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Biochemical and Molecular ToxicologyUNC ENVR/TOXC 442UNC ENVR/TOXC 44230nov2010www.unc.edu/courses/2010fall/envr/442/001/
Toxic Effects of Pesticides
David J. Dix, Ph.D.National Center for Computational ToxicologyNational Center for Computational Toxicology Office of Research & DevelopmentU.S. Environmental Protection AgencyResearch Triangle Park, NC 27711Email: [email protected]://www.epa.gov/ncct/
Pesticides in the News
A t 23 2009August 23, 2009Debating How Much Weed Killer Is Safe in Your Water GlassBy CHARLES DUHIGGFor decades, farmers, lawn care workers and professional green thumbs have relied on the popular weed killer atrazine to protect their crops, golf courses and manicured lawns. But atrazine often washes into water supplies and has become among the most common contaminants in American reservoirs and other sources of drinking water. Now, new research suggests that atrazine may be dangerous at lower concentrations than previously thought. Recent studies suggest that, even at concentrations meeting current federal standards, the chemical may be associated with birth defects, low birth weights and menstrual problems. Laboratory experiments suggest that when animals are exposed to brief doses of atrazine before birth, they may become more vulnerable to cancer later. An investigation by The New York Times has found that in some towns, atrazine concentrations in drinking water have spiked, sometimes for longer than a month. But the reports produced by local water systems for residents often fail to reflect those higher
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concentrations.
Pesticides in the News
August 23 2009
Pesticides in the News
August 23, 2009
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Pesticides in the News
October 7 2009October 7, 2009Regulators Plan to Study Risks of AtrazineBy CHARLES DUHIGGThe Environmental Protection Agency plans to conduct a new study about the potential health risks of atrazine a widely used weedkiller that recent research suggests may be more health risks of atrazine, a widely used weedkiller that recent research suggests may be more dangerous to humans than previously thought.Atrazine — a herbicide often used on corn fields, golf courses and even lawns — has become one of the most common contaminants in American drinking water. For years the E P A has decided against acting on calls to ban the chemical from For years, the E.P.A. has decided against acting on calls to ban the chemical from environmental activists and some scientists who argued that runoff was polluting ecosystems and harming animals. More recently, new studies have suggested that atrazine in drinking water is associated with birth defects low birth weights and reproductive problems among humans even at birth defects, low birth weights and reproductive problems among humans, even at concentrations that meet current federal standards.The E.P.A. is expected to announce on Wednesday that it will conduct a new evaluation of the pesticide to assess any possible links between atrazine and cancer, as well as other health problems such as premature births The E P A may determine that new restrictions are
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problems, such as premature births. The E.P.A. may determine that new restrictions are necessary.
Pesticides in the Scientific LiteraturePesticides in the Scientific Literature
PubMed, “atrazine AND birth defects”, 03nov2009
Agrichemicals in surface water and birth defects in the United States. Winchester PD, Huskins J, Ying J. Acta Paediatr. 2009 Apr;98(4):664-9. Epub 2009 Jan 22.PMID: 19183116
Effects of atrazine and endosulfan sulphate on the ecdysteroid system of Daphnia p y y pmagna. Palma et al. Chemosphere. 2009 Feb;74(5):676-81. Epub 2008 Nov 29.PMID: 19042009
The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors in fish and mammalian cells. Suzawa M, Ingraham HA. PLoS One. 2008 May 7;3(5):e2117 PMID: 184611792008 May 7;3(5):e2117.PMID: 18461179
Atrazine-induced reproductive tract alterations after transplacental and/or lactational exposure in male Long-Evans rats. Rayner JL, Enoch RR, Wolf DC, Fenton SE. Toxicol Appl Pharmacol. 2007 Feb 1;218(3):238-48. Epub 2006 Nov 23.PMID: 17204298
Ch t i ti f t i i d d d l lf ti i Af i l d fCharacterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (cyproterone acetate) and exogenous estrogen (17beta-estradiol): Support for the demasculinization/feminization hypothesis. Hayes et al. Environ Health Perspect. 2006 Apr;114 Suppl 1:134-41.PMID: 16818259
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In Principles and Methods of Toxicology (5th ed), A Wallace Hayes (editior)Pgs 727-840, Informa Healthcare, NY, 2007
Chapter 2 Why is a toxicant poisonous?Why is a toxicant poisonous?
Seven routes to death.
