

Topical antigen application:ID injection w/ or w/o FCA

Days 5-8

Day 20–22 topicalapplication

48, 72 h after challenge

BuehlerAssay


Figure 17.4 The local lymph node assay. (Modified from Sailstad, 2002.)

Agent applied to ears

Isotope incorporation expressed as disintegrationper minute (dpm)

Proliferation measured:

Days 1, 2, 3

Day 6

Day 6: 5 h post IV injection Lymph nodes removed

IV tail injection of isotope


Other Murine Systems

LLNA—Ex Vivo

Mouse Ear Swelling Test (MEST)


SUMMARY OF ACD/CH ANIMAL MODELS

FUTURE

Figure 17.5 Contact hypersensitivity models and endpoint mechanisms.

Elicitation

PHASE

MEST

Induction

GPMT and BA

LLNA


REFERENCES


207

CHAPTER 18

Validating In Vitro Dermal AbsorptionStudies: An Introductory Case Study

CONTENTS

INTRODUCTION


METHODS FOR EVALUATION OF THE REPORTS

VALIDATING IN VITRO DERMAL ABSORPTION STUDIES 209

MATERIALS AND METHODS OF THE INDIVIDUAL STUDIES





RESULTS OF THE STUDIES


Tab

le 1

8.1

Ace

toch

lor

In V

ivo

(Mea

n d

ose

dis

trib

uti

on

as

g e

qu

ival

ents

of

acet

och

lor

per

cm

2 .M

ean

of

fou

r m

ale

rats

.)

Exp

osu

re (

hr)

Was

hC

over

Car

bo

nF

ilter

Ski

nU

rin

eF

eces

Cag

eW

ash

Car

cass

Ab

sorb

edg

/cm

2%

g/c

m2

%

3g

/cm

2

0.5

0.22

0.2

0.1

0.1

4.00

0.01

0.00

10.

001

0.1

0.1

3.79

12.

20.

20.

10.

26.

000.

010.

001

0.00

10.

10.

14.

282

2.0

0.2

0.2

0.2

7.17

0.01

0.00

10.

001

0.2

0.2

7.32

41.

80.

20.

30.

27.

930.

020.

001

0.01

0.3

0.3

9.93

101.

20.

30.

40.

39.

630.

20.

010.

010.

40.

619

.55

240.

90.

40.

70.

412

.53

0.5

0.2

0.02

0.2

0.9

31.3

7

42g

/cm

2

0.5

34.1

3.0

—1.

43.

180.

004

0.00

10.

011.

71.

73.

951

32.3

3.2

—1.

32.

990.

020.

001

0.01

2.5

2.5

5.98

234

.94.

1—

1.2

2.78

0.02

0.00

50.

31.

81.

94.

384

31.1

4.9

—1.

22.

810.

20.

003

0.01

3.6

3.9

9.24

1022

.15.

6—

1.2

2.66

2.1

0.3

0.4

7.0

9.8

23.0

624

15.1

8.1

—0.

91.

835.

52.

10.

64.

412

.629

.64

270

g/c

m2

0.5

212.

024

.2—

9.2

3.40

0.01

0.01

0.04

12.9

12.9

4.79

120

4.9

21.7

—8.

73.

210.

020.

010.

0515

.115

.25.

632

216.

923

.7—

13.3

4.91

0.02

0.01

0.05

8.1

8.2

3.03

421

8.5

21.0

—8.

73.

210.

70.

010.

0612

.313

.04.

8310

190.

428

.2—

10.8

3.99

3.5

0.1

0.56

21.1

25.2

17.5

2

2934

g/c

m2

0.5

2571

.928

5.9

—74

.22.

530.

10.

30.

440

.340

.91.

391

2175

.740

5.4

—95

.23.

240.

20.

30.

215

0.9

153.

045.

222

2322

.735

5.2

—98

.93.

370.

50.

10.

698

.599

.63.

394

2260

.150

2.7

—93

.32.

182.

30.

11.

174

.878

.32.

6710

2261

.443

5.5

—88

.33.

0115

.20.

21.

611

4.3

131.

34.

4724

1788

.962

7.2

—86

.02.

9297

.147

.98.

421

6.7

369.

912

.61

aA

bsor

bed

is s

um o

f ur

ine,

fec

es,

cage

was

h, a

nd c

arca

ss.

Sou

rce:

Lyth

goe,

199

0a.


Table 18.2 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in rat skin. Mean of four to seven skin samples. Results presented as g/cm2.)

Exposure(hr)

AbsorbedWash

SkinDonor Loop

TotalRecoveredg/cm2 % g/cm2 %

3.02 g/cm2

0.05 0.41 13.68 1.73 0.11 3.60 0.08 0.55 2.891 0.82 27.12 1.32 0.11 3.77 0.04 0.44 2.732 1.14 37.78 0.95 0.12 3.84 0.03 0.50 2.734 1.37 45.30 0.80 0.09 2.98 0.05 0.43 2.75

10 2.15 71.19 0.17 0.08 2.52 0.01 0.59 2.9924 2.12 70.13 0.13 0.08 2.75 0.02 0.41 2.77

47.3 g/cm2

0.5 2.02 4.27 25.6 1.09 2.30 1.41 1.03 31.11 3.90 8.25 28.2 1.17 2.47 0.97 1.02 35.32 10.3 21.78 18.7 1.46 3.09 0.57 1.25 32.24 16.0 33.83 13.6 1.08 2.28 0.58 1.38 32.7

10 20.7 43.76 7.1 1.00 2.11 0.78 0.98 30.624 28.7 60.68 2.9 0.79 1.73 0.14 1.21 33.7

318 g/cm2

0.5 6.58 2.07 283 12.9 4.06 10.5 13.4 3251 21.4 6.73 383 16.6 5.22 14.3 16.7 4522 33.4 10.50 261 28.1 8.84 19.4 8.25 3504 80.9 25.44 262 16.2 5.09 4.41 11.6 375

10 182 57.23 158 22.6 7.11 5.59 11.6 37924 246 77.36 67 14.4 4.53 5.52 13.3 347

3095 g/cm2

1 17 0.55 2411 143 4.62 311 118 29992 104 3.36 3146 187 6.04 313 138 38584 137 4.43 2103 111 3.59 316 161 2828

10 264 8.53 2344 196 6.33 294 156 325324 757 24.46 1598 202 6.53 182 127 2866

Note: The 0.5-hr exposure was not performed at the high dose.

Source: Clowes and Scott, 1990a.


DISCUSSION

Table 18.3 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in human skin. Mean of six skin samples. Results expressed as g/cm2.)

Exposure(hr)

AbsorbedWash

SkinDonor Loop

TotalRecoveredg/cm2 % g/cm2 %

3.02 g/cm2

2 0.015 0.486 2.36 0.273 9.04 0.035 0.359 3.0410 0.074 2.44 2.61 0.271 9.98 0.136 0.194 3.2824 0.261 8.63 1.54 0.092 3.03 0.098 0.244 2.24

47.3 g/cm2

2 0.140 0.296 34.2 1.89 3.99 3.12 2.61 41.910 1.70 3.59 30.4 4.43 9.37 3.86 2.31 42.724 3.63 7.68 26.2 1.63 3.45 2.80 3.09 37.4

318 g/cm2

2 0.816 0.256 239 18.9 5.94 70.4 22.4 35210 5.69 1.79 221 12.1 3.82 91.8 15.8 34724 43.6 13.7 245 18.2 5.72 62.3 15.8 384

3095 g/cm2

2 4.57 0.148 2796 51.5 1.67 772 260 388410 22.4 0.722 2057 80.5 2.60 896 259 331424 32.5 1.05 1669 38.4 1.24 671 217 2628

Source: Clowes and Scott, 1990b.


Table 18.4 Comparison of the Percent Absorbed In Vitro and In Vivo in Rat Skin; Data Are from Tables 18.1 and 18.2

Dose( g/cm2) 0.5 1.0 2.0 4.0 10.0 24.0

Exposure Duration (hr)

3.0 in vivo 3.8 4.3 7.3 9.9 19.5 31.43.02 in vitro 13.7 27.1 37.8 45.3 71.2 70.142.5 in vivo 4.0 6.0 4.4 9.2 23.1 29.647.3 in vitro 4.3 8.2 21.8 33.8 43.8 60.7270 in vivo 4.8 5.6 3.0 4.8 9.3 17.5318 in vitro 2.1 6.7 10.5 25.4 57.2 77.42934 in vivo 1.4 5.2 3.4 2.7 4.5 12.63095 in vitro — 0.6 3.4 4.4 8.5 24.5

Ratio in Vitro/in Vivo

3.02/3.0 3.6 6.3 5.2 4.6 3.7 2.247.3/42.4 1.1 1.4 5.0 3.7 1.9 2.1318/270 0.4 1.2 3.5 5.3 6.2 4.43095/2934 — 0.1 1.0 1.6 1.9 1.9

Figure 18.1 The ratio of the in vitro absorption to the in vivo absorption with exposure durationin the rat. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high doseratio. Data are from Table 18.4.

0

1

2

3

4

5

6

7

0 5 10 15 20 25

Dose 1Dose 2

Dose 3Dose 4

Exposure (h)

Rat

io v

itro/

vivo


Table 18.5 Comparison of the Percent Absorbed In Vitro in Rat and Human Epidermal Membrane Preparations; Data Are from Tables 18.2 and 18.3

Dose( g/cm2) 2.0 10.0 24.0

Exposure Duration (hr)

3.03 rat 37.8 71.2 70.1human 0.49 2.44 8.63 47.3 rat 21.8 43.8 60.7human 0.30 3.59 7.68318 rat 10.5 57.2 77.4human 0.26 1.79 13.73095 rat 3.4 8.5 24.5human 0.15 0.72 1.05

Ratio Rat/Human

3.03 77.1 29.2 8.147.3 72.7 12.2 7.9318 40.4 32.0 5.63095 22.7 11.8 23.3

Figure 18.2 The ratio of the rat in vitro absorption to the human in vitro absorption withexposure duration. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to highdose ratio. Data are from Table 18.5.

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25

vitro R/H 47.3 g/cm2

vitro R/H 318 g/cm2

vitro R/H 3095 g/cm2

Duration of Exposure (h)

Rat

io r

at/h

uman


REFERENCES

221

CHAPTER 19

A Molecular Diagnostic Approachto Irritant or Allergic Patch

Testing Using the DermPatch

CONTENTS

BACKGROUND


Table 19.1 Skin Cytokins (adapted from Gerberick et al., 1998)

Constitutive or inducible expression inCytokines Langerhans cells Keratinocytes Fibroblasts

IL-1 – + +IL-1 + – +IL-3 – +IL-6 + + +IL-7 – + –IL-8 – + +IL-10 – + –IL-12 – +IL-15 +G-CSF – + +M-CSF – + –GM-CSF – + +TGF- – + –TGF- + + +TNF- – + –MIP-1 + – –MIP-1 + + –IP-10 – + +

IRRITANT OR ALLERGIC PATCH TESTING USING THE DERMPATCH 223

Table 19.2 mRNA Cytokine Profiles from Human Skin Biopsy or Human Cell Samples

ACD ICD

TNF- increased increasedIFN- increased increasedIL-2 increased increasedGM-CSF increased increasedIL-1 (human cells) dependent on allergen increasedIL-1 (human cells) increased increasedIL-4 increased not determinedIL-6 increased not determinedIL-10 increased no changeIL-12 p35 (human cells) no change no changeIL-12 p40 (human cells) increased no change

Source: Adapted from Wakem and Gaspari, 2000.

Table 19.3 Mechanisms of Irritant versus Allergic Contact Dermatitis

Feature Allergic Irritant

Chemical agents low molecular weight, lipid soluble

acids, alkalies, surfactants, solvents, oxidants, enzymes

Concentration of the agent less critical more criticalGenetic predisposition ++++ ++Sensitization and lag period necessary not necessaryTrigger interaction of antigen with

primed T cellsdamage to keratinocytes

Cytokine release ++++ +++T-cell activation early ++++ later ++++Mast-cell activation ++ ++Langerhans’ cells increased decreasedEicosanoid production ++ ++

Source: Adapted from Marks and DeLeo.



Tape Stripping and Extraction of Total RNA

Ribonuclease Protection Assay

Table 19.4 Clinical and Histological Aspects of Contact Dermatitis

Feature Allergic Irritant

Itch ++++ (early) +++ (late)Pain, burning ++ ++++ (early)Erythema ++++ ++++Vesicles ++++ +Pustules + +++Hyperkeratosis ++ +++Fissuring ++ ++++Sharp demarcation yes yesReaction delay after contact days minutes to hoursSpongiosis ++++ ++++Dermal edema ++++ ++++Necrotic keratinocytes ++++ ++++Ballooning degeneration + +++Lymphocytic infiltrate ++++ ++++Neurotrophilic infiltrate + +++

Source: Adapted from Marks and DeLeo.


Induction of Erythematous Reactions on the Skin

RESULTS


DISCUSSION


REFERENCES

229

CHAPTER 20

In Vitro Skin Equivalent Modelsfor Toxicity Testing

CONTENTS


INTRODUCTION

AIR–LIQUID INTERFACE TISSUE CULTURES

IN VITRO SKIN EQUIVALENT MODELS FOR TOXICITY TESTING 231

Figure 20.1 Schematic representation of the air–liquid interface (ALI) tissue culture technique.

Table 20.1 Epithelial Tissues Successfully Cultured at the ALI

Skin equivalentsa

Keratinocytes onlyKeratinocytes plus fibroblastsb

Keratinocytes plus melanocytesKeratinocytes plus Langerhans cells

Ocular corneal epitheliuma

Tracheal/bronchiala epitheliumTracheal/bronchial submucosal glandsVaginal epitheliuma,b

Gingival epitheliuma,b

a Cultured at MatTek Corp.b In development.

Tissue Culture Well

Culture Insert

ALI Tissue

Medium

Membrane

Tissue Culture Well

Culture Insert

ALI Tissue

Medium

Membrane

Tissue Culture Well

Culture Insert

ALI Tissue

Medium

Membrane


(A)

(B)

Figure 20.2 (A) Histological cross section of H&E stained EpiDerm-200. Magnification = 440 .(B) Transmission electron micrograph of ruthenium tetroxide stained intercellularlamellar lipid sheets (150,000 ).

Figure 20.3 Top macroscopic view of MelanoDerm tissues containing normal human melano-cytes derived from Black donor skin (400 magnification).


Figure 20.4 Development of pigmentation in MelanoDerm tissue produced with melanocytesderived from various skin phototypes. The figure shows the top macroscopic viewof tissue inserts. Day 0 indicates the day of shipment of a fully differentiated tissue.Pigmentation develops during additional culture of the fully differentiated tissueby the end user.

Figure 20.5 Pigmentation of MelanoDerm tissue (Black melanocytes) induced by -MSH and-FGF as shown by top macroscopic view of tissue inserts. Tissues were cultured

in media containing the indicated concentrations of growth factors for up to 20days following shipment of commercial MelanoDerm tissue.

• Asian (A)

• Black (B)

• Caucasian (C)

10-7M MSH, 3 ng/ml FGF

5x10-8 MSH, 1.5 ng/ml FGF

10-8 MSH, 0.3 ng/ml FGF

No MSH or FGF added

10-7M MSH, 3 ng/ml FGF

5x10-8 MSH, 1.5 ng/ml FGF

10-8 MSH, 0.3 ng/ml FGF

No MSH or FGF added


Figure 20.6 Lightening effect of topical kojic acid on MelanoDerm tissue. Tissues containingBlack melanocytes were treated topically with 1% kojic acid for the indicatednumber of days (25 l applied topically every other day). The top macroscopicview of the tissues reveals the visually observable lightening effect.

Table 20.2 Lightening of MelanoDerm Tissue by Topical Treatment with 1% Kojic Acid

Melanin content ( g/tissue)Treatment Day 10 Day 14

H2O 19.9 35.31% kojic acid 11.4 18.3

Figure 20.7 EpiDerm-FT tissue contains normal human fibroblasts cultured within a collagentype I dermal matrix. A fully differentiated epidermis derived from normal humankeratinocytes is cultured on the top of the dermis.

Day: 3 7 10 14

Kojic Acid—topical

Control


SURVEY OF USES AND ENDPOINTS

Corrosivity

Phototoxicity

Figure 20.8 Immunohistochemical staining of HLA-DR on human Langerhans cells within (A)ImmunoDerm tissue and (B) excised human skin.


Irritation

Melanogenesis

Figure 20.9 Phototoxicity of chlorpromazine. EpiDerm tissues were topically treated with the indi-cated concentrations of chlorpromazine and incubated overnight. On the followingday tissues were either irradiated with 6 J/cm2 of UVA or kept in the dark. Tissueswere then rinsed, fed with fresh medium, and incubated overnight. Finally, tissueviability was determined by the MTT assay. A decrease in tissue viability of 30% orgreater between the irradiated and nonirradiated tissues indicates a phototoxic effect.

0

20

40

60

80

100

120

0.001 0.003 0.010 0.032 0.100

[Concentration %]

MT

T (

% u

ntre

ated

con

trol

)

Chloropromazine

68%

42%

87%74%

-2%

-UVA+UVA


Percutaneous Absorption

Drug Metabolism

MISCELLANEOUS STUDIES

QUALITY CONTROL AND VALIDATION ISSUES


HISTOLOGICAL EVALUATION OF EACH TISSUE LOT

QUANTITATIVE ASSESSMENT OF FUNCTIONAL RESPONSE TO TOPICAL TRITON X-100 TREATMENT


EMERGING APPLICATIONS OF ALI TISSUES

Gene Microarray Technology

Table 20.3 EpiDerm-200 Triton X-100 ET50 Database Summary

YearEPI-200

Triton ET-50EPI-200

Triton C.V. Lots Avg. C.V.

2000a 6.76 16.4 89 6.21999 6.75 18.2 146 5.71998 7.24 17.9 175 9.21997 6.78 15.9 228 9.91996 6.74 14.6 184 9.61995 6.65 77.8 112 4.9

a Through September 2000.



Figure 20.10 Changes in cancer-related gene expression following UVB-irradiation of Epi-Derm tissue. AtlasTM human cancer cDNA expression array analysis of geneexpression changes induced in EpiDerm tissues 6 hr after irradiation with 175mJ/cm2 UVB.

Table 20.4 UVB-Irradiation of EpiDerm Tissue

WAF-1 (+)MAP Kinase p38 (+)Growth-arrest-specific protein (+)c-Myc binding protein MM-1 (+)TRAF-interacting protein (+)Caspases (+)Death-associated protein kinase (DAP kinase 1, +)p53-induced protein (+)GADD45 (+)DNA excision repair protein ERCC1 (+)DNA-repair protein XRCC1 (+)Placenta growth factors 1+2 (+)TIMP-1 (+)T-plasminogen activator (+)Rho GDP dissociation inhibitor 1 (+)Endothelin 2 (–)IL-6 (–)Leukocyte interferon-inducible peptide (–)60S ribosomal protein (–)


High-Throughput ALI Tissue Formats

Figure 20.11 Induction of PlGF expression following UVB-irradiation of EpiDerm tissue. Aga-rose gel electrophoresis of products obtained by RT-PCR of total RNA isolatedfrom EpiDerm tissue 20 hours following UVB-irradiation. (–) no irradiation. (+)UVB-irradiated. The expected PlGF PCR product is 273 bp.

Figure 20.12 EpiDerm tissues cultured in 24 well (EpiDerm-224) or 96 well (EpiDerm-296)high throughput ALI formats. Each tissue has its own individual media reservoirto avoid cross-contamination of samples.

20 25 30 35

Cycles

– + – +– + – +

273 bp

Markers


SUMMARY

Figure 20.13 Total RNA-96 isolation kit designed for high throughput total RNA isolation fromEpiDerm-296.

Figure 20.14 Agarose gel electrophoresis of total RNA isolated from EpiDerm-296 with theTotal RNA-96 isolation kit. Average yield is approximately 10 g DNA-free totalRNA/EpiDerm-296 tissue.


ACKNOWLEDGMENTS

REFERENCES




PART IV

A Case Study in the Use of Alternativesto Determine the Mechanism

of Sulfur Mustard Action


251

CHAPTER 21

Cellular Resistance of Tetrahymenato Sulfur Mustard

CONTENTS

INTRODUCTION



Cell Culture and SM Exposures

Coulter Counter Studies

CELLULAR RESISTANCE OF TETRAHYMENA TO SULFUR MUSTARD 253

RESULTS

Figure 21.1 Cell viability vs. [SM]. Viability of six different cell lines was analyzed over a rangeof SM concentrations. Symbols are represented within the graph.

10 -110 -210 -310 -410 -510 -610 -710 -80

20

40

60

80

100

CHO and HeLa

PBL

HEK

Tetrahymena

Yeast

SM Concentration [M]

Per

cent

age

Via

ble


DISCUSSION

Figure 21.2 Effects of SM on cell number over time. The number of Tetrahymena was deter-mined after exposure to a range of SM concentrations and over a period of 72hr. Symbols are represented within the graph.

807060504030201000

10

20

30

40

50

60

8.0 mM4.0 mM2.0 mM

0.5 - 1.0 mM

0.3 mM & Positive Control

Time (h)

Cel

l Num

ber



ACKNOWLEDGMENTS

REFERENCES


259

CHAPTER 22

Studies of Cellular Biochemical ChangesInduced in Human Cells by Sulfur Mustard

CONTENTS


INTRODUCTION


Cell Cultures

HEK

HeLa

BIOCHEMICAL CHANGES INDUCED IN CELLS BY SULFUR MUSTARD 261

Lymphocytes

DNA Isolation

Reagents

Phenol-Chloroform Extraction

Commercial Kits


Modified Kit Usage

Pulse Field Gel Electrophoresis (PFGE)

PARP Assay

Calcium Measurement

Interleukin ELISA


Measurement of Fc and C1q Receptors

RESULTS

Table 22.1 Problems Seen with DNA Isolation from HEK

Procedure Date Sample

Purity Level of DNA (260/280

Ratio)

DNAYield

(mg/ml)

Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells

8/4/98 Control 1.35 608/18/98 Control 1.65 458/24/98 Control 1.35 25

Purification of lymphocyte DNA using Boehringer Mannheim Kit for mammalian blood


Phenol/chloroform extraction of HEK DNA


Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells (modified procedure)



Table 22.2 Purification of HEK DNA using Boehringer Mannheim Kit for Mammalian Cells Exposed to Sulfur Mustard (HD)

Date SamplePurity Level of DNA

(260/280 Ratio)DNAYield

11/19/98 Control 1.81 12250 mM HD 2.10 163100 mM HD 1.89 150300 mM HD 1.82 199




DISCUSSION

Figure 22.1 Time-dependent response of PARP activity following HD exposure of (top) HeLacells and (bottom) HEK cells. Two concentrations of HD were used, 10 and 100 M,and PARP activities are presented as percent maximal response.

HeLa PARP enzyme activity

Time post-HD (h)1 2 64 9 24

% o

f max

imum

res

pons

e

0

20

40

60

80

100

120

100 µM HD10 µM HD

HEK PARP enzyme activity

Time post-HD (h)1 2 4 6 24

% o

f max

imum

res

pons

e

0

20

40

60

80

100

120

140

x-axis vs. 10 µM HDx-axis vs. 100 µM HD


Figure 22.2 Increase in intracellular calcium in HEK (passage 2) exposed to 300 M HD asmeasured by 340:380 nm ratio of Fura-2 AM.

Table 22.3 Binding in HD-Exposed HEKa

Hrs post HD Dose HD ( M)C1q 0 100 200 300

8 – – – –16 – – NT ++24 – + +++ +++

CD32 0 50 100 200

8 – + + +24 – + ++ +++

a + = weak; ++ = moderate; +++ = intense; NT = not tested. Grading of stainingwas judged visually by fluorescence microscope and HEK controls not exposedto HD were negative for fluorescence.

Time (s)

0 200 400 600 800 1000 1200 1400

Rat

io (

340:

380

nm)

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

2.6


REFERENCES

Figure 22.3 Concentration-dependent increase in the secretion of IL-8 from HEK exposed to HD.

HD Concentration

-50 0 50 100 150 200 250 300 350

pg/m

l IL-

8

0

500

1000

1500

2000

2500

3000

3500

4000

HD-exposed HEK

269

CHAPTER 23

Human Keratinocyte InflammatoryTranscript Gene Activity

Following Sulfur Mustard*

CONTENTS

INTRODUCTION



Materials

HUMAN KERATINOCYTE SM INFLAMMATORY TRANSCRIPT GENE ACTIVITY 271

Cell Culture and Exposure

RNA Preparation

Probing cDNA Array Blot


Table 23.1 HEK Housekeeping Gene Transcripts at 16 Hr Following Sulfur Mustard

Gene Ratioa

Gene TranscriptControl

Intensityb 25 M SM 200 M SM

14-3-3 zeta protein 317 0.7 0.4423 kDa Highly Basic Protein 1689 2.0 2.3

-Tubulin 307 0.57 1.4-Actin 236 1.2 1.2

Glyceraldehyde 3-phosphate dehydrogenase 612 0.44 1.1HLA class I histocompatibility antigen C-4 alpha chain

487 0.94 1.0

Hypoxanthine-guanine phosphoribosyltransferase

59 0.9 3.7

Ribosomal Protein S9 978 1.2 2.1Ubiquitin 2064 0.8 0.73

a Induction ratio from phosphorimage densitometeric measurements of listed gene transcriptnormalized to the sum total of the detected transcript intensity/pixel density.

b Control intensity (grayscale pixel density) adjusted from background. Background was setat the median intensity of the blank space between the six array panels. The subtractedbackground intensity ranged from 19 to 43.

Table 23.2 HEK Inflammation-Associated Transcripts at 16 Hr Following Sulfur Mustard

Gene Ratioa

Gene TranscriptControl

Intensityb 25 M SM 200 M SM

CD40 86 4.9 0.3Interleukin 1 67 0.1 1.4Interleukin 1 101 0.6 3.5Interleukin 2 receptor subunit 148 0.3 0.4Interleukin 6 494 0.7 0.6Interleukin 7 receptor subunit 131 0.4 0.7Interleukin 8 16 —c 20Interleukin 13 349 3.0 0.7Interleukin 15 215 — 0.03Macrophage inflammatory protein 2 11 — 13S19 ribosomal protein 886 0.9 2.1Tumor necrosis factor 50 — 1.4

a Induction ratio from phosphorimage densitometeric measurements of listed gene transcriptnormalized to the sum total of the detected transcript density.

b Control intensity (grayscale pixel density) adjusted from background. Background was setat the median intensity of the blank space between the six array panels. The subtractedbackground intensity ranged from 19 to 43.

c Transcript not detected above background.


Subtraction Library Construction

Subtraction Library Sequence and Analysis

Figure 23.1 Atlas cDNA array nylon blot image. HEK were exposed to 200 M sulfur mustardfor 16 h and mRNA isolated and 32P labeled as cDNA. The array contains double-dot blots of cDNA from 588 transcriptionally regulated genes and 9 housekeepinggenes at the bottom of the array (see 3 housekeeping genes in rectangular box).Boxes 1 and 2 show expression of macrophage inflammatory protein 2 andinterleukin 8, respectively. These inflammatory transcripts were at very low expres-sion levels in control HEK.

1

2


RESULTS


DISCUSSION AND CONCLUSIONS




REFERENCES


281

CHAPTER 24

Evaluation of Cytotoxicity Assays of HumanEpidermal Keratinocytes Exposed

In Vitro to Sulfur Mustard

CONTENTS

INTRODUCTION


EXPERIMENT

Reagents

Keratinocyte Growth

Agent Exposure

Calcein AM

Alamar Blue

CYTOTOXICITY ASSAYS OF KERATINOCYTES EXPOSED TO SULFUR MUSTARD 283

Neutral Red

MTS

Propidium Iodide

RESULTS


DISCUSSION

REFERENCES

Figure 24.1 HEKs were exposed to the indicated HD concentrations and then incubated for24 hr at 37 C. Viabilities were determined by using these dyes as described inthe Experimental section.

CYTOTOXICITY ASSAYS OF KERATINOCYTES EXPOSED TO SULFUR MUSTARD 285

287

CHAPTER 25

Comparison of Spectrophotometric andFluorometric Assays of Proteolysis in

Cultured Human Cells Exposedto Sulfur Mustard

CONTENTS


INTRODUCTION


Reagents

Lymphocyte Isolation

ASSAYS OF PROTEOLYSIS INDUCED BY SULFUR MUSTARD 289

Keratinocyte Growth

HD Exposure

Chromozym® Assay (24 hr Postexposure to HD)

Keratinocytes

Lymphocytes

Intracellular Protease Activity

RESULTS


Chromozym® Assay

CellProbe® Assay

Figure 25.1 Substrate descriptions.

Chromozym® SubstratesChromozym® TH Tosyl-glycyl-prolyl-arginine-4-nitranilide acetateChromozym® t-PA N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranilide acetateChromozym® TRY Carbobenzoxy- valyl-glycyl-arginine-4-nitrilanilide acetateChromozym® U Benzoyl-β-alanyl-glycyl-arginine-4-nitranilide acetate

CellProbe® Enzyme Substrates:Nonfluorescent (aa)x -Fl Fluorescent Dye +Substrate-Dye complex ➾➾➾ Nonfluorescent Substrate leaving group

Enzyme SubstrateAAPV- Elastase (AAPV) Dl-Alanyl-Alanyl-Prolyl-Rho 110D-aminopeptidase A (DAA) Asp-Asp-Rho 110K-Aminopeptidase B (KAB) Lysine-Lysine-Rho 110G-Aminopeptidase (GA) Gly-Gly-Rho 100L- Aminopeptidase (LA) Leu-Leu-RhoA- Aminopeptidase M (AAM) Alanine-Alanine-Rho 110R- Aminopeptidase B (RAB) Arginine-Arginine-Rho 110


Figure 25.2 Protease activity was measured in two donors as a function of cell number.Increasing the cell concentration above 2 106/well did not increase proteaseactivity and this concentration was used in all subsequent studies.

Figure 25.3 PBL from five different donors were exposed to 250 M HD and then incubatedfor 24 hr. Protease activity using the Chromozym® TH substrate was measuredas described in Material and Methods. Significant protease activity occurredreproducibly. All assays were run in triplicate and all donors were evaluatedmultiple times on different days.


SUMMARY

Figure 25.4 HEK were exposed to indicated HD concentrations and incubated for 20 hr. Chro-mogenic protease assays were performed as described in the Materials and Methods.The protease activity was only evident at the highest concentration of HD, and onlythe Chromozym® U and Chromozym® t-PA substrates were significantly hydrolyzed.

Figure 25.5 HEK were exposed to HD at the indicated concentrations, and protease activitywas measured fluorometrically 24 hr later as described in the Material and Meth-ods section. KAB and GA substrates were significantly hydrolyzed above thecontrol values while elastase (AAPV) and DAA were decreased.


REFERENCES

295

CHAPTER 26

Effects of Low Dose Sulfur Mustardon Growth and DNA Damage

in Human Cells in Culture

CONTENTS

INTRODUCTION


PURPOSE


PBL Isolation

PBL Exposure

EFFECTS OF LOW DOSE SULFUR MUSTARD ON HUMAN CELLS 297

HEK Cultures

HEK Exposure

Comet Assay

Viability Assays

Growth Curve Assay

RESULTS


Figure 26.1 PBLs were exposed to buffer or to buffer plus HD. Four hours following exposure,cells were harvested and treated with 0.001% H2O2 or buffer. HD concentrationsof 5 M or greater inhibited expression of SSB in the comet assay. Points representmean ±SEM from two experiments.

Figure 26.2 HEKs were exposed to buffer or to buffer plus HD. Four hours following exposure,cells were harvested and treated with 0.002% H2O2 or buffer. Results are similarto those seen with PBLs. Points represent mean ±SEM from two experiments.

µM HD

0 10 20 30 40 50 60

Com

et M

omen

t

0

10

20

30

40

HDHD-HP

µM HD

0 10 20 30 40 50 60

Com

et M

omen

t

0

10

20

30

40

HDHD-HP


Figure 26.3 PBLs were exposed to buffer or to buffer plus CEES. Four hours following expo-sure, cells were harvested and treated with 0.001% H2O2 or buffer. CEES did notinhibit expression of H2O2-induced SSB. Points represent mean ±SEM from twoexperiments.

Figure 26.4 HEKs were exposed to buffer or to buffer plus CEES. Four hours following exposure,cells were harvested and treated with 0.002% H2O2 or buffer. Results are similar tothose seen with PBLs. Points represent mean ±SEM from two experiments.

µM CEES

0 10 20 30 40 50 60

Com

et M

omen

t

0

10

20

30

40

CEESCEES-HP

µM CEES

0 200 400 600 800 1000 1200

Com

et M

omen

t

0

10

20

30

40

CEESCEES-HP


CONCLUSIONS

Figure 26.5 HEKs were exposed to buffer or to buffer plus HD. Eighteen hours followingexposure, cells were harvested and treated with 0.002% H2O2 or buffer. Pointsrepresent mean ±SEM from two experiments.

µM HD

0 10 20 30 40 50 60

Com

et M

omen

t

10

20

30

40

50

60

70

HDHD and H2O2


Figure 26.6 HEKs were exposed to buffer or to buffer plus HD. Twenty-four hours followingexposure, cells were harvested and treated with 0.002% H2O2 or buffer. Pointsrepresent mean ±SEM from two experiments.

Figure 26.7 Growth curves for HEKs following exposure to HD in culture. Lowest line is for5 M concentration.

µM HD

0 10 20 30 40 50 60

Com

et M

omen

t

0

20

40

60

80

100

HDHP and H2O2

Time (h)

20 30 40 50 60 70 80 90 100

Via

ble

Cel

ls/m

L

100x103

1x106

10x106

0.00 M HD 0.01 M HD0.10 M HD0.50 M HD1.00 M HD5.00 M HD


REFERENCES

303

CHAPTER 27

Imaging Sulfur Mustard Lesions in BasalCells and Human Epidermal Tissues

by Confocal and MultiphotonLaser Scanning Microscopy

CONTENTS

INTRODUCTION



Figure 27.1 Basal cell adhesion complex. A microvesicle (lower left panel) showing details ofthe dermal–epidermal separations characteristic of a sulfur mustard blister.Hemidesmosomes (arrowhead), at the roof of the blister, are well displaced fromthe basement membrane (bm) and lamina densa (ld). An expanded model of theintact adhesion complex (circumscribed area) includes the intracellular keratinfilaments K5 and K14 and their facilitated attachments to the transmembrane 6 4

integrin receptors. The exodomains of 6 4 are shown linked by laminin 5 to thebasement membrane zone.

IMAGING SULFUR MUSTARD LESIONS 305

RESULTS


Figure 27.2 Keratin 5 images from control cultures of HEK recorded by multiphoton imaging(A), showed the elaborate cytoskeletal matrix and distribution of these filamentswithin the cell cytoplasm. Image intensity and K5 concentration were greatestaround the nucleus of each cell, and a lacy network of delicate filaments projectedout toward the cell extremities. Analysis of confocal images from K5 controls (B)and HD-exposed populations (C) showed a statistically significant (p < 0.01) 29.2%decrease in intensity at 1 hr postexposure to sulfur mustard.


Figure 27.3 Multiphoton keratin 14 images from control HEK cultures (A) showed elaboratecytoskeletal distribution comparable to that of keratin 5. Image intensity and K14concentration were greatest around the nucleus. Lacy networks of filaments pro-jected out to the cell extremities where they interfaced closely with those ofadjacent cells (arrow). At 1 hr postexposure to sulfur mustard, K14 images (B)indicated a disruption of organization, resulting in withdrawal of filaments from theplasma membrane margins, appearance of punctate nodules (arrows), and asubstantial loss of cytoskeletal definition.

Figure 27.4 Analysis of K14 confocal images from replicate cultures of HEK sham-treatedcontrols (A and C) and HD-exposed populations (B and D) showed a statisticallysignificant (p < 0.01) 30.14% decrease in image intensity (K14 expression) at 1hr postexposure and a nearly complete loss of expression (79% decrease in imageintensity) at 2 hr.


Figure 27.5 A multiphoton montage showing the organization of 6 integrins on an explant ofhuman epidermis. Serial slices (A and B) illustrate the receptor outline on suc-cessive cross sections through the epidermal rete pegs. The three-dimensionalreconstruction (C) and the stereo image (D) are from the same Z-series of serialslices. Together, they show the topographic complexity of ventral epidermis plusthe circular shape and extensive distribution of 6 4-integrin receptors.

Figure 27.6 A multiphoton image (slice) ofhuman epidermis showing incross section the distributionand circular shape of 4 inte-grins (arrows) on the basalcell surface.


DISCUSSION

Figure 27.7 Multiphoton images of 6 4 receptors in human epidermis exposed to sulfurmustard indicated unraveling and loss of circular shape at 1 hr postexposure (A)and an almost total loss of 6 4 expression at the basal cell surface by 2 hrpostexposure (B, C). At 2 hr postexposure, only a basolateral pattern of residualfluorescence remained to outline the constituent basal cells. Loss of 6 and 4

integrin also occurred spontaneously in epidermal tissues following dermal–epi-dermal separation; therefore, the effects may not be strictly related to sulfurmustard exposure.


Figure 27.8 Analysis of confocal images from HEK in replicate control and HD-exposed cul-tures indicated a statistically significant (p < 0.01) decrease of 27.3% and 26.3%in image intensity of 6 and 4 integrins, respectively, at 1 hr postexposure. Thedecrease was characterized by a loss of fluorescence from the surface of attachedbasal cells, resulting in a honeycomb pattern of residual, basolateral fluorescence.Postexposure image patterns from cultures were very similar to those recordedfrom intact epidermal tissues (see Figures 27.7B,C).


REFERENCES


313

CHAPTER 28

Suppression of Sulfur Mustard-IncreasedIL-8 in Human Keratinocyte Cell Cultures

by Serine Protease Inhibitors: Implicationsfor Toxicity and Medical Countermeasures*

CONTENTS

INTRODUCTION


METHODS

Reagents

MUSTARD-INDUCED IL-8 315

Sulfur Mustard Exposure

Protease Inhibitors

Table 28.1 Candidate Antivesicant Drug Screening: Statistically Positive Reduction of at Least 50% in Edema or Histopathology in the Mouse Ear or Hairless Mouse

Edema Histopathology

Anti-Inflammatory Drugs

Fluphenazine dihydrochloride 50Indomethacin 63 96Olvanil 53 91Hydrocortisone 65 71Olvanil (saturated) 53Retro olvanil 62 84Olvanil (urea analog) 81Octyl homovanillamide 65 100Dexamethasone 72

Scavenger Drugs

2-Mercaptopyridine-1-oxide 656-Methyl-2-Mercaptopyridine-1-oxide 564-Methyl-2-Mercaptopyridine-1-oxide 57 94Dimercaprol 43 92

Protease Inhibitor

1-(4-aminophenyl)-3-(4-chlorophenyl)urea HCl 54N-(OP)-L-Ala-L-Ala-benzy ester hydrate 631(G-T)-4-(4-methyl phenylsemithiocarbazide) 50

PADPRP Inhibitor

3-(4 -Bromophenyl)ureidobenzamide 74Benzoylene urea 54

Other

Hydrogen peroxide gel, 3% 58

Data generated by the U.S. Army Medical Research Institute of Chemical DefenseAnti-vesicant Drug Screen.


DATA ANALYSIS

RESULTS

DISCUSSION

Figure 28.1 Percent IL-8 of HD-exposed HEK following treatment with TLCK.

TLCK (µM)

Control 0.0 62.5 125.0 250.0 500.0 1000.0

Per

cent

of I

L-8

0

20

40

60

80

100

120


Figure 28.2 Percent IL-8 of HD-exposed HEK following treatment with 1579.

1579 (µM)

Control 0.0 62.5 125.0 250.0 500.0 1000.0

Per

cent

of I

L-8

0

20

40

60

80

100

120

31.25



REFERENCES



323

CHAPTER 29

Development of Medical Countermeasuresto Sulfur Mustard Vesication

CONTENTS

INTRODUCTION


EXPERIMENTAL DESIGN

Decision Tree Network (DTN)

In Vitro Screening Modules

In Vivo Screening Modules

MUSTARD COUNTERMEASURES 325


Basic Research


Candidate Compound Screening

Figure 29.1 The cellular and tissue alterations induced by HD that are proposed to result inblister formation. HD can have many direct effects such as alkylation of proteinsand membrane components (Memb), as well as activation of inflammatory cells.One of the main macromolecular targets is DNA, with subsequent activation ofpoly(ADP-ribose) polymerase (PARP). Activation of PARP can initiate a series ofmetabolic changes culminating in protease activation. Within the tissue, the pen-ultimate event is the epidermal–dermal separation that occurs in the lamina lucidaof the basement membrane zone. Accompanied by a major inflammatory responseand changes in the tissue hydrodynamics (Hyd), fluid fills the cavity formed at thiscleavage plane and presents as a blister.

Table 29.1 Strategies for Pharmacologic Intervention of the HD Lesion

Biochemical Event Pharmacologic Strategy Example

DNA alkylation Intracellular scavengers N-acetyl cysteineDNA strand breaks Cell cycle inhibitors MimosinePARP activation PARP inhibitors NiacinamideDisruption of calcium Calcium modulators BAPTAa

Proteolytic activation Protease inhibitors AEBSFa

Inflammation Antiinflammatories Indomethacin; Olvanil

a BAPTA is a calcium chelator; AEBSF is a sulfonyl fluoride compound.

Proposed Mechanism of HD Action

DNA

Proteaserelease

Breakdown ofepithelial

attachment

Epidermal-dermalseparation

VESICATION

Memb

MetabolicDisruption

Strand Breaks PARP

Toxicity

HydInflam

NAD+

depletion

ProteinsInflammatory

Cells

Inhibition ofglycolysis

Membranes

InflamCa++

Ca++

CELLULAR

TISSUE

HD

MUSTARD COUNTERMEASURES 327

Future

Table 29.2 Candidate Countermeasures with Greater Than 50% Efficacy in Mouse Ear Model

Percentage Reduction in Pathology

Anti-Inflammatory Drugs

Fluphenazine dihydrochloride 50Indomethacin 96Olvanil 91Olvanil (saturated) 53Retro olvanil 84Olvanil (urea analog) 81Octyl homovanillamide 100

Scavenger Drugs

2-Mercaptopyridine-1-oxide 666-Methyl-2-mercaptopyridine-1-oxide 564-Methyl-2-mercaptopyridine-1-oxide 94Dimercaprol 78Na 3-sulfonatopropyl glutathionyl disulfide 64Hydrogen peroxide gel, 3% 58

Protease Inhibitors

1-(4-aminophenyl)-3-(4-chlorophenyl) urea 54N-(OP)-L-Ala-L-Ala-benzy ester hydrate 62Ethyl p-guanidino benzoate hydrochloride 62

PARP Inhibitors

3-(4 -Bromophenyl)ureidobenzamide 74Benzoylene urea 544-Amino-1-naphthol hydrochloride tech 80Total number of positive compounds = 19


CONCLUSIONS

REFERENCES

PART V

Neurotoxicology: Molecular Biomarkers,Transgenics, and Imaging Technologies

SUMMARY


GENOMICS/PROTEOMICS

NEUROTOXICOLOGY: MOLECULAR BIOMARKERS AND IMAGING TECHNOLOGIES 331

IMAGING

SECTION OUTLINE


REFERENCES

333

CHAPTER 30

Molecular Neurotoxicologyof 6-Hydroxydopamine

and Methamphetamine: LessonsDerived from Transgenic Models

CONTENTS

INTRODUCTION


SIX-OHDA-INDUCED NEURODEGENERATION

NEUROTOXICOLOGY OF 6-OHD AND METHAMPHETAMINE 335

METHAMPHETAMINE-INDUCED NEURODEGENERATION

CONCLUSIONS


REFERENCES

Figure 30.1 Molecular neurotoxicology of methamphetamine.

METHAMPHETAMINE

Mitochondria

DNA Damage

Cell Death DNA Fragmentation

Pig3 ROS

↑ Bax, ↓ Bcl2 Caspases

O 2,Cytochrome c∑

∑ O 2 , H2O2,NO

p53




341

CHAPTER 31

A Microassay Method Usinga Neuroblastoma Cell Line to Examine

Neurotoxicity of Organophosphate Mixtures

CONTENTS

INTRODUCTION



IN VITRO ASSESSMENT OF ORGANOPHOSPHATE NEUROTOXICITY 343

RESULTS

Figure 31.1 Determination of optimal cell number per well and reaction time for AChE activity.

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

0 15 30 45 60

Time (min)

Abs

orba

nce

(405

nm

)

500,000

250,000

125,000

62,500

31,000

16,000

8,000


Figure 31.2 Effect of dichlorvos or paraoxon on AChE activity of SH-SY5Y neuroblastomas.*p < 0.05.

Figure 31.3 Effect of dichlorvos (D) or paraoxon (Px) alone or in combination on AChE activityof SH-SY5Y neuroblastomas. *p < 0.05.

***

*

**

*

**

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Control 1.E-07 1.E-06 1.E-05 5.E-05 1.E-04 5.E-04 1.E-03 5.E-03 1.E-02 5.E-02 1.E-01

Concentration Paraoxon (µM)

Abs

orba

nce

(405

nm

)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Control 1.E-02 5.E-02 1.E-01 5.E-01 1.E+00 5.E+00 1.E+01

Concentration Dichlorvos (µM)

Paraoxon Dichlorvos

*

*

*

*

**

* **

*

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

cont

rol

1.0

D

10-1

D

10-2

D

10-3

Px

10-4

Px

10-5

Px

1.0

D/1

0-3

Px

1.0

D/1

0-4

Px

1.0

D/1

0-5

Px

10-1

D/1

0-3

Px

10-1

D/1

0-4

Px

10-1

D/1

0-5

Px

10-2

D/1

0-3

Px

10-2

D/1

0-4

Px

10-2

D/1

0-5

Px

µM Dichlorvos (D) or Paraoxon (Px)

Abs

orba

nce

(405

nm

)

*

IN VITRO ASSESSMENT OF ORGANOPHOSPHATE NEUROTOXICITY 345

DISCUSSION

Table 31.1 Effect of Paraoxon (Px) or Dichlorvos (D) on AChE Activity of SH-SY5Y Neuroblastomas, Significance Level 0.05

OPconcentration

( M)

Absorbanceat 405 nm ±

SEMMeasured

inhibition (%)Predicted

inhibition (%)Significant

vs. control?

control 1.269 ± 0.031 10–2 D 1.373 ± 0.091 –8 10–1 D 1.198 ± 0.033 6 1.0 D 0.831 ± 0.023 34 x 10–5 Px 1.352 ± 0.081 –7 10–4 Px 0.811 ± 0.086 36 x 10–3 Px 0.362 ± 0.024 72 x10–2 D/10–5 Px 1.332 ± 0.042 –5 –15 10–2 D/10–4 Px 0.630 ± 0.077 50 28 x10–2 D/10–3 Px 0.418 ± 0.017 67 63 x10–1 D/10–5 Px 1.370 ± 0.167 –8 –1 10–1 D/10–4 Px 0.647 ± 0.104 49 42 x 10–1 D/10–3 Px 0.453 ± 0.027 64 77 x1.0 D/10–5 Px 0.432 ± 0.151 66 28 x1.0 D/10–4 Px 0.590 ± 0.156 54 71 x1.0 D/10–3 Px 0.339 ± 0.052 73 106 x


REFERENCES

347

CHAPTER 32

Development of Integrin Expressionas a Molecular Biomarker for Early,

Sensitive Detection of Neurotoxicity

CONTENTS

INTRODUCTION


INTEGRIN EXPRESSION AS A BIOMARKER FOR NEUROTOXICITY 349

Figure 32.1 Integrin heterodimers transduce signals both into and out of the cell via interactionswith extracellular ligands and cytoplasmic accessory or regulatory proteins, thecytoskeleton, and signal transduction proteins. Abbreviations: arginine-glycine-aspartic acid peptide (RGD), calreticulin (Cal), focal adhesion kinase (Fak), paxillin(Pax), phosphatidylinositol-3 kinase (PI3), vinculin (Vin).


Figure 32.2 Integrin-mediated cell functions.

TissueOrganization

Cell - CellCommunication

Activation of SignalTransduction Pathways

CellMigration

NeuriteElongation

CellCycle

GeneExpression

CellSurvival

Re-Organization ofCytoskeletal Proteins

INTEGRIN

ReceptorActivation/Clustering

IntracellularAccessory Protein

ExtracellularLigand

+ +



APPROACH


Figure 32.3 Approach to validation of integrin subunit expression levels as molecular biomar-kers of neurotoxicity. Abbreviations: trimethyltin (TMT), methylmercury (MeHg),3,4-methylenedioxymethamphetamine (MDMA), RNAse protection assay (RPA).

Synaptopathic

Cell Lines

Tissue Culture

Function

Expression Levels

Neuropathic

Axonpathic

In Vivo

In Vitro

In Vivo

MDMA

Glial(C6)

Neuronal (PC12, N2A)

Isolated Cells(Astrocytes, Oligoden)

Primary Cultures/Co-Cultures

Proliferation/Differentiation

Cell AdhesionAssay

mRNA(Northerns, RPA)

Protein(Westerns)

Unknown Mechanisms

SuspectedNeurotoxicants

TMT, MeHg

Acrylamide

TMT, MeHgValidateKnownAgents

DevelopIn Vitro

Test Systems

IdentifyIntegrinSubunits

TestKnownAgents

TestNew

Compounds


DISCUSSION



ACKNOWLEDGMENTS

REFERENCES




361

CHAPTER 33

Two-Photon Fluorescence Microscopy:A Review of Recent Advances

in Deep-Tissue Imaging

CONTENTS

INTRODUCTION


Figure 33.1 Jablonski diagrams for (a) one-photon and (b) two-photon excitation. One-photonexcitation occurs through the absorption of a single photon. The two-photonprocess occurs through the simultaneous absorption of two lower energy photons.After either excitation process, the fluorophore relaxes to the lowest energy levelof the first excited electronic state. The subsequent fluorescence emission processis independent of the mode of excitation.

TWO-PHOTON FLUORESCENCE MICROSCOPY: DEEP-TISSUE IMAGING 363

BASIC TWO-PHOTON MICROSCOPY INSTRUMENTATION


DEEP TISSUE IMAGING BASED ON TWO-PHOTON MICROSCOPY

Figure 33.2 A schematic of two-photon fluorescence microscope design.


RECENT ADVANCES IN TWO-PHOTON MICROSCOPY

Video Rate Two-Photon Microscopy


Figure 33.3 Two-photon autofluorescence images of human skin. Top left: stratum corneum.Top right: basal layer. Bottom: fibrous dermal layer (Zeiss Fluar 100 oil).


Enhancing Image Resolution Based on Maximum Likelihood Deconvolution


Two-Photon Spectral Characterization of Tissue Biochemistry

Figure 33.4 Image restoration of two-photon images of ex vivo human skin using maximumlikelihood approach. Autofluorescence (left) and blind deconvoluted (right) imagesof human basal layer. Top: lateral view. Bottom: axial section (Zeiss Fluar 40 oil).



Figure 33.5 Two independent spectral components isolated in ex vivo human skin based onSMCR: (a) a spectral component corresponds to elastin fibers in the dermis and(b) a spectral component corresponds to melanin (or a fluorophore that colocalizeswith melanin) in the epidermal–dermal junction. For (a) and (b): (left) a two-dimensional image of the concentration distribution of the spectral component;(right, top) the spectrum of this component; (right, bottom) the depth distributionprofile of this component.


CONCLUSIONS

REFERENCES



PART VI

Role of Transgenics and Toxicogenomics inthe Development of Alternative Toxicity Tests


377

CHAPTER 34

The Application of Genomics andProteomics to Toxicological Sciences

CONTENTS

INTRODUCTION


TOXICOGENOMICS AND PROTEOMICS 379

MICROARRAY TECHNOLOGIES



PROTEOMIC TECHNOLOGIES


Figure 34.1 Schematic of genomic and proteomic technologies: The first step in toxicoge-nomics (right) is the construction of a microarray that involves the amplificationby PCR and the immobilization of known DNA sequences (either cDNA oroligonucleotides) on a solid support. The mRNA prepared from a biologicalmodel can be labeled and hybridized to the microarray and visualized usingphosphorimager scanning. Subsequent bioinformatic analyses using appropriatesoftware allows determination of the extent of hybridization of the labeled probesto the corresponding arrayed cDNA spots, and a comparison of control with testsamples permits quantitative assessment of changes in gene expression asso-ciated with treatment. Total protein content from a biological model treated witha toxicant is separated on two-dimensional gel electrophoresis according toisoelectric point (first dimension) and molecular weight (second dimension),allowing bioinformatic analysis of differences in protein expression of treatedversus untreated samples. Therefore, there is no preliminary work such as arrayconstruction for proteomic studies (left), but proteins with altered expressionhave to be identified subsequently. Individual proteins of interest are excisedfrom two-dimensional-gels, digested with trypsin, and applied to a mass spec-trometer. Identification of these proteins is obtained by searching protein data-bases with mass spectrometry data.

Biological model

Sample preparationTreated vs. untreated

SDS-PAGE (2nd D)

Mass spectrometry

Protein database search

Protein identity

Bioinformatic analysis

Protein sample mRNA sample isolation

Research lead Toxicological marker

Protein expression and posttranslational modification

cDNA labeling

Hybridization to microarray

cDNA selection

PCR amplification

Microarray construction

Iso electrofocusing (1st D)

mRNA expressed

Toxicant signature


TOXICOGENOMICS APPLICATIONS


PROTEOMICS APPLICATIONS


THE CHALLENGE OF PATTERN RECOGNITION


OPPORTUNITIES IN COMBINATION: A PERSPECTIVE


REFERENCES




391

CHAPTER 35

Use of Transgenic Animals in RegulatoryCarcinogenicity Evaluations*

CONTENTS

INTRODUCTION


TRANSGENIC ANIMALS IN REGULATORY CARCINOGENICITY EVALUATIONS 393


COMMERCIAL COMPLICATIONS

OVERVIEW OF INDIVIDUAL MODELS


Tg.AC

Table 35.1 IARC Class I or 2A Human Carcinogen, or NTP Reasonably Anticipated Human Carcinogen

Tg.ACTopical TgrasH2 p53+/– XPA–/–/p53+/–

GenotoxicBenzene + + + nd*Benzo(a)pyrene + nd + +Cyclophosphamide – + + nd7,12-Dimethylbenzanthracene + nd + +**Melphalan – +/– + ndPhenacetin – + – –Procarbazine nd + nd nd

NongenotoxicCyclosporin A + +/– + +Diethylstilbestrol + + + +17- -estradiol (or ethinyl estradiol#) +# – +/– +Oxymetholone + nd – nd2,3,7,8-TCDD + nd nd

* nd: no adequate data available on the performance of the compound in that model.**Positive in 6 month XPA–/– and positive in 6 month p53+/–; not tested in XPA–/–/P53+/–

bitransgenic.

Table 35.2 Genotoxic Trans-Species Rodent Carcinogens

Tg.ACTopical TgrasH2 P53+/– XPA–/–/P53+/–

p-Cresidine – + + +2,4-Diaminotoluene + nd – ndDiethylnitrosamine nd + nd ndDimethylnitrosamine nd + + ndN-Ethylnitrosourea nd + + ndGlycidol – + – ndN-Methylnitrosourea nd + + ndPhenolphthalein nd – + ndThiotepa nd + nd ndUrethane nd + + nd4-Vinyl-1-cyclohexene-diepoxide* – + + nd

* Applied dermally to each model tested.


Table 35.3 Nongenotoxic Rodent Carcinogens and Human Carcinogenicity Unlikely or Uncertain


Chlorpromazine nd – nd ndClofibrate + + – ndDieldrin nd nd – ndDiethylhexylphthalate – + +/– –Haloperidol nd – – –D-Limonene nd nd – ndMetaproterenol nd – – ndPentachlorophenol + nd – ndPhenobarbital – – – –Reserpine – – – –Sulfamethoxazole – – – –WY-14643 – + – +*

* Positive in 6 month XPA–/–, not tested in XPA–/–/p53+/– bitransgenic.

Table 35.4 Rodent Noncarcinogens


Genotoxicp-Anisidine – – – nd2-Chloroethanol – nd nd nd1-Chloro-2-propanol – nd – nd2,6-Diaminotoluene – nd – nd8-Hydroxy-quinoline – – – nd

NongenotoxicAmpicillin nd – nd ndBenzethonium chloride – nd nd ndD-Mannitol nd – nd –Oleic acid diethanolamine – nd – ndPhenol – nd nd ndResorcinol + – – ndRotenone – – – ndSulfisoxazole – – nd nd



TgrasH2



P53+/– MODEL



XPA–/–/P53+/–



REGULATORY EXPERIENCE


SUMMARY AND CONCLUSIONS



REFERENCES





413

CHAPTER 36

Changes in Gene Expression after Exposureto Organophosphorus (OP) Agents

CONTENTS


INTRODUCTION

CHANGES IN GENE EXPRESSION AFTER EXPOSURE TO AGENTS 415

METHODS

Dosing Regimen

Collection of Tissue


Preparation of Total RNA and mRNA

Figure 36.1 Rat toxicology U34 gene array. This gene expression display was created byGeneSpring® (Silicon Genetics) gene array analysis software from our data (ratbrain RNA 1 h postexposure to CPF) read from an Affymetrix Rat Toxicology U34GeneChip®. In the original display (depicted in gray tones here), red and purpleblocks represented up-regulated genes and the blue represented down-regulatedgenes. Gray blocks represented genes whose expression is essentially the sameas in the control animal.


Synthesis of Biotin-Labeled cRNA and Target Preparation

Hybridization, Staining, and Washing of DNA Microarray

Probe Array Scan

Analysis of DNA Microarray Data

Measurement of Butylcholinesterase (BChE) Activity

RESULTS

Selection of Genes to Measure


Analysis of Gene Expression Patterns

Quantification of Gene Expression Patterns in the Brain

Figure 36.2 Illustrations of the six expression pattern categories. The numerals under the x-axis represent hours after CPF exposure. Y-axis represents relative gene expres-sion level. (A) No alteration of gene expression; (B) initial up-regulation, thenreturn to normal by 24 h; (C) initial up-regulation, then return to normal by 4 h;(D) delayed up-regulation by 4 h, then return to normal by 24 h; (E) delayed up-regulation by 24 h; (F) rapid down-regulation, then return to normal by 4 h.

14 24

14 24

14 24

14 24

14 24

14 24

A. B.

C. D.

E. F.


Quantification of Gene Expression Patterns in the Liver

Figure 36.3 The relative percentages of the six patterns of gene expression detected in theCPF-exposed rats.

X12%

F4% E

7% D7%

C13%

B23%

A34%

A.

X23%

F10%

E10%

D7%

C 11%

B 5%

A34%

B.

A33%

B 9%

C 5%

D14%

E6%

F4%

X29%

C.


Identification of Up-Regulated Genes Involved in Key Cellular Functions and Biochemical Pathways

Figure 36.4 Scatterplot of data from rat toxicology U34 GeneChip.


Identification of Down-Regulated Genes Involved in Key Cellular Functions and Biochemical Pathways

Measurement of BChE Activity in Blood of CPF-Exposed Rats

Figure 36.5 In vivo effects of CPF on BuChE activity.

Effect of CPF on Rat BuChE Activity

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Control 1 4 24

Hours Post-Exposure

Bu

Ch

e A

ctiv

ity

(O.D

. at

412

nm

)


DISCUSSION





REFERENCES


PART VII

Recent Innovations in Alternatives

431

CHAPTER 37

Archival Data in Toxicology: MinimizingNeed for Animal Experiments

CONTENTS


INTRODUCTION

Perspective

PROBLEM SOLVING

ARCHIVAL DATA IN TOXICOLOGY: MINIMIZING NEED FOR ANIMAL EXPERIMENTS 433

INHALATION TOXICOLOGY OF HYDROGEN CYANIDE

Modeling Approaches

Relevant Toxicological Features and Methodology


Figure 37.1 Comparison of toxicological features of HCN and nerves gases, such as sarin.The larger arrow represents inhaled HCN. The smaller arrow represents theeffective dosage, after detoxification and loss of the 30% of inhaled HCN that isexhaled (Moore and Gates, 1946), leaving 0.7 retained. HCN concentrations upto 30 mg/m3 are normally neutralized by human detoxification systems beforeeffects become observable (Prentiss, 1937).


Figure 37.2 Contrasting plots of data from the same rat exposures to HCN (after Ballantyne,1987). One plot (left) reflects use of exposures with fixed duration and varied HCNconcentrations. The steep initial slope shows that HCN must be very concentratedfor a small number of breaths to deliver a lethal dosage. The second plot presentsthe data results as customarily displayed, given a fixed toxic gas concentrationand varied exposure durations. LCt50 values tend to increase as more timebecomes available for detoxification.

Figure 37.3 Comparison of responses from different strains of rats under similar HCN exposureconditions (after Levin et al., 1985). Log–log plots of the data reveal differencesof effective HCN concentrations and probit slopes of lines but do not show whichstrain better represents the human race.


Mechanisms and Rationales

Extrapolations of Human Data

Figure 37.4 Relationships of anatomical and pharmacological factors leading to lethality formammals that inhale HCN. Nonionized HCN is rapidly absorbed into blood tomove the short distance from alveoli to the sensor. The sensor response (or lackof a signal) communicates a need for oxygen to respiratory neurones that initiatehyperventilation. Increased minute volumes multiply HCN intake until respiratorycenter poisoning leads to apnea.


A Biological Common Denominator

Table 37.1 Hydrogen Cyanide Concentrations and Effects Associated with Various HCN Exposure Conditions in Animals and Man

Mg/m3 Effects Ref.

30,000a Rat, inhalation LC50 Levin et al., 19859,300b Men, 11/11 hyperventilate Bodansky and Helm, 19447,740b Men, “most hyperventilate” Cope and Abramovitz, 19594,000a Rat, inhalation LC50 Ballantyne, 19873,200b Men, “50% hyperventilate” Wexler et al., 19472,230 Pig, LC50 within 2 min of inhalation Stemler et al., 1994

a Lethal concentration for 50% of subject rats, 0.1 min inhalation exposure.b Equivalent HCN mg/m3 dosage calculated for intravenous sodium cyanide solution.


Figure 37.5 Observed breathing rates and blood cyanide concentrations of three miniaturepigs exposed to HCN for 2 min (after Stemler et al., 1994). Values for bloodconcentrations of cyanide, at the top and right side of Figure 37.5, are presentedwith the corresponding respiratory rate values. Although data acquired after theonset of HCN exposure for 2 min are very limited, they are consistent with otherindicators that an aortic blood HCN concentration of 4 mg/m3 at 5 min is a thresholdvalue for a lethal outcome.

Figure 37.6 Average whole blood cyanide levels in dogs after four continuous intravenousslow infusion trials (1 mg/kg/min) with NaCN solution at 4.0 mg/ml (after Vicket al., 2000). RA indicates respiratory arrest and cessation of NaCN infusion.Methemoglobin formation by 20 mg/ml hydroxylamine hydrochloride solution wasinitiated 30 sec after RA. Although 3.6 g/kg was the average value observedat RA, it appears that survival is dependent upon avoidance of blood cyanideconcentrations above 4 g/kg.


Acute and Peracute Exposures


Lessons Learned

PROJECTION OF DELAYED EYE EFFECTS OF MUSTARD EXPOSURES

Vive la Difference!


Dose Responses—with Time

Dosage-Degree-Duration


Applications of Figure 37.7

Figure 37.7 Triaxial depiction that was hand drawn to integrate (1) time course informationreported hourly or by the day; (2) mustard vapor dosage information; and (3) levelsof human effect severity for the given time and dosage. The dashed line representsdata from one particularly relevant experiment (Unde and Dunphy, 1944) amongmore than 100 experimental reports that were considered.


ESTIMATION OF PERFORMANCE DEGRADATION BY “FOOD POISONING”

Traveler’s Diarrhea


From Cases to Predictions

Figure 37.8 Bar chart designed to illustrate relative severity of enteric disease effects ascorrelated with time after arrival of a mixed population in Mexico City. Each of the19 represented cases involved isolation of enterotoxigenic Escherichia coli organ-isms that accounted for 45% of traveler’s diarrhea cases observed during thisprospective study (Merson et al., 1976).


SUMMARY

ACKNOWLEDGMENTS

Table 37.2 Estimates of Percent of Military Personnel with Indicated Degree of Performance-Degradation per Day after Onset of Gastrointestinal Symptoms/Signs from Infection with Enterotoxigenic Escherichia coliBacteriaa

Percentage of cases with degraded military performanceDay of Onsetb

% New Casesc No.d (25%)c No.e (50%)c No.f (100%)c

Recoveredg

(%)c

1 (14.6) 9 (47.4) 7 (36.8) 3 (15.5) 0 (0)2 (19.0) 7 (36.8) 7 (36.8) 3 (15.8) 2 (10.5)3 (19.3) 4 (21.1) 10 (52.6) (0) 5 (26.3)4 (17.1) 4 (21.1) 8 (42.1) (7) (36.8)5 (9.8) 2 (10.5) 8 (42.2) (9) (47.4)6 (6.1) 2 (10.5) 6 (31.6) (11) (57.9)7 (4.1) 4 (21.1) 3 (15.8) (12) (63.2)8 (2.9) 4 (21.1) 2 (10.5) (13) (68.4)9 (2.0) 3 (15.8) 1 (5.3) (15) (78.9)

10 (1.5) 3 (15.8) (0) (16) (84.2)11 (1.3) 2 (10.5) (17) (89.5)12 (1.1) 1 (5.3) (18) (94.7)13 (0.7) 0.5 (2.6) (18.5) (97.4)14 (0.5) (0) (19) (100.0)

a Percentages based upon data from 19 proven cases of enterotoxigenic Escherichia coliinfection acquired in Mexico City (Merson et al., 1976).

b Onset day 1 is day 3 from exposure to infection.c Calculated from daily rate curve (Fischer and Mershon, 1994).d Cases (9) with uncomplicated traveler’s diarrhea, performance 25% degraded on day 1.e Cases (7) with changed activities, performance 50% degraded during onset day 1.f Cases (3) with recovery time in bed; performance 100% degraded during onset day 1.g Cases (19) all had some degree of incapacitation during onset day 1.


REFERENCES

Figure 37.9 This cartoon is included to suggest that modeling represents a combination of artand science. The art of modeling methodology is applied when conventional testingmethods are not applicable for collection of experimental results. In each case,previously collected data were reacquired and analyzed to provide a best possibleestimate of reality.


449

CHAPTER 38

Information Management at the Library ofCongress: An Overview with Special

Reference to Biomedicine*

CONTENTS


INTRODUCTION

A HISTORICAL SKETCH

INFORMATION MANAGEMENT AT THE LIBRARY OF CONGRESS 451


CURRENT HOLDINGS

SCIENCE AND TECHNOLOGY


CLIENT SERVICES STATISTICS

INFORMATION MANAGEMENT

In the Beginning…


Current LC Practices


A Historical Perspective


SUBJECT-BASED ACCESS TO INFORMATION

Preamble


Subject Headings: General Information


Rationale for Subject Headings


The Subject Authority Record

Subject Analysis: General Principles

Tag 010053150450450550550550

Field Data_a sh 85064594_a RC582.17_a Immunotoxicology_a Immunologic toxicology_a Immunotoxicity _w g _a Immunopathology_w g _a Toxicology_a Immunopharmacology

ExplanationsRecord NumberClass NumberSubject HeadingUF (Use For Term)UF (Use For Term)BT (Broader Term)BT (Broader Term) RT (Related Term)

Figure 38.1 LC authority record for Immunotoxicology, supplemented with explanatory notations.



Subject Headings: Select Biomedical Examples


Select Subdivision Examples


LCSH: Coda

INFORMATION RETRIEVAL


The Futility Point


THE MARC RECORD*

Genetic toxicology / editors, Albert P. Li, Robert H. Heflich. -- Boca Raton : CRC Press, cl991.x, 493 p. : ill. ; 24 cm.

Includes bibliographical references. Includes index. ISBN 0-8493-8815-3

1. Genetic toxicology. I. Li, A. P. II. Heflich, Robert U., 1946-

[DNLM: 1. Carcinogens. 2. Chromosome Abnormalities--chemically in-duced. 3. Mutagens--adverse effects. 4. Mutation. QH 465.C5 G328] RA1224.3.G457 1991 615.9'02--dc20 90-11279

DNLM/DLCfor Library of Congress

Figure 38.2 Traditional LC catalog card for Genetic Toxicology, Li, AP, and Heflich, RH,editors, 1991.


Tag Ind I Ind 2 Field Data 000 01037pam__2200325_a_4500 001 996520 005 9910520155750.7 008 900824sl991____njua_____b____001_0_eng_c

035 _9 (DLC) 90011279 906 _a 7 _b cbc _c orignew _d 1 _e ocip _f 19 _g y-gencatlg 955 _a CIP ver. ea10 to SL 05-13-91 010 _a 90011279 020 _z 0849388153 040 _a DNLM/DLC _c DLC _d DLC 050 0 0 _a RA1224.3 _b .G457 1991 060 _a QH 465.C5 G328 082 0 0 _a 615.9/02 _2 20 245 0 0 _a Genetic toxicology / _c editors, Albert P. Li, Robert H. Heflich. 260 _a Boca Raton : _b CRC Press, _c cl991. 300 _a x, 493 p. : _b ill. ; _c 24 cm. 504 _a Includes bibliographical references. 500 _a Includes index. 650 0 _a Genetic toxicology. 650 2 _a Carcinogens. 650 2 _a Chromosome Abnormalities _x chemically induced. 650 2 _a Mutagens _x adverse effects. 650 2 _a Mutation. 700 1 _a Li, A. P. 700 1 _a Heflich, Robert H., _d 1946- 991 _b c-GenColl _h RA1224.3 _i .G457 1991 _t Copy 1 _w

Figure 38.3 LC MARC record for Genetic Toxicology, Li, AP, and Heflich, RH, editors, 1991.



EPILOGUE

Genetic toxicology / editors, Albert P. Li, Robert H. Heflich

LC Control Number: 90011279Main Title: Genetic toxicology / editors, Albert P. Li, Robert H. Heflich

Published/Created: Boca Raton : CRC Press, c1991.Related Names: Li, A. P.

Heflich, Robert H., 1946-Description: x, 493 p.,: ill.; 24 cm.

ISBN: 0849388153Notes: Includes index.

Includes bibliographical references.Subjects: Genetic toxicology.

Carcinogens.Chromosome Abnormalities--chemically induced.Mutagens--adverse effects.Mutation.

LC Classification: RA1224.3 .G457 1991NLM Class No.: QH465.C5 G328

Dewey Class No.: 615.9/02 20

Figure 38.4 LC OPAC Full Record display for Genetic Toxicology, AP Li, and RH Heflich,editors, 1991.



REFERENCES



473

CHAPTER 39

World Wide Web Biomedical, Chemical,and Toxicological Information Resources

from the National Library of Medicine

CONTENTS

INTRODUCTION AND BACKGROUND


BIBLIOGRAPHIC DATABASES

FACTUAL DATABASES

WEB RESOURCES FROM THE NATIONAL LIBRARY OF MEDICINE 475


Figure 39.1 TEHIP homepage.

WEB RESOURCES FROM THE NATIONAL LIBRARY OF MEDICINE 477

REFERENCES

Figure 39.2 TOXNET’s page search on “acrylamide.”


NIH/NLM URL PAGES

479

CHAPTER 40

In Silico Approaches for PhysiologicallyBased Pharmacokinetic Modeling

CONTENTS


INTRODUCTION

IN SILICO APPROACHES FOR PBPK MODELING 481

METHODOLOGICAL BASIS OF IN SILICO APPROACHES

QSARs

LFE-Type Models

Electrostatic Features in LFE-Type Models


Steric Features in LFE-Type Models

Hydrophobic Features in LFE-Type Models


Free–Wilson Type Models


Biologically Based Algorithms

IN SILICO APPROACHES FOR PBPK MODEL PARAMETERS

In Silico Approaches for Tissue:Air Partition Coefficients


Tab

le 4

0.1

In S

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Ap

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ach

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lass

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ipos

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MW

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es; L

MW

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and

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(199

4)

log

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dney

:air

= –

1.08

4 +

0.4

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26 +

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24 +

2.9

26 +

0.

534

log

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es; L

MW

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(199

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+ 0

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he:a

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es; L

MW

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Cs

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aham

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(199

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log

Plu

ng:a

ir=

–1.

300

+ 0

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+ 3

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.35

+ 0

.458

lo

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es; L

MW

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Abr

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HIn

ert

Gas

es; L

MW

VO

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

Ste

ric

des

crip

tors

log

Pad

ipos

e:ai

r=

(0.

7341 x

v ) –

(0.

029

) –

(1.5

7(1/

1 x))

– (

0.55

9(1/

1 xv )

) –

0.09

83+

2.2

13

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

(con

tinue

d)


Tab

le 4

0.1

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e T

issu

e:A

ir P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

log

Pad

ipos

e:ai

r=

0.7

341 X

v –

0.02

91 –

1.5

70/1 X

v–

0.55

91 Xv

– 0.

0984

+ 2

.213

RH

aloa

lkan

esC

sana

dy a

nd L

aib

(199

0)

log

Pad

ipos

e:ai

r=

0.5

63N

Cl+

1.0

28N

Br+

0.4

67N

C+

0.2

70Q

H–

0.19

9NF

–0.

097

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

log

Pad

ipos

e:ai

r=

1.0

371 x

v –

(0.0

07(1

/))

+ 0

.022

QH

– 0.

1773

– 0.

199N

F

– 0.

0036

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

log

Pliv

er:a

ir=

(1.

0721 x

v ) –

(0.

021(

1/))

+ (

0.64

7(1/

1 xv )

) –

(0.3

044

) –

1.21

2

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

log

Pliv

er:a

ir=

0.3

66N

Cl–

0.58

8NB

r+

0.3

45Q

H–

0.17

9NF

– 0.

007

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

log

Pliv

er:a

ir=

–0.

6851 x

v –

(0.0

20(1

/))

+ 0

.232

QH

+ (

0.29

8(1/

1 xv )

) +

0.

104N

Cl–

0.72

6

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

log

Pliv

er:a

ir=

1.0

721 X

v –

0.02

1/ +

0.6

47/1 X

v–

0.30

44–

1.21

2R

Hal

oalk

anes

Csa

nady

and

Lai

b (1

990)

log

Pm

uscl

e:ai

r=

0.3

79Q

H–

0.27

8NC

l+

0.5

36N

Br–

0.19

0NF

+ 0

.169

NC

l–

0.43

9R

Hal

oalk

anes

Gar

gas

et a

l. (1

988)

log

Pm

uscl

e:ai

r=

0.3

991 x

v –(0

.007

(1/

)) +

0.2

95Q

H+

0.2

594

– 0.

194N

F

– 0.

217

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

log

Pm

uscl

e:ai

r=

(0.

9951 x

v ) –

(0.

018(

1/))

– (

0.42

44)

– (0

.559

(1/1 x

v ) +

(0.6

02(1

/1 xv )

) –

1.33

4

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

Hyd

rop

ho

bic

des

crip

tors

log(

Pad

ipos

e:w

ater

–V

wt)

= 0

.9P

o:w

+ 0

.31

FC

hlor

oeth

anes

; Ben

zene

Ber

tels

en e

t al

. (19

88)

log(

Pki

dney

:wat

er–

Vw

t) =

0.7

2Po:

w–

0.56

FC

hlor

oeth

anes

; Ben

zene

Ber

tels

en e

t al

. (19

88)

log(

Pliv

er:w

ater

–V

wt)

= 1

.06P

o:w

– 1.

43F

Chl

oroe

than

es; B

enze

neB

erte

lsen

et

al. (

1988

)lo

g(P

mus

cle:

wat

er–

Vw

t) =

0.6

3Po:

w–

0.60

FC

hlor

oeth

anes

; Ben

zene

Ber

tels

en e

t al

. (19

88)

lnP

adip

ose:

air=

0.0

32T

b –

5.45

6H

Hal

oalk

anes

Csa

nady

and

Lai

b (1

990)


lnP

liver

:air

= 0

.022

Tb

–4.

638

HH

aloa

lkan

esC

sana

dy a

nd L

aib

(199

0)lo

gP

adip

ose:

air=

0.2

09 +

0.0

628

log

Pw

:a+

0.8

868

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pad

ipos

e:ai

r=

0.2

1 lo

g P

o:a

+ 0

.24

log

Pw

:aH

Hyd

roph

ilic

VO

Cs

Tic

hy (

1991

b)lo

gP

adip

ose:

air=

0.7

82 lo

g P

o:a

+ 0

.201

log

Pw

:a+

0.4

32H

Hyd

roph

obic

VO

Cs

Tic

hy (

1991

a)lo

gP

adip

ose:

air=

0.9

01 lo

g P

o:a

+ 0

.150

HLM

WV

OC

sF

iser

ova-

Ber

gero

va e

t al

. (1

984)

log

Pad

ipos

e:ai

r=

0.1

74 +

0.9

10 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

brai

n:ai

r=

–0.

16 lo

g P

o:a

+ 0

.82

log

Pw

:a+

0.4

7H

Hyd

roph

ilic

VO

Cs

Tic

hy (

1991

b)lo

gP

brai

n:ai

r=

0.2

74 +

0.5

37 lo

g P

w:a

+ 0

.444

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pbr

ain:

air=

0.3

94 +

1.0

96 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

brai

n:ai

r=

0.4

71 lo

g P

o:a

+ 0

.630

log

Pw

:a–

0.30

5H

Hyd

roph

obic

VO

Cs

Tic

hy (

1991

a)lo

gP

brai

n:ai

r=

0.8

44 lo

g P

o:a

– 1.

124

HLM

WV

OC

sF

iser

ova-

Ber

gero

va e

t al

. (1

984)

log

Pbr

ain:

air=

–0.

850

+ 0

.773

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pbr

ain:

air=

–3.

692

+ 1

.253

RG

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

kidn

ey:a

ir=

–0.

18 lo

g P

o:a

+ 0

.82

log

Pw

:a+

0.5

3H

Hyd

roph

ilic

VO

Cs

Tic

hy (

1991

b)lo

gP

kidn

ey:a

ir=

0.2

77 +

1.1

11 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

kidn

ey:a

ir=

0.4

66 lo

g P

o:a

+ 0

.379

log

Pw

:a–

0.33

2H

Hyd

roph

obic

VO

Cs

Tic

hy (

1991

a)lo

gP

kidn

ey:a

ir=

0.7

00 lo

g P

o:a

– 0.

877

HLM

WV

OC

sF

iser

ova-

Ber

gero

va e

t al

. (1

984)

log

Pki

dney

:air

= –

0.92

0 +

0.7

64 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

liver

:air

= –

0.38

8 +

0.5

02 lo

g P

w:a

+ 0

.497

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pliv

er:a

ir=

0.4

32 +

1.0

64 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

liver

:air

= 0

.746

log

Po:

a+

0.1

78 lo

g P

w:a

– 0.

767

HH

ydro

phob

ic V

OC

sT

ichy

(19

91a)

log

Pliv

er:a

ir=

0.8

71 lo

g P

o:a

– 1.

044

HLM

WV

OC

sF

iser

ova-

Ber

gero

va e

t al

. (1

984)

log

Pliv

er:a

ir=

–0.

875

+ 0

.773

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Plu

ng:a

ir=

–0.

21 lo

g P

o:a

+ 0

.91

log

Pw

:a+

0.4

1H

Hyd

roph

ilic

VO

Cs

Tic

hy (

1991

b)lo

gP

lung

:air

= –

0.05

7 +

0.8

70 lo

g P

w:a

+ 0

.146

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Plu

ng:a

ir=

0.0

57 +

0.9

78 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

) (con

tinue

d)


Tab

le 4

0.1

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e T

issu

e:A

ir P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

log

Plu

ng:a

ir=

0.3

73 lo

g P

o:a

+ 0

.416

log

Pw

:a–

0.21

6H

Hyd

roph

obic

VO

Cs

Tic

hy (

1991

a)lo

gP

lung

:air

= 0

.644

log

Po:

a–

0.81

5H

LMW

VO

Cs

Fis

erov

a-B

erge

rova

et

al.

(198

4)lo

gP

lung

:air

= –

0.83

3 +

0.9

11 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

mus

cle:

air=

–0.

19 lo

g P

o:a

+ 0

.82

log

Pw

:a+

0.5

4H

Hyd

roph

ilic

VO

Cs

Tic

hy (

1991

b)lo

gP

mus

cle:

air=

0.4

9 lo

g P

o:a

+ 0

.39

log

Pw

:a–

0.31

HH

ydro

phob

ic V

OC

sT

ichy

(19

91b)

log

Pm

uscl

e:ai

r=

–0.

263

+ 0

.575

log

Pw

:a+

0.4

23 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

mus

cle:

air=

0.3

51 +

1.1

08 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

mus

cle:

air=

0.6

52 lo

g P

o:a

– 0.

702

HLM

WV

OC

sF

iser

ova-

Ber

gero

va e

t al

. (1

984)

log

Pm

uscl

e:ai

r=

–0.

852

+ 0

.768

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pm

uscl

e:ai

r=

–3.

247

+ 0

.965

RG

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)P

adip

ose:

air=

0.4

47P

o:a

+ 0

.075

Pw

:a+

6.5

9H

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

brai

n:ai

r=

(0.

026S

o+

0.5

1Sw)/

Sa

HLM

WV

OC

sP

ater

son

and

Mac

kay

(198

9)P

brai

n:ai

r=

0.0

20P

o:a

+ 0

.380

Pw

:a+

0.9

4H

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

kidn

ey:a

ir=

(0.

014S

o+

0.5

1Sw)/

Sa

HLM

WV

OC

sP

ater

son

and

Mac

kay

(198

9)P

kidn

ey:a

ir=

0.0

11P

o:a

+ 0

.400

Pw

:a+

0.6

9H

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

kidn

ey:a

ir=

–0.

391

+ 0

.550

log

Pw

:a+

0.4

40 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)P

liver

:air

= (

0.02

8So

+ 0

.51S

w)/

Sa

HLM

WV

OC

sP

ater

son

and

Mac

kay

(198

9)P

liver

:air

= 0

.028

Po:

a+

0.7

9H

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

mus

cle:

air=

0.0

14P

o:a

+ 0

.384

Pw

:a+

0.9

4H

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)ln

Pad

ipos

e:ai

r=

0.0

32T

b–

5.45

6R

LMW

VO

Cs

Csa

nady

and

Lai

b (1

990)

lnP

liver

:air

= 0

.022

Tb

– 4.

638

RLM

WV

OC

sC

sana

dy a

nd L

aib

(199

0)lo

gP

adip

ose:

air=

0.9

20 lo

g P

o:a

+ 0

.136

RLM

WV

OC

sG

arga

s et

al.

(198

9)lo

gP

adip

ose:

air=

0.9

27 lo

g P

o:a

– 0.

032

log

Pw

:a+

0.1

20R

LMW

VO

Cs

Gar

gas

et a

l. (1

989)


log

Pad

ipos

e:ai

r=

1.0

27 lo

g P

o:a

– 0.

046

log

Pw

:a–

0.11

9R

Hal

oalk

anes

Gar

gas

et a

l. (1

988)

log

Pliv

er:a

ir=

0.5

74 lo

g P

o:a

+ 0

.302

log

Pw

:a–

0.27

8R

Hal

oalk

anes

Gar

gas

et a

l. (1

988)

log

Pliv

er:a

ir=

0.7

30 lo

g P

o:a

+ 0

.128

log

Pw

:a–

0.55

0R

LMW

VO

Cs

Gar

gas

et a

l. (1

989)

log

Pm

uscl

e:ai

r=

0.4

77 lo

g P

o:a

+ 0

.365

log

Pw

:a–

0.37

4R

Hal

oalk

anes

Gar

gas

et a

l. (1

988)

log

Pm

uscl

e:ai

r=

0.6

44 lo

g P

o:a

+ 0

.180

log

Pw

:a–

0.72

5R

LMW

VO

Cs

Gar

gas

et a

l. (1

989)

Pad

ipos

e:ai

r=

0.5

94P

o:a

+ 0

.085

Pw

:a+

9.4

0R

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

brai

n:ai

r=

0.0

54P

o:a

+ 0

.832

Pw

:aR

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

kidn

ey:a

ir=

0.0

97P

o:a

+ 0

.826

Pw

:aR

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

liver

:air

= 0

.026

Po:

a+

0.8

78P

w:a

+ 2

.36

RLM

WV

OC

s; C

FC

sM

eule

nber

g an

d V

ijver

berg

(2

000)

Pm

uscl

e:ai

r=

0.0

10P

o:a

+ 0

.772

Pw

:a+

0.2

9R

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)

Bio

log

ical

ly b

ased

alg

ori

thm

s

Ptis

sue:

air=

(S

sVw

t+

SvV

nt+

0.7

SsV

pt+

0.3

SvV

pt)/

Sa

R,

HLM

WV

OC

sP

oulin

and

Kris

hnan

(19

96a)

Ptis

sue:

air=

Po:

wP

w:a(V

nt+

0.3

Vpt)

+ P

w:a(V

wt+

0.7

Vpt)

R,

HLM

WV

OC

sP

oulin

and

Kris

hnan

(19

96c)

a=

dip

olar

ity/p

olar

izab

ility

, =

ove

rall

hydr

ogen

-bon

d ac

idity

, =

ove

rall

hydr

ogen

-bon

d ba

sici

ty,

1 Xv ,

,1 X,4

,3

,4 X

vpc

= c

onne

ctiv

ityin

dice

s, N

Br=

num

ber

of b

rom

ide

atom

s in

the

mol

ecul

e, N

C=

num

ber

of c

arbo

n at

oms

in th

e m

olec

ule,

NC

l=

num

ber

of c

hlor

ide

atom

s in

the

mol

ecul

e,N

F=

num

ber

of fl

uorid

e at

oms

in t

he m

olec

ule,

Phe

:a=

hex

adec

ane:

air

part

ition

coe

ffici

ent,

Po:

a=

n-oc

tano

l:air

part

ition

coe

ffici

ent

(or

vege

tabl

e oi

l:air)

,P

o:w

= n

-oct

anol

:wat

er p

artit

ion

coef

ficie

nt (

or v

eget

able

oil:

wat

er),

Pw

:a=

wat

er:a

ir pa

rtiti

on c

oeffi

cien

t, Q

H=

var

iabl

e de

pend

ant

on t

he p

olar

ity o

f th

em

olec

ule

due

to t

he p

rese

nce

of h

ydro

gen

atom

s, R

2=

Exc

ess

mol

ar r

efra

ctio

n, R

g=

ave

rage

sol

ubili

ty in

ent

ire s

et o

f so

lven

t sy

stem

s, S

a=

sol

ubili

tyin

air,

So

= s

olub

ility

in

n-oc

tano

l (o

r ve

geta

ble

oil),

Ss

= s

olub

ility

in

salin

e, S

v=

sol

ubili

ty i

n ve

geta

ble

oil,

Sw

= s

olub

ility

in

wat

er,

Tb

= b

oilin

g po

int,

Vnt

= v

olum

e fr

actio

n of

neu

tral

lipi

ds in

tis

sues

, V

pt=

vol

ume

frac

tion

of p

hosp

holip

ids

in t

issu

es,

and

Vw

t=

vol

ume

frac

tion

of w

ater

in t

issu

es.

bF

= fi

sh,

H =

hum

an,

and

R =

rat

s.c

CF

Cs

= c

hlor

ofluo

roca

rbon

s, L

MW

VO

Cs

= lo

w m

olec

ular

wei

ght

vola

tile

orga

nic

chem

ical

s, a

nd V

OC

s =

vol

atile

org

anic

che

mic

als.


In Silico Approaches for Blood:Air PCs


Tab

le 4

0.2

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e B

loo

d:A

ir P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ele

ctro

stat

ic d

escr

ipto

rs

log

Pbl

ood:

air=

–1.

269

+ 0

.612

R2

+ 0

.916

+ 3

.614

+ 3

.381

+0.

362

log

Phe

:a

HIn

ert

Gas

es; L

MW

VO

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

log

Ppl

asm

a:ai

r=

–1.

48 +

0.4

90R

2+

2.0

4 +

3.5

074

+ 3

.911

+

0.15

7 lo

g P

he:a

HIn

ert

Gas

es; L

MW

VO

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

Ste

ric

des

crip

tors

log

Pbl

ood:

air=

0.0

072M

W+

0.1

97H

Trih

alom

etha

nes

Bat

term

an e

t al

. (20

02)

log

Pbl

ood:

air=

0.3

21N

Br+

1.0

6H

Trih

alom

etha

nes

Bat

term

an e

t al

. (20

02)

Pbl

ood:

air=

0.0

7MW

+ 5

.59

HA

lipha

tic h

ydro

carb

ons

Per

belli

ni e

t al

. (19

85)

log

Pbl

ood:

air=

0.4

43Q

H–

0.30

3NF

+ 0

.225

NC

l+

0.5

10N

BR

+ 0

.155

NC

– 0.

104

RH

aloa

lkan

esG

arga

s et

al.

(198

8)

Hyd

rop

ho

bic

des

crip

tors

log

(Pbl

ood:

wat

er–

Vw

b) =

0.7

Po:

w–

0.75

FC

hlor

oeth

anes

; ben

zene

Ber

tels

en e

t al

. (19

98)

lnP

bloo

d:ai

r=

0.0

38T

b–

13.3

HA

lipha

tic h

ydro

carb

ons

Csa

nady

and

Lai

b (1

990)

log

Pbl

ood:

air=

0.0

109T

b–

2.58

4H

Trih

alom

etha

nes

Bat

term

an e

t al

. (20

02)

log

Pbl

ood:

air=

–0.

14 lo

g P

o:a

+ 0

.86

log

Pw

:a+

0.4

7H

Hyd

roph

ilic

VO

Cs

Tic

hy (

1991

b)

log

Pbl

ood:

air=

0.6

85 lo

g P

o:a

– 0.

6565

HTr

ihal

omet

hane

sB

atte

rman

et

al. (

2002

)lo

gP

bloo

d:ai

r=

0.4

5 lo

g P

w:a

+ 1

.21

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

–0.

003

log

Pw

:a+

1.4

7H

VO

Cs

Laas

s (1

987)

lo

gp b

lood

:air

= –

0.07

4 +

0.8

02 lo

g P

w:a

+ 0

.218

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pbl

ood:

air=

–0.

07 lo

g S

w+

1.2

1H

VO

Cs

Laas

s (1

987)

(c

ontin

ued)


Tab

le 4

0.2

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e B

loo

d:A

ir P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

log

Pbl

ood:

air=

–0.

09 lo

g P

o:a

+ 2

.45

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

–0.

102

+ 0

.675

log

Pw

:a+

0.3

15 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

bloo

d:ai

r=

–0.

295

+ 0

.588

log

Pw

:a+

0.4

11 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

bloo

d:ai

r=

–0.

338

log

Po:

a+

3.1

21H

Hal

ogen

ated

hyd

roca

rbon

sT

ichy

et

al. (

1984

)lo

gP

bloo

d:ai

r=

–0.

6737

+ 0

.531

9 lo

g P

o:a

log

Pw

:aH

VO

Cs

Sat

o an

d N

akaj

ima

(197

9)lo

gP

bloo

d:ai

r=

0.6

95 lo

g P

o:a

– 1.

076

HLM

WV

OC

sF

iser

ova-

Ber

gero

va e

t al

. (1

984)

log

Pbl

ood:

air=

–0.

820

+ 0

.754

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Pbl

ood:

air=

0.0

9 lo

g S

w+

8.2

5 lo

g V

o–

11.0

9H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.1

1 lo

g S

w+

1.9

1H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.1

80 lo

g P

o:a

+ 0

.889

log

Pw

:a+

0.0

54H

Hyd

roph

obic

VO

Cs

Tic

hy (

1991

a)lo

gP

bloo

d:ai

r=

0.2

0 lo

g S

w+

1.2

9H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.2

2 lo

g P

w:a

+ 0

.67

log

Po:

a–

0.98

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

0.2

2 lo

g S

w+

10.

78 lo

g V

w–

40.9

9H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.2

62 +

0.9

96 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

bloo

d:ai

r=

0.2

7 lo

g 10

00/P

+ 5

.10

log

Vo

– 6.

67H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.3

1 lo

g S

w+

3.9

0 lo

g V

o–

4.53

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

0.3

5 lo

g 10

00/P

+ 1

.01

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

0.3

5 lo

g S

w+

0.7

9 lo

g 10

00/P

+ 1

.34

log

Vo

– 2.

23H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.3

7 lo

g S

w+

10.

09 lo

g V

w–

38.4

0H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.3

8 lo

g S

w+

0.9

1 lo

g 10

00/P

–0.

45H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.4

5 lo

g S

w+

0.8

1 lo

g 10

00/P

–0.

40H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.4

8 lo

g S

w+

0.7

5 lo

g 10

00/P

+ 1

.67

log

Vo

– 2.

77H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.5

1 lo

g 10

00/P

+ 0

.37

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

0.5

81 lo

g P

o:a

+ 0

.332

log

Pw

:a–

0.59

9H

LMW

VO

Cs

Gar

gas

et a

l. (1

989)

log

Pbl

ood:

air=

0.6

3 lo

g 10

00/P

+ 0

.38

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

0.6

5 lo

g P

o:a

– 0.

84H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

0.8

51 lo

g S

w+

1.7

8H

VO

Cs

Laas

s (1

987)


log

Pbl

ood:

air=

0.9

84 lo

g P

w:a

+ 0

.053

HK

eton

es; e

ther

s; g

ases

Tic

hy e

t al

. (19

84)

log

Pbl

ood:

air=

1.0

7 lo

g P

w:a

+ 0

.27

log

Po:

a–

0.79

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

1.2

1 lo

g V

o–

0.17

HV

OC

sLa

ass

(198

7)

log

Pbl

ood:

air=

3.0

5 –

0.34

Po:

nH

Ket

ones

Cab

ala

et a

l. (1

992)

lo

gP

bloo

d:ai

r=

–3.

922

+ 1

.369

RG

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

) lo

gP

bloo

d:ai

r=

5.8

9 lo

g V

w–

21.4

3H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

7.8

6 lo

g V

o–

10.4

0H

VO

Cs

Laas

s (1

987)

lo

gP

bloo

d:ai

r=

8.9

0 lo

g V

w–

33.4

0H

VO

Cs

Laas

s (1

987)

lo

gP

milk

:air

= 0

.900

log

Po:

a–

1.09

5H

Trih

alom

etha

nes

Bat

term

an e

t al

. (20

02)

log

Ppl

asm

a:ai

r=

–0.

079

+ 0

.896

log

Pw

:a+

0.1

49 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

) lo

gP

plas

ma:

air=

–0.

082

+ 0

.894

log

Pw

:a+

0.1

52 lo

g P

o:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

plas

ma:

air=

–0.

848

+ 0

.890

log

Po:

aH

Iner

t ga

ses;

LM

WV

OC

sA

brah

am e

t al

. (19

85)

log

Ppl

asm

a:ai

r=

–3.

696

+ 1

.208

RG

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)lo

gP

plas

ma:

air=

0.0

38 +

1.0

19 lo

g P

w:a

HIn

ert

gase

s; L

MW

VO

Cs

Abr

aham

et

al. (

1985

)P

bloo

d:ai

r=

0.0

072P

o:a

+ 0

.898

Pw

:a+

0.0

3H

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)P

bloo

d:ai

r=

0.0

8e0.

0308

Tb

HA

lipha

tic h

ydro

carb

ons

Per

belli

ni e

t al

. (19

85)

Pbl

ood:

air=

0.0

0442

Po:

aH

Alip

hatic

hyd

roca

rbon

sP

erbe

llini

et

al. (

1985

)P

bloo

d:ai

r=

0.8

8Pw

:a+

0.0

12H

VO

Cs

Fei

ngol

d (1

976)

Pbl

ood:

air=

0.8

9Pw

:a+

0.0

11P

o:a

HLM

WV

OC

sT

ichy

et

al. (

1984

) P

bloo

d:ai

r=

0.9

0 lo

g P

w:a

– 46

1H

Est

ers;

alc

ohol

sK

anek

o et

al.

(199

4)P

bloo

d:ai

r=

Pw

:a+

(P

o:a/

100)

HA

naes

thet

ics

Ege

r an

d La

rson

(19

64)

Pbl

ood:

air=

Sw(1

+ 0

.003

5Po:

w)/

Sa

HLM

WV

OC

sP

ater

son

and

Mac

kay

(198

9)lo

gP

bloo

d:ai

r=

Pw

:a[V

lbP

o:w

0.85

+V

prb(

86.2

/Po:

w+

3.7

0)]

+ V

wb

H,

RLM

WV

OC

sC

onne

ll et

al.

(199

3)

log

Pbl

ood:

air=

0.4

26lo

gP

o:a

+ 0

.515

log

Pw

:a–

0.07

0R

Hal

oalk

anes

Gar

gas

et a

l. (1

988)

lo

gP

bloo

d:ai

r=

0.5

53 lo

g P

o:a

+ 0

.351

Pw

:a–

0.28

6R

LMW

VO

Cs

Gar

gas

et a

l. (1

989)

P

bloo

d:ai

r=

0.0

054P

o:a

+ 0

.931

Pw

:a+

1.1

6R

LMW

VO

Cs;

CF

Cs

Meu

lenb

erg

and

Vijv

erbe

rg

(200

0)(c

ontin

ued)


Tab

le 4

0.2

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e B

loo

d:A

ir P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

QS

AR

s: F

ree–

Wils

on

-typ

e eq

uat

ion

s

Pbl

ood:

wat

er=

BS

(C-C

)(2

8.4)

+ n

CL 2

(–12

.9)

+ n

CL 3

(12.

9)F

Chl

oroe

than

esF

ouch

écou

rt e

t al

. (20

00)

Pbl

ood:

air=

BS

(C-C

)(2

6.2)

+ n

H3(

–34.

9) +

nC

L(–4

.51)

+ n

CL 2

(29.

4) +

nC

L 3(1

1.5)

HC

hlor

oeth

anes

Fou

chéc

ourt

and

Kris

hnan

(2

000)

Pbl

ood:

air=

BS

(C-C

)(4

5.6)

+ n

H3(

–51.

5) +

nC

L(–8

.86)

+ n

CL 2

(36.

4) +

nC

L 3(1

1.1)

RC

hlor

oeth

anes

Fou

chéc

ourt

and

Kris

hnan

(2

000)

Bio

log

ical

ly b

ased

alg

ori

thm

s

Pbl

ood:

air=

Po:

wP

w:a(V

nb+

0.3

Vpb

) +

Pw

:a(V

wb

+ 0

.7V

pb)

R,

HLM

WV

OC

sP

oulin

and

Kris

hnan

(199

6c)

Pbl

ood:

air=

[f e

(SsV

we

+S

vVne

+ 0

.7S

sVpe

+ 0

.3S

vVpe

) +

fp(

SsV

wp

+S

vVnp

+0.

7SsV

pp+

0.3

SvV

pp)]

/Sa

R,

HLM

WV

OC

sP

oulin

and

Kris

hnan

(199

6b)

a=

dip

olar

ity/p

olar

izab

ility

, =

ove

rall

hydr

ogen

-bon

d ac

idity

, =

ove

rall

hydr

ogen

-bon

d ba

sici

ty, B

S =

Bas

ic s

truc

ture

, fe

= fr

actio

n of

ery

thro

cyte

sin

blo

od,

f p=

fra

ctio

n of

pla

sma

in b

lood

, M

W =

mol

ecul

ar w

eigh

t, N

Br

= n

umbe

r of

bro

mid

e at

oms

in t

he m

olec

ule,

NC

= n

umbe

r of

car

bon

atom

s in

the

mol

ecul

e, N

Cl=

num

ber

of c

hlor

ide

atom

s in

the

mol

ecul

e, n

CL

= n

umbe

r of

CL

frag

men

ts,

nCL 2

= n

umbe

r of

CL 2

frag

men

ts,

nCL 3

= n

umbe

r of

CL 3

frag

men

ts,

NF

= n

umbe

r of

fluo

ride

atom

s in

the

mol

ecul

e, n

H3

= n

umbe

r of

H3

frag

men

ts,

P=

vap

or p

ress

ure,

Phe

:a=

hex

adec

ane:

air

part

ition

coef

ficie

nt,

Po:

a=

n-o

ctan

ol:a

ir pa

rtiti

on c

oeffi

cien

t (o

r ve

geta

ble

oil:a

ir),

Po:

n=

veg

etab

le o

il:ni

trog

en p

artit

ion

coef

ficie

nt,

Po:

w=

n-o

ctan

ol:w

ater

par

titio

nco

effic

ient

(or

veg

etab

le o

il:w

ater

), P

w:a

= w

ater

:air

part

ition

coe

ffici

ent,

QH

= v

aria

ble

depe

ndan

t on

the

pol

arity

of

the

mol

ecul

e du

e to

the

pre

senc

eof

hyd

roge

n at

oms,

R2

= e

xces

s m

olar

ref

ract

ion,

Rg

= p

aram

eter

s re

lativ

e to

the

sol

vent

, S

a=

sol

ubili

ty i

n ai

r, S

s=

sol

ubili

ty i

n sa

line,

Sv

= s

olub

ility

in v

eget

able

oil,

Sw

= s

olub

ility

in w

ater

, Tb

= b

oilin

g po

int,

Vlb

= v

olum

e fr

actio

n of

lipi

ds in

blo

od,

Vnb

= v

olum

e fr

actio

n of

neu

tral

lipi

ds in

blo

od,

Vne

=vo

lum

e fr

actio

n of

neu

tral

lipi

ds in

ery

thro

cyte

s, V

np=

vol

ume

frac

tion

of n

eutr

al li

pids

in p

lasm

a, V

o=

sur

face

tens

ion,

Vpb

= v

olum

e fr

actio

n of

pho

spho

lipid

sin

blo

od,

Vpe

= v

olum

e fr

actio

n of

pho

spho

lipid

s in

ery

thro

cyte

s, V

pp=

vol

ume

frac

tion

of p

hosp

holip

ids

in p

lasm

a, V

prb

= v

olum

e fr

actio

n of

pro

tein

s in

bloo

d,V

w=

hea

t re

leas

ed d

ue t

o ev

apor

atio

n of

the

sub

stan

ce a

t bo

iling

tem

pera

ture

, V

wb

= v

olum

e fr

actio

n of

wat

er in

blo

od, V

we

= v

olum

e fr

actio

n of

wat

er in

ery

thro

cyte

s, a

nd V

wp

= v

olum

e fr

actio

n of

wat

er in

pla

sma.

bF

= fi

sh,

H =

hum

an,

and

R =

rat

s.c

CF

Cs

= c

hlor

ofluo

roca

rbon

s, L

MW

VO

Cs

= lo

w m

olec

ular

wei

ght

vola

tile

orga

nic

chem

ical

s, a

nd V

OC

s =

vol

atile

org

anic

che

mic

als.



In Silico Approaches for Tissue:Blood PCs


Tab

le 4

0.3

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e T

issu

e:B

loo

d P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ste

ric

des

crip

tors

log

Pad

ipos

e:bl

ood

= 0

.168

+ 0

.198

R2

+ 0

.130

– 1

.211

–

3.26

7 +

2.2

75V

x

HIn

ert

gase

s; L

MW

VO

Cs;

CF

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

log

Pbr

ain:

bloo

d=

–0.

166

+ 0

.239

R2–

0.62

6 –

0.3

68 –

0.6

15

+ 1

.072

Vx

HIn

ert

gase

s; L

MW

VO

Cs;

CF

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

log

Pbr

ain:

bloo

d=

–0.

0148

PS

A +

0.1

52 lo

g P

o:w

+ 0

.139

HIn

ert

gase

s; H

MW

OC

s; L

MW

VO

Cs

Cla

rk (

1999

) lo

gP

brai

n:bl

ood

= 1

.359

+ 0

.338

log

Pcy

h–

0.00

618V

mH

H2-

R a

ntag

onis

tsK

aliz

an a

nd M

arku

szew

ski

(199

6)

log

Phe

art:b

lood

= –

0.34

6 +

0.2

04 –

2.1

50 –

0.8

53 +

0.9

31V

xH

Iner

t ga

ses;

LM

WV

OC

s; C

FC

sA

brah

am a

nd W

eath

ersb

y (1

994)

log

Pki

dney

:blo

od=

–0.

188

+ 0

.226

R2

– 0.

559

– 0

.433

+ 0

.832

Vx

HIn

ert

gase

s; L

MW

VO

Cs;

CF

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

log

Pliv

er:b

lood

= –

0.27

0 +

0.2

33R

2–

0.37

5 –

1.0

04 –

1.1

18

+ 0

.832

Vx

HIn

ert

gase

s; L

MW

VO

Cs;

CF

Cs

Abr

aham

and

Wea

ther

sby

(199

4)

log

Plu

ng:b

lood

= –

0.15

0 –

0.19

5 +

0.3

89V

xH

Iner

t ga

ses;

LM

WV

OC

s; C

FC

sA

brah

am a

nd W

eath

ersb

y (1

994)

log

Pm

uscl

e:bl

ood

= –

0.22

2 –

0.47

9 –

0.5

17 +

0.9

99V

xH

Iner

t ga

ses;

LM

WV

OC

s; C

FC

sA

brah

am a

nd W

eath

ersb

y (1

994)

Pad

ipos

e:pl

asm

a=

1.9

988

– 0.

5004

UN

S +

0.1

793N

PL

+ 0

.059

31D

IFF

2H

PC

Bs

(48)

log

Pbr

ain:

bloo

d=

0.0

88 +

0.2

64R

2–

0.96

6 –

0.7

05–

0.75

6+

1.1

89V

x

RH

2-R

ant

agon

ists

Nor

inde

r an

d H

aebe

rlein

(2

002)

(con

tinue

d)


Tab

le 4

0.3

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e T

issu

e:B

loo

d P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

log

Pbr

ain:

bloo

d=

–0.

088

+ 0

.272

log

Po:

w–

0.00

116M

WR

H2-

R a

ntag

onis

tsK

aliz

an a

nd M

arku

szew

ski

(199

6)lo

gP

brai

n:bl

ood

= 0

.001

16M

W +

0.2

72 lo

g P

o:w

– 0.

088

RIn

ert

gase

s; v

olat

ile h

ydro

carb

ons

Nor

inde

r an

d H

aebe

rlein

(2

002)

log

Pbr

ain:

bloo

d=

–0.

01V

m+

0.3

5 lo

g P

o:w

+ 0

.99I

3+

1.2

5R

Dru

g-lik

e m

olec

ules

Nor

inde

r an

d H

aebe

rlein

(2

002)

log

Pbr

ain:

bloo

d=

–0.

021P

SA

– 0

.003

MV

+ 1

.643

RIn

ert

gase

s; H

MW

OC

s; L

MW

VO

Cs

Cla

rk (

1999

) lo

gP

brai

n:bl

ood

= –

0.03

22D

PS

A +

1.3

3R

HM

WO

Cs

Nor

inde

r an

d H

aebe

rlein

(2

002)

log

Pbr

ain:

bloo

d=

–0.

038

+ 0

.198

R2

– 0.

687

– 0

.715

–

0.69

8+

0.9

95V

x

RH

2-R

ant

agon

ists

; Ine

rt g

ases

; SO

Ms

Nor

inde

r an

d H

aebe

rlein

(2

002)

log

Pbr

ain:

bloo

d=

–0.

218(

NN

+N

O)

+ 0

.235

log

Po:

w–

0.02

7R

HM

WO

Cs

Nor

inde

r an

d H

aebe

rlein

(2

002)

log

Pbr

ain:

bloo

d=

0.4

76 +

0.5

41 lo

g P

o:w

– 0.

0079

4MW

RH

2-R

ant

agon

ists

Kal

izan

and

Mar

kusz

ewsk

i (1

996)

log

Pbr

ain:

bloo

d=

1.2

96 +

0.3

09 lo

g P

cyh

– 0.

0057

0MW

RH

2-R

ant

agon

ists

Kal

izan

and

Mar

kusz

ewsk

i (1

996)

Hyd

rop

ho

bic

des

crip

tors

log

Pbr

ain:

bloo

d=

0.3

9 lo

g P

o:w

+ 0

.68

HD

rugs

, ho

rmon

esS

eyde

l and

Sch

aper

(19

82)

log

Pbr

ain:

bloo

d=

0.0

54G

o+

0.4

3H

H2-

R a

ntag

onis

ts; L

MW

VO

Cs

Lom

bard

o et

al.

(199

6)

Pad

ipos

e:bl

ood

=(V

lt+

Vw

t)/(V

lb+

Vw

b)+

BH

, R

LMW

VO

Cs

DeJ

ongh

et

al. (

1997

)

Pbr

ain:

bloo

d=

(Vlt

+V

wt)/

(Vlb

+V

wb)

+B

H,

RLM

WV

OC

sD

eJon

gh e

t al

. (19

97)

Pki

dney

:blo

od=

(Vlt

+V

wt)/

(Vlb

+V

wb)

+B

H,

RLM

WV

OC

sD

eJon

gh e

t al

. (19

97)

Pliv

er:b

lood

=(V

lt +

Vw

t)/(V

lb+

Vw

b)+

BH

, R

LMW

VO

Cs

DeJ

ongh

et

al. (

1997

)


Pm

uscl

e:bl

ood

=(V

lt+

Vw

t)/(V

lb+

Vw

b)+

BH

, R

LMW

VO

Cs

DeJ

ongh

et

al. (

1997

)

LnP

kidn

ey:b

lood

= 0

.006

5o

RH

MW

OC

sYa

mag

uchi

et

al. (

1996

) Ln

Pliv

er:b

lood

= 0

.025

iR

HM

WO

Cs

Yam

aguc

hi e

t al

. (19

96)

LnP

mus

cle:

bloo

d=

0.0

069

iR

HM

WO

Cs

Yam

aguc

hi e

t al

. (19

96)

log

Pbr

ain:

bloo

d=

0.0

35G

solv

+ 0

.259

RH

2-R

ant

agon

ists

; LM

WV

OC

sN

orin

der

and

Hae

berle

in

(200

2)lo

gP

brai

n:bl

ood

= 0

.427

5 –

0.38

73n a

cc,s

olv+

0.1

092

log

Po:

w–

0.00

17A

pol

RD

rugs

; LM

WV

OC

s; a

naes

thet

ics

Feh

er e

t al

. (20

00)

log

Pbr

ain:

bloo

d=

1.9

79 +

0.3

73 lo

g P

cyh

– 0.

0027

5Vw

avR

H2-

R a

ntag

onis

tsK

aliz

an a

nd M

arku

szew

ski

(199

6)lo

gP

brai

n:pl

asm

a=

–0.

48

log

Poc

t-cy

c+

0.8

9R

H2-

R a

ntag

onis

tsTe

sta

et a

l. (2

000)

ln

Pad

ipos

e:bl

ood

= 0

.05

i + 0

.021

RH

MW

OC

sYa

mag

uchi

et

al. (

1996

)

Pad

ipos

e:bl

ood

= 0

.915

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(199

0)

Pad

ipos

e:pl

asm

a=

0.0

16R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Pbo

ne m

arro

w:b

lood

= 1

.975

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(199

0)

Pbo

ne:p

lasm

a=

0.0

36R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Pbr

ain:

bloo

d=

3.1

57R

bB

asic

dru

gsYo

koga

wa

et a

l. (1

990)

Pbr

ain:

plas

ma

= 0

.062

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(200

2)

Pgu

t:blo

od=

3.0

02R

bB

asic

dru

gsYo

koga

wa

et a

l. (1

990)

Pgu

t:pla

sma

= 0

.058

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(200

2)

Phe

art:b

lood

= 1

.678

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(199

0)

Phe

art:p

lasm

a=

0.0

32R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Pki

dney

:pla

sma

= 0

.075

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(200

2)

(con

tinue

d)


Tab

le 4

0.3

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e T

issu

e:B

loo

d P

arti

tio

n C

oef

fici

ents

(P

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

Pliv

er:p

lasm

a=

0.0

64R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Plu

ng:b

lood

= 1

.158

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(199

0)

Plu

ng:p

lasm

a=

0.0

31R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Pm

uscl

e:bl

ood

= 4

.928

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(199

0)

Pm

uscl

e:pl

asm

a=

0.0

99R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Psk

in:b

lood

= 2

.997

Rb

Bas

ic d

rugs

Yoko

gaw

a et

al.

(199

0)

Psk

in:p

lasm

a=

0.0

58R

bB

asic

dru

gsYo

koga

wa

et a

l. (2

002)

Psp

leen

:blo

od=

3.0

02R

bB

asic

dru

gsYo

koga

wa

et a

l. (1

990)

QS

AR

s: F

ree–

Wils

on

-typ

e eq

uat

ion

s

Pad

ipos

e:bl

ood

= B

S(C

-C)(9

4.5)

+ n

CL 2

(–29

.2)

+ n

CL 3

(29.

2)F

Chl

oroe

than

esF

ouch

écou

rt e

t al

. (20

00)

Pliv

er:b

lood

= B

S(C

-C)(2

.93)

+ n

CL 2

(–0.

238)

+ n

CL 3

(0.2

38)

FC

hlor

oeth

anes

Fou

chéc

ourt

et

al. (

2000

)P

mus

cle:

bloo

d=

BS

(C-C

)(3.0

2) +

nC

L 2(–

0.17

5) +

nC

L 3(0

.175

)F

Chl

oroe

than

esF

ouch

écou

rt e

t al

. (20

00)

Pad

ipos

e:bl

ood

= B

S(C

-C)(4

9.2)

+ n

H3(

–0.4

40)

+ n

CL(

–14.

54)

+

nCL 2

(–6.

65)

+ n

CL 3

(26.

5)H

Chl

oroe

than

esF

ouch

écou

rt a

nd K

rishn

an

(200

0)P

liver

:blo

od=

BS

(C-C

)(2.6

4) +

nH

3(–0

.61)

+ n

CL(

–0.6

6) +

nC

L 2(–

0.18

)+

nCL 3

(1.6

8)H

Chl

oroe

than

esF

ouch

écou

rt a

nd K

rishn

an

(200

0)P

mus

cle:

bloo

d=

BS

(C-C

)(1.1

1) +

nH

3(0.

08)

+ n

CL(

–0.0

2) +

nC

L 2(–

0.21

)+

nCL 3

(0.1

5)H

Chl

oroe

than

esF

ouch

écou

rt a

nd K

rishn

an

(200

0)P

adip

ose:

bloo

d=

BS

(C-C

)(30.

1) +

nH

3(–9

.88)

+ n

CL(

–6.0

2) +

nC

L 2(–

3.90

)+

nCL 3

(17.

3)R

Chl

oroe

than

esF

ouch

écou

rt a

nd K

rishn

an

(200

0)P

liver

:blo

od=

BS

(C-C

)(1.7

9) +

nH

3(–0

.9)

+ n

CL(

–0.3

8) +

nC

L 2(–

0.21

) +

nC

L 3(1

.27)

RC

hlor

oeth

anes

Fou

chéc

ourt

and

Kris

hnan

(2

000)


Pm

uscl

e:bl

ood

= B

S(C

-C)(0

.69)

+ n

H3(

–0.1

2) +

nC

L(0.

04)

+ n

CL 2

(–0.

12)

+nC

L 3(0

.17)

RC

hlor

oeth

anes

Fou

chéc

ourt

and

Kris

hnan

(2

000)

Bio

log

ical

ly b

ased

alg

ori

thm

s

Ptis

sue:

bloo

d=

(S

oVnt

+S

w0.

7Vpt

+S

o0.3

Vpt

+S

wV

wt)/

(SoV

nb+

Sw0.

7Vpb

+S

o0.3

Vpb

+S

wV

wb)

HLM

WV

OC

sP

oulin

and

Kris

hnan

(19

95a)

Ptis

sue:

bloo

d=

(P

o:wV

nt+

Vw

t+

Po:

w0.

3Vpt

+ 0

.7V

pt)/

[f e(P

o:wV

ne+

Vw

e+

Po:

w0.

3Vpe

+ 0

.7V

pe)

+ f

p(P

o:wV

np+

Vw

p+

Po:

w0.

3Vpp

+ 0

.7V

pp)]

RK

eton

es; A

lcoh

ols;

Est

ers

Pou

lin a

nd K

rishn

an (

1995

b)

Ptis

sue:

bloo

d=

[P

o:w(V

nt+

0.3

Vpt)

+ (

Vw

t+

0.7

Vpt)]

/[Po:

w(V

nb+

0.3

Vpb

) +

(V

wb

+ 0

.7V

pe)]

R,

HLM

WV

OC

sP

oulin

and

Kris

hnan

(19

96b)

a =

dip

olar

ity/p

olar

izab

ility

, =

ove

rall

hydr

ogen

-bon

d ac

idity

, =

ove

rall

hydr

ogen

-bon

d ba

sici

ty,

Gso

lv =

free

ene

rgy

of s

olva

tion

in h

exad

ecan

e,i =

mol

ecul

ar s

truc

ture

Fuj

ita v

alue

, o

= m

olec

ular

str

uctu

re F

ujita

val

ue,

A1,

A2

= C

olla

nder

-typ

e co

effic

ient

, A

pol=

pol

ar s

urfa

ce a

rea,

B=

cor

rect

ion

fact

or,

BS

= b

asic

str

uctu

re,

DIF

F =

var

iabl

e de

pend

ant

on t

he n

umbe

r of

chl

orid

e at

oms

in t

he a

rom

atic

cyc

le,

DP

SA

= d

ynam

ic p

olar

sur

face

are

a,f e

= f

ract

ion

of e

ryth

rocy

tes

in b

lood

, f p

= f

ract

ion

of p

lasm

a in

blo

od,

I 3=

var

iabl

e de

pend

ant

on t

he p

rese

nce

of a

n am

ino

nitr

ogen

or

carb

oxyl

gro

up,

MV

= m

olec

ular

vol

ume,

MW

= m

olec

ular

wei

ght,

n acc

,sol

v=

num

ber

of s

olva

ted

hydr

ogen

-bon

d ac

cept

ors,

nC

L =

num

ber

of C

L fr

agm

ents

, nC

L 2 =

num

ber

of C

L 2fr

agm

ents

, nC

L 3=

num

ber

of C

L 3fr

agm

ents

, nH

3=

num

ber

of H

3fr

agm

ents

, N

N=

num

ber

of n

itrog

ens,

NO

= n

umbe

r of

oxy

gens

, N

PL

= v

aria

ble

depe

ndan

t on

the

num

ber

of c

hlor

ide

atom

s in

the

mol

ecul

e in

ort

ho p

ositi

on,

o G =

Gib

bs fr

ee e

nerg

y re

late

d to

the

solv

atio

n of

the

subs

tanc

ein

wat

er,

Pcy

h=

cyc

lohe

xane

:wat

er p

artit

ion

coef

ficie

nt,

Po:

w=

n-o

ctan

ol:w

ater

par

titio

n co

effic

ient

(or

veg

etab

le o

il:w

ater

), P

oct-

cyc

= o

ctan

ol-c

yclo

hexa

ne,

PS

A =

pol

ar s

urfa

ce a

rea,

R2

= E

xces

s m

olar

ref

ract

ion,

So

= s

olub

ility

in n

-oct

anol

(or

veg

etab

le o

il),

Sw

= s

olub

ility

in w

ater

, UN

S =

var

iabl

e de

pend

ant

on t

he n

umbe

r of

ato

ms

in t

he m

olec

ule

that

are

not

chl

orid

es, V

lb=

vol

ume

frac

tion

of li

pids

in b

lood

, Vlt

= v

olum

e fr

actio

n of

lipi

ds in

tis

sue,

Vm

= m

olar

volu

me,

Vnb

= v

olum

e fr

actio

n of

neu

tral

lip

ids

in b

lood

, V

ne=

vol

ume

frac

tion

of n

eutr

al l

ipid

s in

ery

thro

cyte

s, V

np=

vol

ume

frac

tion

of n

eutr

al l

ipid

s in

plas

ma,

Vnt

= v

olum

e fr

actio

n of

neu

tral

lip

ids

in t

issu

es,

Vpb

= v

olum

e fr

actio

n of

pho

spho

lipid

s in

blo

od,

Vpe

= v

olum

e fr

actio

n of

pho

spho

lipid

s in

eryt

hroc

ytes

, Vpp

= v

olum

e fr

actio

n of

pho

spho

lipid

s in

pla

sma,

Vpt

= v

olum

e fr

actio

n of

pho

spho

lipid

s in

tis

sues

, Vw

av=

vol

ume

of w

ater

nee

ded

in o

rder

to s

olub

ilize

the

subs

tanc

e, V

wb

= v

olum

e fr

actio

n of

wat

er in

blo

od, V

wb

= v

olum

e fr

actio

n of

wat

er in

blo

od, V

we

= v

olum

e fr

actio

n of

wat

er in

ery

thro

cyte

s,V

wp

= v

olum

e fr

actio

n of

wat

er i

n pl

asm

a, V

wt

= v

olum

e fr

actio

n of

wat

er i

n tis

sue,

Vw

t=

vol

ume

frac

tion

of w

ater

in

tissu

es,

and

Vx

= M

cGow

anch

arac

teris

tic v

olum

e.b

F =

fish

, H

= h

uman

, an

d R

= r

ats.

cC

FC

s =

chl

orofl

uoro

carb

ons,

HM

WO

Cs

= h

igh

mol

ecul

ar w

eigh

t org

anic

che

mic

als,

LM

WV

OC

s =

low

mol

ecul

ar w

eigh

t vol

atile

org

anic

che

mic

als,

PC

Bs

= p

olyc

hlor

obip

heny

ls,

and

VO

Cs

= v

olat

ile o

rgan

ic c

hem

ical

s.


In Silico Approaches for Protein Binding


Tab

le 4

0.4

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

Pro

tein

Bin

din

g o

f C

hem

ical

sa

Ap

pro

ach

bS

pec

iesc

Ch

emic

al C

lass

Ref

eren

ce

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Hyd

rop

ho

bic

des

crip

tors

log

(1/f u

(pla

sma)

– 1)

= 0

.994

log

Po:

w–

1.10

HA

rom

atic

aci

dsTe

sta

et a

l. (2

000)

lo

g (1

/f u(p

lasm

a)–

1) =

0.9

94 lo

g P

o:w

– 1.

10H

Org

anic

aci

dsLa

znic

ek e

t al

. (19

87)

log

(1/K

a(pl

asm

a))

= –

3.91

log

Po:

w2+

13

log

Po:

w–

13.7

HC

epha

losp

orin

sTe

sta

et a

l. (2

000)

lo

g (1

– f

u(br

ain))

= 0

.36

log

Po:

w–

1.07

HB

arbi

tura

tes

Sey

del a

nd S

chap

er (

1982

) lo

g (1

– f

u(pl

asm

a))

= 0

.276

log

Po:

w+

1.2

HP

enic

illin

sS

eyde

l and

Sch

aper

(19

82)

log

(1 –

fu(

plas

ma))

= 0

.30

log

Po:

w–

1.03

HB

arbi

tura

tes

Sey

del a

nd S

chap

er (

1982

)lo

g (1

– f

u(pl

asm

a))

= 0

.33

log

Po:

w+

1.9

4H

Tetr

acyc

lines

Sey

del a

nd S

chap

er (

1982

)lo

g 1/

Ka(

albu

min

bin

ding

)=

–0.

85 lo

g P

o:w

+ 2

.73

HS

ulfa

pyrim

idin

es; s

ulfa

pyrid

ines

Sey

del a

nd S

chap

er (

1982

)lo

g 1/

Ka(

albu

min

bin

ding

)=

–0.

97 lo

g P

o:w

+ 3

.24

HS

ulfa

pyrid

ines

Sey

del a

nd S

chap

er (

1982

)lo

g 1/

Ka(

albu

min

bin

ding

)=

–0.

97 lo

g P

o:w

– 0.

70I

+ 3

.24

HS

ulfa

pyrim

idin

es; s

ulfa

pyrid

ines

Sey

del a

nd S

chap

er (

1982

)lo

g 1/

Ka(

albu

min

bin

ding

)=

–0.

99 lo

g P

o:w

+ 2

.49

HS

ulfa

pyrim

idin

esS

eyde

l and

Sch

aper

(19

82)

log

Kal

bum

in b

indi

ng=

0.8

9 lo

g P

o:w

+ 1

.47

HS

ulfa

pyrim

idin

esS

eyde

l and

Sch

aper

(19

82)

log

Kal

bum

in b

indi

ng=

1.1

5 lo

g P

o:w

+ 1

.23

HS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

)lo

gK

albu

min

bin

ding

= 1

.23

log

Po:

w–

0.05

6H

Ste

roid

bis

guan

ylhy

draz

ones

Sey

del a

nd S

chap

er (

1982

)lo

gK

albu

min

bin

ding

= 1

.32

log

Po:

w+

0.3

7H

Pen

icill

ins

Sey

del a

nd S

chap

er (

1982

)lo

gK

albu

min

bin

ding

= 1

.39

log

Po:

w–

1.19

HC

arde

nolid

esS

eyde

l and

Sch

aper

(19

82)

log

Kal

bum

in b

indi

ng=

1.6

5 lo

g P

o:w

– 2.

57H

Ste

roid

hor

mon

esS

eyde

l and

Sch

aper

(19

82)

log

Kpl

asm

a pr

otei

n bi

ndin

g=

0.7

3R

mui

+ 1

.46

HS

ulfa

pyrid

ines

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(bl

ood

prot

ein

bind

ing)

= 0

.504

– 0.

665

HP

enic

illin

sB

ird a

nd M

arsh

all (

1967

)lo

g (1

/f u(p

lasm

a)–

1) =

1.0

11 lo

g P

o:w

– 1.

745

RO

rgan

ic a

cids

Lazn

icek

et

al. (

1987

) lo

g (1

– f

u)/f u

(adi

pose

)=

log

0.75

0 +

0.9

36 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(bra

in)=

log

0.07

3 +

0.8

60 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)(c

ontin

ued)


Tab

le 4

0.4

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

Pro

tein

Bin

din

g o

f C

hem

ical

sa

Ap

pro

ach

bS

pec

iesc

Ch

emic

al C

lass

Ref

eren

ce

log

(1 –

fu)

/f u(g

ut)=

log

0.09

9 +

0.8

24 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(hea

rt)=

log

0.13

5 +

0.7

80 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(kid

ney)

= lo

g 0.

676

+ 0

.619

log

Po:

wR

Bar

bitu

ric a

cids

N

este

rov

et a

l. (1

998)

log

(1 –

fu)

/f u(li

ver)

= lo

g 1.

775

+ 0

.504

log

Po:

wR

Bar

bitu

ric a

cids

N

este

rov

et a

l. (1

998)

log

(1 –

fu)

/f u(lu

ng)=

log

0.16

4 +

0.8

41 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)

log

(1 –

fu)

/f u(m

uscl

e)=

log

0.08

0 +

0.8

35 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(pan

crea

s)=

log

0.02

2 +

1.0

95 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)

log

(1 –

fu)

/f u(p

lasm

a)=

log

0.01

6 +

0.9

75 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(red

blo

od c

ell)

= lo

g 0.

178

+ 0

.677

log

Po:

wR

Bar

bitu

ric a

cids

N

este

rov

et a

l. (1

998)

log

(1 –

fu)

/f u(s

kin)

= lo

g 0.

271

+ 0

.736

log

Po:

wR

Bar

bitu

ric a

cids

N

este

rov

et a

l. (1

998)

log

(1 –

fu)

/f u(s

plee

n)=

log

0.12

6 +

0.8

41 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(sto

mac

h)=

log

0.05

8 +

0.9

39 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

g (1

– f

u)/f u

(tes

tis)=

log

0.12

0 +

0.7

47 lo

g P

o:w

RB

arbi

turic

aci

ds

Nes

tero

v et

al.

(199

8)lo

gK

plas

ma

prot

ein

bind

ing

= 0

.33

Rm

ui–

0.53

I +

4.0

8R

Sul

fapy

ridin

esS

eyde

l and

Sch

aper

(19

82)

log

(1/f u

(pla

sma)

– 1)

= 1

.016

log

Po:

w–

1.27

5R

bO

rgan

ic a

cids

Lazn

icek

et

al. (

1987

)

aK

= p

rote

in a

ffini

ty c

onst

ant

(Fre

undl

ich

isot

herm

), K

a=

pro

tein

affi

nity

con

stan

t (S

catc

hard

isot

herm

) an

d f u

= u

nbou

nd f

ract

ion.

bP

o:w

= o

ctan

ol:w

ater

par

titio

n co

effic

ient

, =

mol

ecul

ar h

ydro

phob

icity

con

stan

t, I

= fa

mily

indi

cato

r va

riabl

e,

Rm

ui=

var

iabl

e de

pend

ant

on t

here

sist

ance

con

stan

t du

e to

diff

usio

n of

the

non

ioni

zed

form

in t

he li

pid

mem

bran

e.c

H =

hum

ans,

Rb

= r

abbi

t, an

d R

= r

at.



In Silico Approaches for Clearance Constants


Tab

le 4

0.5

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

Cle

aran

ces

(CL

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ele

ctro

stat

ic d

escr

ipto

rs

log

CL (

hepa

tic)=

0.6

4 lo

g P

o:w

– 0.

98IP

+ 9

.33

HB

enzo

diaz

epin

esLe

wis

(20

00)

log

CL (

hepa

tic)=

0.0

55E

nerg

y –

0.95

IP–

0.53

HB

D +

10.

63H

Ben

zodi

azep

ines

Lew

is (

2000

)lo

g C

L (he

patic

)=

0.0

67E

nerg

y –

1.01

IP–

0.34

HB

D –

0.4

3E

+ 1

4.66

HB

enzo

diaz

epin

esLe

wis

(20

00)

log

CL (

hepa

tic)=

0.0

94E

nerg

y –

1.18

IP–

0.74

E+

18.

65H

Ben

zodi

azep

ines

Lew

is (

2000

)lo

g C

L (he

patic

)=

0.6

5 lo

g P

o:w

– 0.

40IP

– 0.

37H

BD

+ 0

.002

5Hf

+ 3

.63

HB

enzo

diaz

epin

esLe

wis

(20

00)

Met

abol

ic r

atio

= 2

.72Q

6+

1.9

6EH

+ 0

.014

SN

+ 6

.43

RD

ichl

orob

iphe

nyls

Lew

is a

nd D

icki

ns (

2002

)

Ste

ric

des

crip

tors

1/lo

g C

L (in

trin

sic;

hep

atic

)=

3.5

8 –

0.05

8S_s

Cl –

0.5

7S_a

aO –

0.4

7Sha

dow

Z le

ngth

–

0.75

CIC

HC

omm

erci

ally

ava

ilabl

e dr

ugs

Eki

ns a

nd O

bach

(20

00)

1/lo

g C

L (in

trin

sic;

hep

atic

)=

–3.

11 –

0.1

0Dip

ole

– m

ag +

13.

25Ju

rs –

RP

CG

+ 0

.57J

urs

– R

PC

S +

0.0

0013

Apo

l

HC

omm

erci

ally

ava

ilabl

e dr

ugs

Eki

ns a

nd O

bach

(20

00)

CL (

intr

insi

c; h

epat

ic)=

25S

teric

+ 4

4Ele

ctro

stat

ic +

20L

UM

O +

11H

INT

RH

aloa

lkan

esW

alle

r et

al.

(199

6)

Hyd

rop

ho

bic

des

crip

tors

CL

(intr

insi

c; h

epat

ic)=

0.0

555

Po:

w1.

05H

Bas

ic d

rugs

Yoko

gaw

a et

al.

(200

2)

log

CL (

rena

l)=

–0.

24(lo

g P

o:w)2

– 0.

04 lo

g P

o:w

+ 0

.58

HP

robe

neci

d an

alog

sS

eyde

l and

Sch

aper

(19

82)

log

CL (

rena

l)=

–lo

g(0.

35 +

0.0

13)

HP

robe

neci

d an

alog

sS

eyde

l and

Sch

aper

(19

82)

log

CL (

rena

l)=

–0.

5 lo

g P

o:w

+ 3

HN

SA

IDS

mith

et

al. (

1996

)lo

g C

L (re

nal)

= –

0.5

log

Po:

w+

13

Hß

-blo

cker

sS

mith

et

al. (

1996

)lo

g C

L (he

patic

)=

–0.

54R

mui

– 0.

51R

Sul

fona

mid

esS

eyde

l and

Sch

aper

(19

82)

(con

tinue

d)


Tab

le 4

0.5

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

Cle

aran

ces

(CL

) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

log

CL (

rena

l)=

–0.

41R

mui

– 0.

80R

Sul

fona

mid

esS

eyde

l and

Sch

aper

(19

82)

log

CL (

rena

l)=

–0.

51 lo

g P

o:w

– 0.

33R

Sul

fapy

ridin

esYa

mag

uchi

et

al. (

1996

) lo

g C

L (re

nal)

= –

log

[0.0

48 +

6.9

8 10

–4(1

0)1.

394 ]

RX

ylid

ines

Sey

del a

nd S

chap

er (

1982

)lo

g C

L (to

tal)

= –

0.74

Rm

ui+

0.2

2pK

a–

1.73

RS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

)lo

g E

(hep

atic

)=

0.0

45 lo

g P

o:w

– 0.

32R

HM

WO

Cs

Yam

aguc

hi e

t al

. (19

96)

CL (

intr

insi

c; h

epat

ic)=

3.8

28R

bB

asic

dru

gsIs

hiza

ki e

t al

. (19

97)

CL (

intr

insi

c; h

epat

ic)=

0.0

875

Rb

Bas

ic d

rugs

Ishi

zaki

et

al. (

1997

)

CL (

intr

insi

c; h

epat

ic)=

0.2

48R

bB

asic

dru

gsIs

hiza

ki e

t al

. (19

97)

QS

AR

S:

Fre

e–W

ilso

n-t

ype

equ

atio

ns

log

CL (

rena

l)=

0.4

17R

2(C

H3)

– 0

.744

R1(

OC

3H7)

+ 1

.33

RX

ylid

ines

Sey

del a

nd S

chap

er (

1982

) lo

g C

L (to

tal)

= 0

.49R

2(C

H3)

+ 0

.57

R2(

C2H

5) +

0.2

5R1(

OC

3H7)

+ 1

.76

RX

ylid

ines

Sey

del a

nd S

chap

er (

1982

)

aE

= v

aria

ble

rela

ted

to m

olec

ular

orb

itals

, R

mui

= v

aria

ble

depe

ndan

t on

the

res

ista

nce

cons

tant

due

to

diffu

sion

of

the

noni

oniz

ed f

orm

in t

he li

pid

mem

bran

e,

= m

olec

ular

hyd

roph

obic

ity c

onst

ant,

Apo

l=

pol

ar s

urfa

ce a

rea,

CIC

= c

ompl

emen

tary

info

rmat

ion

cont

ent,

Dip

ole-

mag

= d

ipol

e m

omen

t,E

H=

HO

MO

ene

rgy,

Ele

ctro

stat

ic =

Cou

lom

bic

inte

ract

ion

ener

gy,

Ene

rgy

= m

inim

um in

tern

al e

nerg

y, H

BD

= p

oten

tial h

ydro

gen

bond

don

or a

tom

s in

the

mol

ecul

e, H

f =

ent

halp

y of

form

atio

n, H

INT

= h

ydro

phob

ic fi

eld

ener

gy,

IP =

ioni

zatio

n po

tent

ial,

Jurs

-RP

CG

= r

elat

ive

posi

tive

char

ge,

Jurs

-RP

CS

= r

elat

ive

posi

tive

char

ge s

urfa

ce a

rea,

LU

MO

= l

owes

t un

occu

pied

mol

ecul

ar o

rbita

l en

ergy

, P

o:w

= n

-oct

anol

:wat

er p

artit

ion

coef

ficie

nt (

or v

eget

able

oil:w

ater

), P

o:w

, ap

p=

app

aren

t oc

tano

l:wat

er p

artit

ion

coef

ficie

nt,

Q6

= n

et a

tom

ic c

harg

e on

car

bon

atom

at

biph

enyl

rin

g po

sitio

n, R

2(C

H3)

= m

ethy

lfr

agm

ent

at R

2po

sitio

n,R

2(C

2H5)

= e

thyl

fra

gmen

t at

R2

posi

tion,

R1(

OC

3H7)

= p

ropy

l eth

er f

ragm

ent

at R

1po

sitio

n, S

_aaO

= E

-sta

te in

dice

s fo

r ox

ygen

atom

s w

ith tw

o ar

omat

ic b

onds

, S_s

Cl =

E-s

tate

indi

ce fo

r ch

lorin

e at

oms

with

a s

ingl

e bo

nd, S

hado

w Z

leng

th =

leng

th o

f the

mol

ecul

e in

Zdi

men

sion

,S

N=

tot

al n

ucle

ophi

llic

supe

rdel

ocal

izab

ility

, an

d S

teric

= V

an d

er W

aals

inte

ract

ion

ener

gy.

bF

= fi

sh,

H =

hum

an,

and

R =

rat

s.c

CF

Cs

= c

hlor

ofluo

roca

rbon

s, H

MW

OC

s =

hig

h m

olec

ular

wei

ght

orga

nic

chem

ical

s, L

MW

VO

Cs

= lo

w m

olec

ular

wei

ght

vola

tile

orga

nic

chem

ical

s, a

ndV

OC

s =

vol

atile

org

anic

che

mic

als.


Tab

le 4

0.6

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

Rea

ctio

n R

ates

of

Ch

emic

alsa

Ap

pro

ach

bS

pec

iesc

Ch

emic

al C

lass

dR

efer

ence

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ele

ctro

stat

ic d

escr

ipto

rs

log

V(o

xida

tion)

= 0

.894

– 0

.111

diam

eter

– 0

.007

EH

Nitr

iles

Lew

is a

nd D

icki

ns (

2002

) lo

gk c

at (

oxid

atio

n)=

19.

97 –

0.0

24H

– 0.

95IP

HTo

luen

esLe

wis

and

Dic

kins

(20

02)

log

V(o

xida

tion)

= 2

6.90

– 2

.58I

PH

Hal

otha

nes

Lew

is a

nd D

icki

ns (

2002

) lo

gk c

at (

oxid

atio

n)=

0.0

24V

ol –

0.2

3–

1.14

HB

arbi

tura

tes

Lew

is a

nd D

icki

ns (

2002

) lo

gk c

at (

oxid

atio

n)=

1.3

3 –

0.15

HA

nilin

esLe

wis

and

Dic

kins

(20

02)

log

k cat

(de

met

hyla

tion)

= –

0.68

–

+ 1

.06

RX

-C6H

4N(C

H3)

2H

ansc

h an

d Le

o (1

995)

Ste

ric

des

crip

tors

log

k cat/K

m(O

xida

tion)

= 0

.034

7SA

– 2

.29

E+

1.9

2H

Tolu

enes

Lew

is a

nd D

icki

ns (

2002

) lo

gV

max

(n-

dem

ethy

latio

n)=

0.1

8 Le

ngth

– 1

.94

HE

thyl

amin

esLe

wis

(20

01)

log

Vm

ax (

n-de

met

hyla

tion)

= 3

.50

Leng

th –

0.1

3 Le

ngth

2–

23.9

HE

thyl

amin

esLe

wis

(20

01)

log

V(o

xida

tion)

= 2

.486

1 –

0.13

64N

PL

* N

SID

E +

0.5

694U

NS

– 0

.243

3NO

M *

NM

C

+ 0

.001

227M

W *

NU

NS

TOT

+ 0

.824

2IN

D –

1.1

493M

OD

RP

CB

sP

arha

m a

nd P

ortie

r (1

998)

log

Vm

ax (

oxid

atio

n)=

–1.

6764

+ 0

.424

3–

0.13

4 +

1.6

22R

Hal

oalk

anes

Gar

gas

et a

l. (1

988)

V(n

-dem

ethy

latio

n)=

0.0

05S

A–

0.52

RA

min

esLe

wis

and

Dic

kins

(20

02)

V(n

-dem

ethy

latio

n)=

0.0

38S

A–

0.00

001S

A2

– 25

.64

RA

min

esLe

wis

and

Dic

kins

(20

02)

Hyd

rop

ho

bic

des

crip

tors

log

k cat/K

m(d

emet

hyla

tion)

= 0

.53

log

Po:

w+

3.4

7R

X-C

6H4N

(CH

3)2

Han

sch

and

Leo

(199

5)

log

V=

0.5

5 lo

g P

o:w

RB

arbi

tura

tes

Han

sch

and

Leo

(199

5)(c

ontin

ued)


Tab

le 4

0.6

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

Rea

ctio

n R

ates

of

Ch

emic

alsa

Ap

pro

ach

bS

pec

iesc

Ch

emic

al C

lass

dR

efer

ence

QS

AR

S:

Fre

e–W

ilso

n-t

ype

equ

atio

ns

Vm

axc

= B

S(C

-C)(5

1.6)

+ n

H3(

14.6

) +

nC

L(–4

.84)

+ n

CL 2

(10.

2) +

nC

L 3(–

16.9

)R

, H

Chl

oroe

than

esF

ouch

écou

rt a

nd K

rishn

an

(200

0)

ak c

at=

cat

alyt

ic r

ate,

Km

= e

nzym

e af

finity

con

stan

t, V

= m

etab

olic

rat

e, V

max

= m

axim

al v

eloc

ity o

f m

etab

olis

m,

and

Vm

axc

= b

ody

wei

ght

norm

aliz

edm

axim

al v

eloc

ity o

f m

etab

olis

m.

bE

= L

UM

O e

nerg

y –

HO

MO

ene

rgy,

H

= h

ydro

gen

abst

ract

ion

ener

gy,

= d

ipol

ar m

omen

t of

the

mol

ecul

e, 4

,3

, =

con

nect

ivity

indi

ces,

BS

= b

asic

str

uctu

re,

diam

eter

= d

iam

eter

of

the

mol

ecul

e, I

ND

= v

aria

ble

depe

ndan

t on

exp

erim

enta

l da

ta u

sed,

IP

= i

oniz

atio

n po

tent

ial,

Leng

th =

leng

th o

f the

mol

ecul

e, M

OD

= v

aria

ble

depe

ndan

t on

expe

rimen

tal d

ata

used

, MW

= m

olec

ular

wei

ght,

nCL

= n

umbe

r of

CL

frag

men

ts,

nCL 2

= n

umbe

rof

CL 2

frag

men

ts,

nCL 3

= n

umbe

r of

CL 3

frag

men

ts,

nH3

= n

umbe

r of

H3

frag

men

ts,

NM

C =

num

ber

of m

eta

chlo

rines

, N

OM

= n

umbe

r of

adj

acen

tun

subs

titut

ed o

rtho

-met

a ca

rbon

pai

rs, N

PL

= v

aria

ble

depe

ndan

t on

the

num

ber

of c

hlor

ide

atom

s in

the

mol

ecul

e in

ort

ho p

ositi

on, N

SID

E =

var

iabl

ede

pend

ant

on t

he n

umbe

r of

chl

orid

e at

oms

in t

he m

olec

ule

in m

eta

posi

tion,

NU

NS

TOT

= v

aria

ble

depe

ndan

t on

the

num

ber

of c

hlor

ide

atom

s in

the

mol

ecul

e, P

o:w

= n

-oct

anol

:wat

er p

artit

ion

coef

ficie

nt (

or v

eget

able

oil:

wat

er),

SA

= s

urfa

ce a

rea,

UN

S =

var

iabl

e de

pend

ant o

n th

e nu

mbe

r of

ato

ms

in t

he m

olec

ule

that

are

not

chl

orid

e, V

ol =

vol

ume

of t

he m

olec

ule,

and

–

= H

amm

et c

onst

ant.

cF

= fi

sh,

H =

hum

an,

and

R =

rat

s.d

PC

Bs

= p

olyc

hlor

obip

heny

ls.


Tab

le 4

0.7

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e M

ich

aelis

–Men

ten

Affi

nit

y C

on

stan

t (K

m)

of

Ch

emic

als

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ele

ctro

stat

ic d

escr

ipto

rs

log

1/K

m(d

emet

hyla

tion)

= 0

.46

log

Po:

w+

0.6

3–

+ 2

.62

HX

-C6H

4N(C

H3)

2H

ansc

h an

d Le

o (1

995)

lo

g 1/

Km

(sul

fatio

n)=

0.9

2 lo

g P

o:w

– 1.

48M

R4

– 0.

64M

R3

+ 1

.04M

R2

+ 0

.67

–+

4.0

1H

Phe

nols

Han

sch

and

Leo

(199

5)lo

gK

m(a

cety

latio

n)=

–0.

42 lo

g P

ui+

0.1

4pK

a–

2.89

HS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

) K

m(o

xida

tion)

= [

(ia

d() i)

/|IP

i–

b|]

+ c

RA

lken

esC

sana

dy e

t al

. (19

95)

log

Km

(ace

tyla

tion)

= 0

.17p

Ka

– 0.

69R

Sul

fona

mid

esS

eyde

l and

Sch

aper

(19

82)

log

Km

(ace

tyla

tion)

= –

0.42

Rm

u:i+

0.1

5pK

a–

1.39

RS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

)lo

gK

m(a

cety

latio

n)=

0.0

7pK

a+

0.3

1 lo

g P

o:w

– 0.

33R

bS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

)

Hyd

rop

ho

bic

des

crip

tors

log

1/K

m(o

xida

tion)

= 1

.39

log

Po:

w–

0.22

log

Po:

w2

– 0.

50H

Bar

bitu

rate

sLe

wis

and

Dic

kins

(20

02)

log

1/K

m(s

ulfa

tion)

= 2

.93F

2+

1.1

62

+ 0

.91

3+

0.8

2MR

2–

0.59

IO

H+

1.2

9IE

T+

2.5

9H

Phe

nols

Han

sch

and

Leo

(199

5)

–log

Km

(oxi

datio

n)=

43.

27 –

4.0

3E

– 0.

60 lo

g P

o:w

HTo

luen

esLe

wis

and

Dic

kins

(20

02)

log

1/K

m(g

luco

roni

datio

n)=

0.8

3 lo

g P

o:w

+ 1

.37

RP

heno

lsH

ansc

h an

d Le

o (1

995)

log

1/K

m(h

ydro

lysi

s)=

0.0

56Z

1 H2O

+ 0

.051

Z2 H

2O+

0.0

26Z

3 H2O

+ 0

.04Z

4 H2O

+ 4

.616

RP

heny

lhip

pura

tes

Kim

(19

93)

log

1/K

m(h

ydro

lysi

s)=

0.0

66Z

1 H2O

+ 4

.259

RP

heny

lhip

pura

tes

Kim

(19

93)

log

1/K

m(h

ydro

lysi

s)=

0.4

4+

4.0

8R

Phe

nylh

ippu

rate

sK

im (

1993

)lo

g 1/

Km

(hyd

roly

sis)

= 0

.40

+ 4

.40

RP

heny

lhip

pura

tes

Kim

(19

93)

log

1/K

m(N

AD

P-o

xida

tion)

= 0

.69

log

Po:

w+

2.9

0R

Dru

gsS

eyde

l and

Sch

aper

(19

82)

log

Km

(n-d

emet

hyla

tion)

= –

0.55

log

Po:

w+

2.6

7R

Mor

phin

esH

ansc

h an

d Le

o (1

995)

log

Km

(oxi

datio

n)=

0.6

1 lo

g P

o:w

+ 2

.23

RC

arba

mat

esH

ansc

h an

d Le

o (1

995)

log

Km

(oxi

datio

n)=

1.0

2 lo

g P

o:w

+ 2

.98

RP

yraz

oles

Han

sch

and

Leo

(199

5)(c

ontin

ued)


Tab

le 4

0.7

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e M

ich

aelis

–Men

ten

Affi

nit

y C

on

stan

t (K

m)

of

Ch

emic

als

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

log

Km

(oxi

datio

n)=

1.0

5 lo

g P

o:w

+ 1

.22

R4-

nitr

ophe

nyl a

lkyl

et

hers

Han

sch

and

Leo

(199

5)

log

Km

(oxi

datio

n)=

0.7

9 lo

g P

o:w

+ 1

.46

RA

lkyl

benz

enes

Han

sch

and

Leo

(199

5)lo

gK

m(o

xida

tion)

= 1

.04

log

Po:

w+

1.1

0R

bTo

luen

esH

ansc

h an

d Le

o (1

995)

QS

AR

s: F

ree–

Wils

on

-typ

e eq

uat

ion

s

Km

= B

S(C

-C)(3

.8)

+ n

H3(

–2.5

9) +

nC

L(–0

.37)

+ n

CL 2

(0.7

9) +

nC

L 3(0

.19)

R,

HC

hlor

oeth

anes

Fou

chéc

ourt

and

Kris

hnan

(2

000)

aE

= L

UM

O e

nerg

y –

HO

MO

ene

rgy,

–

= H

amm

et c

onst

ant,

= d

ipol

ar m

omen

t of

the

mol

ecul

e,

,2,

3=

mol

ecul

ar h

ydro

phob

icity

con

stan

ts,

Rm

ui

= v

aria

ble

depe

ndan

t on

the

resi

stan

ce c

onst

ant d

ue to

diff

usio

n of

the

noni

oniz

ed fo

rm in

the

lipid

mem

bran

e, a

= o

rbita

l ava

ilabi

lity,

b=

LU

MO

ene

rgy,

BS

= b

asic

str

uctu

re,

c=

var

iabl

e de

pend

ant

on t

he m

olec

ular

siz

e, d

()

= n

orm

aliz

ed e

lect

ron

dens

ity,

F2

= v

aria

ble

depe

ndan

t on

the

ele

ctric

al fi

eld

indu

ced

by o

rtho

pos

ition

ed a

tom

s, l

OH

= v

aria

ble

depe

ndan

t on

the

num

ber

of

OH

gro

ups

in t

he m

olec

ule,

lE

T=

var

iabl

e de

pend

ant

on t

he f

amily

of t

he s

ubst

ance

, IP

= io

niza

tion

pote

ntia

l, M

R2,

3,4

= m

olar

ref

ract

ivity

indi

ces,

nC

L =

num

ber

of C

L fr

agm

ents

, nC

L 2=

num

ber

of C

L 2fr

agm

ents

, nC

L 3=

num

ber

of C

L 3fr

agm

ents

, nH

3=

num

ber

of H

3fr

agm

ents

, pK

a=

log

diss

ocia

tion

cons

tant

of a

n ac

id in

wat

er, P

o:w

= n

-oct

anol

:wat

er p

artit

ion

coef

ficie

nt(o

r ve

geta

ble

oil:w

ater

), P

ui=

n-o

ctan

ol:w

ater

par

titio

n co

effic

ient

for

the

non

ioni

zed

form

, an

d Z

1, 2

, 3,

4H

2O=

var

iabl

es c

orre

spon

ding

to

the

pote

ntia

len

ergy

for

the

inte

ract

ion

betw

een

the

mol

ecul

e an

d w

ater

.b

F =

fish

, H

= h

uman

, an

d R

= r

ats.

cP

CB

s =

pol

ychl

orob

iphe

nyls

.


In Silico Approaches for Skin Permeability Constants


Tab

le 4

0.8

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e S

kin

Per

mea

bili

ty C

oef

fici

ent

(Kp)

of

Ch

emic

als

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

cR

efer

ence

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ele

ctro

stat

ic d

escr

ipto

rs

log

Kp

= –

0.62

6C

a –

23.8

(Q+

)/0.

289S

sssC

H –

0.0

357S

sOH

–

0.48

2IB

+ 0

.405

BR

+ 0

.834

HLM

WV

OC

s; H

MW

OC

sM

oss

et a

l. (2

002)

log

Kp

= 0

.44R

2–

0.49

– 1

.48

– 3.

44+

1.9

4Vx

– 5.

13H

LMW

VO

Cs;

HM

WO

Cs

Mos

s et

al.

(200

2)

log

Kp

= –

0.59

– 0

.63

– 3.

48+

1.7

9Vx

– 5.

05H

LMW

VO

Cs;

HM

WO

Cs

Mos

s et

al.

(200

2)

log

Kp

= –

5.33

– 0

.62

– 0

.38

– 3.

34+

1.8

5Vx

HA

lcoh

ols,

ste

roid

sG

hafo

uria

n an

d F

oola

di (

2001

)

Ste

ric

des

crip

tors

Kp

= (

b 1+

0.0

025/

(b2

+ b

3+

))–1

MW

b5H

LMW

VO

Cs;

HM

WO

Cs

Mos

s et

al.

(200

2)

Kp

= (

b 1+

b 2P

o:w)e

(b3M

W)

HLM

WV

OC

s; H

MW

OC

sM

oss

et a

l. (2

002)

log

Kp

= –

5.14

– 0

.47

Ca

+ 0

.23

Cd

+ 0

.038

Pol

HA

lcoh

ols,

ste

roid

sR

aevs

ky a

nd S

chap

er (

1998

)lo

gK

p=

–6.

14 –

0.4

2C

a +

0.2

3C

d +

0.2

1L –

0.1

1WH

Alc

ohol

s, s

tero

ids

Rae

vsky

and

Sch

aper

(19

98)

log

Kp

= –

7.29

+ 0

.15P

olH

Alc

ohol

sR

aevs

ky a

nd S

chap

er (

1998

)lo

gK

p=

b 1+

b 2lo

gP

o:w

+b 3

MW

0.5

HLM

WV

OC

s; H

MW

OC

sM

oss

et a

l. (2

002)

log

Kp

= –

0.42

8–

4.80

+ 2

8.06

HH

ydro

cort

icon

e es

ters

Gha

four

ian

and

Foo

ladi

(20

01)

log

Kp

= 0

.652

log

Po:

w–

0.00

603M

W –

0.6

23A

BS

Qon

– 0

.313

Sss

sCH

– 2

.3H

Der

mal

dru

gs; L

MW

VO

Cs;

H

MW

OC

sP

atel

et

al. (

2002

)

log

Kp

= 0

.77

log

Po:

w–

0.01

03M

W –

2.3

3H

LMW

VO

Cs;

HM

WO

Cs

Mos

s et

al.

(200

2)

log

Kp

= –

0.78

6OT

+ 0

.252

2–

1.61

7 –

5.7

67H

Alc

ohol

s, s

tero

ids

Gha

four

ian

and

Foo

ladi

(20

01)

log

Kp

= 0

.82

log

Po:

w–

0.00

93V

m–

0.03

9MP

t–

2.36

HS

tero

ids

Mos

s et

al.

(200

2)lo

gK

p=

0.8

4 lo

g P

o:w

– 0.

07(lo

g P

o:w)2

– 0.

27H

b –

1.84

log

MW

+ 4

.39

HLM

WV

OC

s; H

MW

OC

sM

oss

et a

l. (2

002)


log

Kp

= 2

8.4q

–+

0.0

18V

m+

2.8

24H

Bar

bitu

rate

s; I

soqu

inol

ine;

S

alic

yclic

aci

dG

hafo

uria

n an

d F

oola

di (

2001

)

log

Kp

= 3

.99

log

TA +

4.5

3 –

0.7

62O

T –

11.

364

HA

lcoh

ols,

Ste

roid

sG

hafo

uria

n an

d F

oola

di (

2001

)

Hyd

rop

ho

bic

des

crip

tors

Kp

= 1

.17

10–7

+ 2

.73

10–8

HP

harm

aceu

tical

sM

oss

et a

l. (2

002)

Kp

=b 1

(/(

b 3+

))H

HM

WO

Cs

Mos

s et

al.

(200

2)

log

Kp

= –

0.20

7 lo

g +

1.4

9 lo

g P

o:w

– 5.

42H

Ste

roid

sS

eyde

l and

Sch

aper

(19

82)

log

Kp

= –

0.37

log

+ 2

.39

log

Po:

w–

8.71

HP

heno

lsTe

sta

et a

l. (2

000)

log

Kp

= 0

.544

log

Po:

w–

2.88

HA

lipha

tic a

lcoh

ols

Sey

del a

nd S

chap

er (

1982

)lo

gK

p=

0.8

0 lo

g P

o:w

– 8.

883

HH

ydro

cort

icon

e es

ters

Gha

four

ian

and

Foo

ladi

(20

01)

log

Kp

= –

1.46

lo

gP

o:w

+ 0

.29

log

Po:

w–

3.75

HA

lcoh

ols,

ste

roid

sTe

sta

et a

l. (2

000)

lo

gK

p=

–4.

36 –

0.3

8C

a +

0.2

4C

dH

Ste

roid

sR

aevs

ky a

nd S

chap

er (

1998

)

Mec

han

isti

cally

bas

ed e

qu

atio

ns

Kp

=

(Pvo

:w*0.

028D

l/0.0

340)

+ (

Pp:

w*0.

88D

p/0.0

018)

HA

cids

; Alc

ohol

s; H

ydro

carb

ons

Pou

lin a

nd K

rishn

an (

2001

)

a=

sol

ubili

ty p

aram

eter

, =

dip

olar

ity/p

olar

izab

ility

, =

ove

rall

hydr

ogen

-bon

d ac

idity

, =

ove

rall

hydr

ogen

-bon

d ba

sici

ty,

Ca

= h

ydro

gen

bond

acce

ptor

fre

e en

ergy

in t

he m

olec

ule,

C

d =

hyd

roge

n bo

nd d

onor

in t

he m

olec

ule,

2=

mol

ecul

ar s

hape

inde

x,

= c

onne

ctiv

ity in

dice

s, A

BS

Qon

= s

um o

f ab

solu

te c

harg

es o

n ox

ygen

and

nitr

ogen

ato

ms,

b1,

b 2,

b 3,

b 4,

b 5=

reg

ress

ion

coef

ficie

nts

with

out

any

assi

gned

rol

e, B

R=

num

ber

of r

otat

able

bond

s, D

l=

coe

ffici

ent

for

diffu

sion

int

o th

e lip

id f

ract

ion

of s

trat

um c

orne

um,

Dp

= c

oeffi

cien

t fo

r di

ffusi

on i

nto

the

prot

ein

frac

tion

of s

trat

um c

orne

um,

Hb

= n

umbe

r of

hyd

roge

n bo

nds

form

ed b

y th

e su

bsta

nce,

IB

= B

alab

an in

dex,

L=

mol

ecul

ar le

ngth

, M

Pt=

mel

ting

poin

t, M

W =

mol

ecul

ar w

eigh

t, O

T=

num

ber

of h

ydro

gen

bond

ing

hete

roat

oms,

Po:

w=

n-o

ctan

ol:w

ater

par

titio

n co

effic

ient

(or

veg

etab

le o

il:w

ater

), P

ol =

des

crib

es b

ulk

or v

olum

e re

late

def

fect

s, P

p:w

= p

rote

in:w

ater

par

titio

n co

effic

ient

for

str

atum

cor

neum

, P

vo:w

= v

eget

able

oil:

wat

er p

artit

ion

coef

ficie

nt,

q–=

the

mos

t ne

gativ

e ch

arge

on

the

hydr

ogen

bon

d ac

cept

ing

hete

roat

oms,

Q+/

= p

ositi

ve c

harg

e pe

r un

it vo

lum

e,

= s

um o

f at

omic

cha

rges

on

hydr

ogen

bon

ding

het

eroa

tom

s, =

sum

of

atom

ic c

harg

es o

n hy

drog

en b

ondi

ng h

ydro

gens

, R

2=

exc

ess

mol

ar r

efra

ctio

n, S

sOH

= s

um o

f E

-sta

te i

ndic

es f

or a

ll hy

drox

y gr

oups

,S

sssC

H =

sum

of

E-s

tate

indi

ces

for

all m

ethy

l gro

ups,

TA

= t

otal

sol

vant

acc

essi

ble

surf

ace,

Vm

= m

olar

vol

ume,

Vx

= M

cGow

an c

hara

cter

istic

vol

ume,

and

W =

mol

ecul

ar w

idth

.b

F =

fish

, H

= h

uman

, an

d R

= r

ats.

cC

FC

s =

chl

orofl

uoro

carb

ons,

HM

WO

Cs

= h

igh

mol

ecul

ar w

eigh

t or

gani

c ch

emic

als,

LM

WV

OC

s =

low

mol

ecul

ar w

eigh

t vo

latil

e or

gani

c ch

emic

als,

and

VO

Cs

= v

olat

ile o

rgan

ic c

hem

ical

s.


In Silico Approaches for Oral Absorption Constants

INTEGRATING IN SILICO APPROACHES INTO RISK ASSESSMENT


Tab

le 4

0.9

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e O

ral

Ab

sorp

tio

n C

on

stan

t (K

a) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

Ref

eren

ce

QS

AR

s: L

FE

-typ

e eq

uat

ion

s

Ele

ctro

stat

ic d

escr

ipto

rs

log

Ka(

abso

rptio

n)=

–0.

58 lo

g P

o:w

+ 0

.35p

Ka

– 1.

77F

Bar

bitu

rate

sS

eyde

l and

Sch

aper

(19

82)

Hyd

rop

ho

bic

des

crip

tors

Ka(

abso

rptio

n)=

k m[(

1/R

f) –

1]n’/(

Q+

[(1

/Rf)

– 1]

n’)

RS

ulfo

nam

ides

Test

a et

al.

(200

0)

log

Ka(

abso

rptio

n)=

–0.

04 (

log

Po:

w)2

+ 0

.22

log

Po:

w+

0.0

4R

Sul

fona

mid

esS

eyde

l and

Sch

aper

(19

82)

log

Ka(

abso

rptio

n)=

0.0

67 +

log

Po:

w–

log(

1.4

+ P

o:w)

RP

harm

aceu

tical

sYa

mag

uchi

et

al. (

1996

) lo

gK

a(ab

sorp

tion)

= –

0.08

2(lo

g P

o:w)2

+ 0

.268

log

Po:

w+

3.9

6R

Org

anic

aci

dsS

eyde

l and

Sch

aper

(19

82)

log

Ka(

abso

rptio

n)=

–0.

09 (

log

Po:

w)2

+ 0

.44

log

Po:

w–

0.39

6R

Sul

fona

mid

esS

eyde

l and

Sch

aper

(19

82)

log

Ka(

abso

rptio

n)=

0.0

9 lo

g P

o:w

+ 0

.83

RX

anth

enes

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(ab

sorp

tion)

= 0

.18

log

Po:

w+

0.2

3R

Car

bam

ates

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(ab

sorp

tion)

= 0

.24

log

Po:

w–

1.37

RA

ntih

ista

min

esS

eyde

l and

Sch

aper

(19

82)

log

Ka(

abso

rptio

n)=

0.3

0 lo

g P

o:w

– 0.

07(lo

g P

o:w)2

– 2.

38R

Sul

fony

lure

asS

eyde

l and

Sch

aper

(19

82)

log

Ka(

abso

rptio

n)=

0.3

log

Po:

w–

0.57

log

(0.3

4 P

o:w

+ 1

) –

0.15

I–

0.74

RC

arba

mat

esS

eyde

l and

Sch

aper

(19

82)

log

Ka(

abso

rptio

n)=

0.4

6 lo

g P

o:w

– 0.

36 lo

g (0

.60P

o:w

+ 1

) –

0.23

RS

ulfo

nylu

reas

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(ab

sorp

tion)

= 0

.5 lo

g P

o:w

–0.6

1 lo

g (0

.07P

o:w

+ 1

) –

0.39

RS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(ab

sorp

tion)

= 0

.502

log

Po:

w–

log

(0.0

53P

o:w

0.08

62+

1)

– 0.

384

RS

ulfo

nam

ides

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(ab

sorp

tion)

= 0

.56

log

Po:

w–

(0.0

4Po:

w0.

84+

1)

– 0.

63R

Phe

nols

; Ani

lines

; Est

ers

Sey

del a

nd S

chap

er (

1982

)lo

gK

a(ab

sorp

tion)

= 1

.36

log

Po:

w+

0.3

6R

Org

anic

ani

ons

Sey

del a

nd S

chap

er (

1982

)(c

ontin

ued)


Tab

le 4

0.9

(co

nti

nu

ed)

In S

ilico

Ap

pro

ach

es f

or

Est

imat

ing

th

e O

ral

Ab

sorp

tio

n C

on

stan

t (K

a) o

f C

hem

ical

s

Ap

pro

ach

aS

pec

iesb

Ch

emic

al C

lass

Ref

eren

ce

QS

AR

s: F

ree–

Wils

on

typ

e eq

uat

ion

s

log

Ka(

abso

rptio

n)=

BS

(BE

N-S

O2-

NH

CO

NH

)(–2.

272)

+ n

H (

0) +

n2-

CH

3(0

.088

) +

n4-

CH

3(0

.074

) +

n 4-C

2H5(0

.163

) +

n4-

OC

H3

(–0.

229)

+ n

2-N

O2

(–0.

324)

+

n3-N

O2

(–0.

207)

+ n

4-N

O2

(–0.

323)

+ n

4-C

l (0.

198)

+ n

4-B

r(0.

122)

+ n

n-C

4H9(

0) +

nC

H3

(–0.

638)

+ n

C2H

5(–

0.36

1) +

nn-

C3H

7(–

0.14

5) +

ni-C

3H7

(–0.

244)

+ n

i-C4H

9(–0

.035

) +

nt-

C4H

9(0

.149

) +

ncy

-C6H

11(0

.135

) +

nal

lyl

(–0.

419)

+ n

C6H

5(–

0.08

8)

RS

ulfo

nylu

reas

Sey

del a

nd S

chap

er (

1982

)

aal

lyl

= a

llyl

grou

p, B

r =

bro

mid

e, B

S =

bas

ic s

truc

ture

, C

2H5

= e

thyl

gro

up,

C6H

5=

aro

mat

ic r

ing

grou

p, C

H3

= m

ethy

l gr

oup,

Cl

= c

hlor

ide

grou

p, c

y-C

6H11

= c

yclo

hexy

l gr

oup,

H =

hyd

roge

n gr

oup,

I=

fam

ily i

ndic

ator

var

iabl

e, i

-C3H

7=

iso

prop

yl g

roup

, i-C

4H9

= i

sobu

tyl

grou

p, k

m=

rat

e co

nsta

nt f

ortr

ansf

er o

ut o

f th

e m

embr

ane,

n=

gro

up o

ccur

renc

e in

mol

ecul

e, n

’= c

onst

ant

spec

ific

to t

he e

quat

ion

with

out

any

give

n ro

le,

n-C

3H7

=n-

prop

yl g

roup

,n-

C4H

9=

n-bu

tyl g

roup

, N

O2

= n

itroo

xide

gro

up,

OC

H3

= m

ethy

l eth

er g

roup

, pK

a=

log

diss

ocia

tion

cons

tant

of

an a

cid

in w

ater

, P

o:w

=n-

octa

nol:w

ater

part

ition

coe

ffici

ent (

or v

eget

able

oil:

wat

er),

Q=

con

stan

t spe

cific

to th

e eq

uatio

n w

ithou

t any

giv

en r

ole,

Rf=

rev

erse

-pha

se T

LC li

poph

ilici

ty p

aram

eter

,an

dt-

C4H

9=

tert

-but

yl g

roup

.b

F =

fish

and

R =

rat

s.


Free–Wilson QSARs for Chloroethanes

Table 40.10Frequency of Occurrence of Molecular Fragments for Each Chloroethane of the Series

Chemical BSa H3 Cl Cl2 Cl3

Chloroethane 1 1 1 0 01,1-dichloroethane 1 1 0 1 01,2-dichloroethane 1 0 2 0 01,1,1-trichloroethane 1 1 0 0 11,1,2-trichloroethane 1 0 1 1 01,1,1,2-tetrachloroethane 1 0 1 0 11,1,2,2-tetrachloroethane 1 0 0 2 0Pentachloroethane 1 0 0 1 1Hexachloroethane 1 0 0 0 2

a BS = basic structure (C-C).


Figure 40.1 Chemical description methodology used in this study. The chemicals are repre-sented as a basic structure (C-C) with substituents on the two carbons. Examplesof the description of 1,1,1 trichloroethane and 1,1,2,2 tetrachloroethane are pre-sented.

Cl

Cl

Cl

H

H

H

Cl

Cl

H

Cl

Cl

H

1,1,1-trichloroethane 1,1,2,2-tetrachloroethane

Basic structure:(-C-C-)

Molecular fragments:1 x (-C-C-)

1 x H3

1 x Cl3

Molecular fragments:1 x (-C-C-)

2 x Cl2H


Table 40.11 Contributionsa of Chloroethane Structural Features to Rat Partition Coefficientsb and Metabolic Constantsc

Fragments Pb Pl Ps Pf Vmaxc Km

BS 56.8 2.02 0.746 28.9 52.7 3.75Cl2 42.7 –0.319 –0.0181 –1.16 9.40 0.863Cl3 7.00 1.60 0.233 14.1 –15.3 –0.0932Cl –9.60 –0.506 0.00710 –7.22 –7.22 –0.234H3 –50.1 –0.653 –0.0770 –8.56 12.9 –1.65r 2 0.98 0.91 0.99 0.96 0.82 0.88

a Contributions were obtained by multiple linear regression from experimental data onchloroethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetra-chloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, and hexachloroethane. BS =basic structure (C-C).

b Pb, Pl,, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:bloodpartition coefficients, respectively.

c Vmaxc ( mol/hr/kg) and Km ( M) refer to maximal velocity of metabolism and affinity con-stant, respectively.

Table 40.12 Contributionsa of Chloroethane Structural Features to Human Partition Coefficientsb

Fragments Pb Pl Ps Pf

BS 37.4 2.72 1.099 38.9Cl2 29.6 –0.365 –0.163 0.105Cl3 7.53 2.16 0.166 12.2Cl –8.92 –0.446 0.0510 –10.6H3 –39.3 –0.699 0.0450 –1.05r 2 0.83 0.98 0.91 0.94

a Contributions were obtained by multiple linear regression from experimental data on chlo-roethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachlo-roethane, 1,1,2,2-tetrachloroethane, and hexachloroethane. BS = basic structure (C-C).

b Pb, Pl, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:bloodpartition coefficients, respectively.


Figure 40.2 Comparison of rat experimental and predicted parameter values. Experimentalvalues from Gargas et al. (1988, 1989).

Figure 40.3 Comparison of human experimental and predicted parameter values. Experimen-tal values were derived from Gargas et al. (1988, 1989).

y = 1.0089xR2 = 0.9159

0.1

1

10

100

1000

0.1 1 10 100 1000

Experimental parameter value

Est

imat

ed p

aram

eter

val

ue

y = 0.8984xR2 = 0.8557

0.1

1

10

100

1000

0.1 1 10 100 1000

Experimental parameter value

Est

imat

ed p

aram

eter

val

ue


Integrating Free–Wilson QSARs into PBPK Models

Table 40.13 Comparison of Experimentala (Exp) and QSAR-Estimated (Est) Values of Rat Partition Coefficientsb and Metabolic Constantsc for1,1,1-Trichloroethane

Parameter Exp Est

Pb 5.67 13.7Pl 1.52 2.97Ps 0.56 0.90Pf 46.4 34.5

Vmaxc 43.1 50.3Km 3.14 2.01

a Experimental data from Gargas et al. (1988, 1989).b Pb, Pl,, Ps, and Pf refer to blood:air, liver:blood, slowly perfused

tissue:blood and fat:blood partition coefficients, respectively.c Vmaxc ( mol/hr/kg) and Km ( M) refer to maximal velocity of

metabolism and affinity constant, respectively.

Table 40.14 Comparison of Experimentala (Exp) and QSAR-Estimated (Est) Values of Human Partition Coefficientsb for 1,1,1-Trichloroethane

Parameter Exp Est

Pb 2.53 5.56Pl 3.40 4.18Ps 1.25 1.31Pf 104 50.1

a Data derived from Gargas et al. (1989).b Pb, Pl, Ps, and Pf refer to blood:air, liver:blood, slowly perfused

tissue:blood and fat:blood partition coefficients, respectively.


QSAR-Based Risk Assessment of Methyl Chloroform


CONCLUSIONS AND FUTURE DIRECTIONS

Figure 40.4 Quantitative structure-activity relationship (QSAR) physiologically based pharma-cokinetic (PBPK) modeling framework. User input consists of the exposure sce-nario and chemical structure information such as the number of fragmentsconstituting the molecule. This information is fed to the program that contains themodel constants, the Free–Wilson type SPR, the contribution values of eachmolecular fragment (Cs) and of the basic structure (BS) to the model parameters(P), and the simulation algorithms. The model can then simulate the pharmaco-kinetics of the chemical in biota and then provide its profile as output. The exampleof 1,1,1-trichloroethane is shown.


Figure 40.5 Comparison of steady-state blood and tissue concentrations of chloroethanes inrats exposed to 1 ppm, as simulated by conventional and QSAR PBPK models.

Table 40.15 Steady-State Tissue Concentrations ( g/L) of 1,1,1-Trichloroethane in Rat and Humans Estimated Using the Conventional (PBPK) and QSAR-Based (QSAR) Physiologic Model Following a Continuous Exposure to 1 ppm

TissueRat Human

QSAR PBPK QSAR PBPK

Blood 22.9 16.6 12.8 8.5Liver 6.77 4.22 6.23 5.61Slowly perfused 20.7 9.31 16.7 10.7Fat 790 770 502 472Richly perfused 68.1 25.2 53.4 28.9

Table 40.16 Steady-State Arterial Blood Concentration (Cass) Obtained Using the Conventional (PBPK) and QSAR-Based (QSAR) Physiological Model in Rats Exposed to the NOAEL of 1,1,1-Trichloroethane (875 ppm) and the Corresponding Environmental Concentration (Ci) in Humans Derived Using the Human Conventional (PBPK) and QSAR-Based (QSAR) Physiological Models

Endpoint QSAR PBPK

Rat Cass (mg/L) 59.3 24.9Human Ci (ppm)a 6342 4252

a Calculated using the QSAR-derived Cass (59.3 mg/L).

y = 1.2184xR2 = 0.9746

0.0001

0.001

0.01

0.1

1

10

0.0001 0.001 0.01 0.1 1 10

Tissue concentrations obtained using QSPR-PBPK type model (mg/L)

Tis

sue

con

cen

trat

ion

s o

bta

ined

usi

ng

co

nven

tio

nal

-typ

e P

BP

Km

od

el (

mg

/L)



REFERENCES





533

CHAPTER 41

In Silico Application of Quantum ChemicalMethods for Relating Toxicity

to Chemical Reactivity

CONTENTS

INTRODUCTION


IN SILICO APPLICATION OF QUANTUM CHEMICAL METHODS 535

Figure 41.1 Structures of commercial pesticides.

N

CH3

OCH

O

P SCH3O

S

OCH3

CH C

CH2 C

O

OCH2CH3

OCH2CH3

O

MalathionCarbaryl


CHEMICAL REACTIVITY

PROGRESS OF THE REACTION

Figure 41.2 Hydrolysis of acetylcholine.

+

Choline

HOCH2CH2N CH3

CH3

CH3+

Acetic acid

CH3C

O

OHAChE

H2O

Acetylcholine

CH3C OCH2CH2N

O

CH3

CH3

CH3+


STRUCTURE/TOXICITY RELATIONS BASED ON CHEMICAL REACTIVITY


Figure 41.3 Progress of the reaction (x-axis) mapped against energy (y-axis).

ENERGY

P R2R1

O

F

PR1R2

O

OH

F

- P R2R1

OH

O

++ F-

OH-

PROGRESS OF THE REACTION


REFERENCES



543

CHAPTER 42

In Silico Cardiac Toxicity: Increasingthe Discovery of Therapeutics through

High-Performance Computing

CONTENTS

INTRODUCTION


IN SILICO CARDIAC TOXICITY 545

IN SILICO MODELS


E-Cell

Virtual Cell Environment


CellML Language and Other Resources

DATA MINING


A PRACTICAL EXAMPLE


In Silico Simulation of Atrial Action Potential Propagation in the Presence of Soman



o


Solution of the Monodomain Equations on the MSRC Assets

RESULTS

DISCUSSION


(a)

(b)

Figure 42.1 Frame sequence showing the evolution of the action potential in an atrial tissueusing the Nygren model for the electrophysiology of the tissue. The times of theframes going clockwise are (a) 50 ms, (b) 100 ms, (c) 150 ms, and (d) 200 ms.The stimulus consisted of a current pulse of 40 cm2 delivered to the leftmostcolumn of atrial cells. The stimulus lasted for 2 ms and VM in the legend is thevoltage in mV. (continued)


(c)

(d)

Figure 42.1 (continued) Frame sequence showing the evolution of the action potential in anatrial tissue using the Nygren model for the electrophysiology of the tissue. Thetimes of the frames going clockwise are (a) 50 ms, (b) 100 ms, (c) 150 ms, and(d) 200 ms. The stimulus consisted of a current pulse of 40 cm2 delivered tothe leftmost column of atrial cells. The stimulus lasted for 2 ms and VM in thelegend is the voltage in mV.


Figure 42.2 Action potential, entering from the left and encountering a region of high potassiumconcentration region at t = 40 and 250 ms, respectively. The hyperkalemic regionis along the tissue central axis, in the center taking up 1 cm2 of the 3 cm 3 cmtissue.


Figure 42.3 This figure contrasts the effect of potassium current blockers on the action poten-tial. The left-hand panel shows the voltage, that is, the action potential in thetissue without modulation of the currents. The effect of reducing the iKr and iKs,such as is effected by newer antiarrhythmic drugs such as azimilide, is shown inthe right-hand panel. Modulation of the wave distortion and erratic behavior shouldbe noted.


ACKNOWLEDGMENTS

REFERENCES


559

CHAPTER 43

Submillimeter-Wave FrequencyStudies of the Vibrational Modes

of Deoxyribonucleic Acid:A Metric for Mutagenicity?

CONTENTS

INTRODUCTION


METHODS AND MATERIALS

Computational Studies

Experimental Measurements

FREQUENCY STUDIES OF THE VIBRATIONAL MODES OF DNA 561


Computational Studies

Figure 43.1 Calculated spectra of Poly(dA)Poly(dT) and of Poly(dAdT)Poly(dTdA) at two line-widths: 2-wavenumber and 7-wavenumber.

0 100 200 300 400 500

Poly(dA)Poly(dT)

γ = 7 cm -1

γ = 2 cm -1

0 100 200 300 400 500

Poly(dAdT)Poly(dTdA)

0 100 200 300 400 5000 100 200 300 400 500

Abs

orpt

ion

Frequency (cm-1)


Experimental Studies

Table 43.1 Comparison of Predicted Vibrational Band Positions (in Wavenumbers) of Poly(dA)Poly(dT) with Values Observed by Experiments in the Literature

CalculationsExperiment

(Powell, 1987) CalculationsExperiment

(Powell, 1987)

20 Unknown 172 17043 Unknown 199 20057 62 211 21471 80 — 23894 95 270 —

106 106 348 —131 136 400 —160 — 459 —

Figure 43.2 Experimental absorbance spectra of herring DNA in the mid-infrared through thevery far infrared region.

3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250 0

IIIIII

Rel

ativ

e A

bsor

banc

e

Wavenumber (cm-1)


Figure 43.3 Fine structure in the absorbance spectra of herring and salmon DNA in the veryfar infrared region.

24 22 20 18 16 14 12 100.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60 Herring DNA Salmon DNA

Tran

smis

sion

Wavenumber (cm-1)


REFERENCES

Figure 43.4 Transmission scattering parameter (S12) measurement of herring and salmon DNAin the microwave (W-band) region.

75.0 80.0 85.0 90.0 95.0 100.0 105.0 110.00.6

0.7

0.8

0.9

SalmonDNA HerringDNA

Sca

tterin

g P

aram

eter

, S12

Frequency, GHz


567

CHAPTER 44

Trophoblast Toxicity Assay (TTA):A Gestational Toxicity Test Using

Human Placental Trophoblasts

CONTENTS

HUMAN TROPHOBLASTS IN VIVO:THREE DIFFERENTIATION PATHWAYS


Figure 44.1 Pathways of trophoblast differentiation. Just as the basal layer of the skin givesrise to keratinocytes, the cytotrophoblast — the stem cell of the placenta — givesrise to the differentiated forms of trophoblasts. (Left) Within the chorionic villi,cytotrophoblasts fuse to form the overlying syncytiotrophoblast. The villous syn-cytiotrophoblast makes the majority of the placental hormones, the most studiedbeing hCG. cAMP, EGF, and even hCG itself have been implicated as stimulatorsof this differentiation pathway. In addition to upregulating hCG secretion, cAMPhas also been shown to down-regulate trophouteronectin (TUN) synthesis. (Cen-ter) At the point where chorionic villi make contact with external extracellularmatrix (decidual stromal ECM in the case of intrauterine pregnancies), a popu-lation of trophoblasts proliferates from the cytotrophoblast layer to form the secondtype of trophoblast — the junctional trophoblast. These cells form the anchoringcell columns that can be seen at the junction of the placenta and endometriumthroughout gestation. Similar trophoblasts can be seen at the junction of thechorion layer of the external membranes and the decidua. The junctional tropho-blasts make a unique fibronectin — trophouteronectin — that appears to mediatethe attachment of the placenta to the uterus. TGF and LIF have been shown toinduce cultured trophoblasts to secrete increased levels of trophouteronectin,while down-regulating hCG secretion. (Right) Finally, a third type of trophoblastdifferentiates toward an invasive phenotype and leaves the placenta entirely —the invasive intermediate trophoblast. In addition to making human placentallactogen, these cells also make urokinase and plasminogen activator inhibitor-1(PAI-1). Phorbol esters have been shown to increase trophoblast invasiveness inin vitro model systems and to upregulate PAI-1 in cultured trophoblasts. Thegeneral theme that comes from these observations is that specific factors arecapable of shifting the differentiation pathway of the cytotrophoblast toward oneof the above directions while turning off differentiation toward the other pathways.See text for details.

Cytotrophoblast

AnchoringTrophoblasts

VillousSyncytiotrophoblast

hCG TUN

Invading Trophoblasts

PAI-1

cAMPhCG

PhorbolEsters

LIFTGFß

HJK

TROPHOBLAST TOXICITY ASSAY (TTA) 569

Villous Syncytiotrophoblast


Anchoring Trophoblasts

Invading Trophoblasts

IN VITRO MODEL SYSTEMS TO STUDY TROPHOBLAST DIFFERENTIATION


TROPHOBLASTS AS ENDOCRINE CELLS

Figure 44.2 Purification of cytotrophoblasts from term placenta. A term placenta is minced anddigested with trypsin and DNAse. The supernatant is passed through calf serumto inactivate the digestive enzymes; then these pellets are pooled and placed ona Percoll gradient to separate out the cytotrophoblasts. (From Kliman et al. (1986)Endocrinology, 118(4), 1567–1582. With permission.)


PROTEIN HORMONES

Chorionic Gonadotropin

Figure 44.3 In vitro morphologic differentiation of cytotrophoblasts. After purification, thecytotrophoblasts are dispersed as individual cells (left). When plated in culturemedia containing serum, these cells flatten out and begin to move toward eachother. After 24 hr in culture, aggregates begin to appear, with some evidence ofcell fusion (center). After 72 hr in culture, most of the trophoblasts have fused andformed large, multinucleated syncytiotrophoblasts. (From Kliman et al. (1986)Endocrinology, 118(4), 1567–1582. With permission.)


Figure 44.4 hCG secretion by trophoblasts in culture. Percoll-gradient purified cytotrophoblastswere cultured in DMEM media for four days. Media was changed daily and assayedfor hCG by radioimmunoassay. hCG was not detectable at the time of initial plating.(From Kliman et al. (1986) Endocrinology, 118(4), 1567–1582. With permission.)

Table 44.1 Regulation of Trophoblast hCG Secretion

FactorTrophoblasts (Trimester)

Effect on hCG Secretion References

CAMP Term Stimulates (Feinman et al., 1986)HCG Term Stimulates (Shi et al., 1993)GnRH Term Stimulates (Belisle et al., 1989;

Szilagyi et al., 1992)GnRH First, Term Not clear (Kelly et al., 1991)-Adrenergic agonists First Stimulates (Oike et al., 1990)

Dexamethasone Term Stimulates (Ringler et al., 1989a)Inhibin Term Inhibits (Petraglia et al., 1987,

1989, 1991)Activin Term Potentiates GnRH

stimulation of hCG secretion

(Petraglia et al., 1991)

Activin First Stimulates (Steele et al., 1993)EGF First, Term Stimulates (Maruo et al., 1987)Thyroid hormone First, Term Stimulates (Maruo et al., 1991)Thyroid stimulating hormone

Term Inhibits (Beckmann et al., 1992)

Interleukin-1 First Stimulates (Yagel et al., 1989b)Interleukin-6 First Stimulates (Nishino et al., 1990)Basement membrane First Stimulates (Truman and Ford,

1986)Decidual protein Term Inhibits (Ren and Braunstein,

1991)Prolactin Term Inhibits (Yuen et al., 1986)


Human Placental Lactogen (hPL)

Figure 44.5 Effects of 8-bromo-cAMP on hCG and progesterone secretion by cultured cytotro-phoblasts. Percoll-gradient purified cytotrophoblasts were cultured for 48 hr in theabsence (�) or presence (�) of 8-bromo-cAMP. hCG (A) and progesterone (B)were quantitated in the medium at 24-hr intervals. Values presented are the mean± SE from six separate experiments. At each time point, 8-bromo-cAMP-treatedcultures secreted significantly more (p < 0.014, by the Wilcoxon signed rank test)progesterone and hCG than did control cultures. (From Feinman et al. (1986) J.Clin. Endocrinol. Metab., 63(5), 1211. With permission.)


TROPHOBLAST TOXICITY ASSAY

CONCLUSIONS


REFERENCES





581

Index

A


B

C

INDEX 583

D


E

INDEX 585

F

G

H


I

J

K

L

INDEX 587

M

N


O

P

Q

INDEX 589

R

S


T

U

V

INDEX 591

W Z

Documents

Alternative Toxicological Methods - H. Salem, S. katz (CRC, 2003) WW