Publisher : CRC | ISBN : 0748409106 | edition 2004 | PDF | 296 pages
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Pesticides prevent, destroy, repel or mitigate any pestranging from insects animals and weeds to ranging from insects, animals and weeds, to microorganisms such as fungi, molds, bacteria and viruses.
insect killers (insecticides) • insect killers (insecticides)
• mold and fungi killers (fungicides)
• weed killers (herbicides)( )
• slug pellets (molluscicides)
• plant growth regulators
• bird and animal repellents
• rat and mouse killers (rodenticides)
• antimicrobials• antimicrobials
• inert ingredients
Pesticides help to manage and prevent pests that spread p g p p pdisease, that damage crops, buildings, and other property, and that are a public nuisance. 2
Pesticidal Classes- FungicidesPesticidal Classes Fungicides
A l l i d O lidi• Acylalanines and Oxazolidinones • Benzimidazoles and Thiophanates • Carboxamides • Methoxyacrylate and Oximinoacetate (Strobilurins)• Methoxyacrylate and Oximinoacetate (Strobilurins)• Organotin Compounds • Anilinopyrimidines • PhenylpyrrolesPhenylpyrroles• Dicarboximides • Demethylase Inhibitors• Inorganic Fungicidesg g• Dithiocarbamates and Ethylenebisdithiocarbamates • Phthalimides • Chloronitriles
9
Pesticidal Classes- InsecticidesPesticidal Classes Insecticides • Carbamates: AChE Inhibitors• Organophosphorus Insecticides: AChE Inhibitors
C l di O hl i GABA A i• Cyclodiene Organochlorines: GABA Antagonists • Organochlorines: Sodium Channel Modulators • Pyrethroids: Sodium Channel Modulators • Nicotine and Neonicotinoids: Acetylcholine Receptor Agonists• Nicotine and Neonicotinoids: Acetylcholine Receptor Agonists• Spinosyns: Acetylcholine Receptor Agonists• Avermectins and Milbemycin: Chloride Channel Activators • Juvenile Hormone Mimics and Selective Feeding BlockerJuvenile Hormone Mimics and Selective Feeding Blocker • Phenyltetrazines/Aminotriazines: Larvacides/ Molt Disruptors • Delta-Endotoxins Derived from Bacillus thuringiensis • Benzoylureas: Chitin Synthesis Inhibitors y y• Diacyhydrazine: Ecdysone Agonists • Octopaminergic Agonists and Monoamine Oxidase Inhibitors • Respiratory Inhibitors and Uncouplers
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• Pheromones
Pesticidal Classes- HerbicidesPesticidal Classes Herbicides • Acetyl–CoA Carboxylase Inhibitors • Aryloxyphenoxypropionates
• Diphenyl Ethers• N-Phenylphthalimides, Thiadiazoles,
d T i li• Aryloxyphenoxypropionates • Cyclohexanediones• Acetolactate Synthase Inhibitors• Sulfonylureas• Imidazolinones
and Triazolinones• Oxadiazole and Pyrimidindione
Herbicides• Bleaching Herbicides• Pyridazinones• Imidazolinones
• Triazolopyrimidines• Pyrimidinylthiobenzoates• Inhibition of Photosynthetic Electron
Transport
• Pyridazinones• Triketones and Isoxazoles• Triazoles and Isoxazolidinones • Synthase Inhibitors• Dinitroaniline Microtubule AssemblyTransport
• Triazines and Triazinone• Uracils and Pyridazinones • Ureas • Nitriles
• Dinitroaniline Microtubule Assembly Inhibitors
• Chloroacetamide Inhibitors of Very-Long-Chain Fatty Acid Synthesis
• Cellulose and Lipid Synthesis Nitriles• Benzothiadiazoles and Phenylpyridazine• Bipyridyliums • Protoporphyrinogen Oxidase Inhibitors
p yInhibitors
• Synthetic Auxin Mimics (Phenoxy, Benzoic, and Pyridine Acids)
• Herbicides with Unknown Mechanisms of Action
11
Mechanisms of Action
Complexities of the Nomenclature: Example
Stenersen, J. Chemical pesticides: Mode of Action and Toxicology. CRC Press 2004
Compendium of Pesticide Common Names
13http://www.alanwood.net/pesticides/
Pesticide MassPesticide Mass
14
Pesticide ToxicityPesticide Toxicity
Toxicity = Hazard = Dose x Response
Environmental Toxicity = Human + Ecological
• acutei
acute• chronic• cancer• mutagenicity
d ti
• aquatic• avian• terrestial• honey bee• reproductive
• endocrine• neurotoxicity• immunotoxicity
honey bee• plant• worms• ecosystems
15
y
Modes of action of pesticides:
• Disturbance in energy production
p
• Inhibition of photosynthesis• Free radical generation & SH-group reactivity• Interference with cell divisionInterference with cell division• Inhibition of nucleic acid synthesis• Inhibition of enzymes:
l hErgosterol synthesisAmino acid synthesisChitin synthesisyCholinesterase
• Hormone-like and behavior-modifying agents
Pesticide ExposuresPesticide Exposures
• RoutesRisk = Hazard x Exposure
• Routes– Oral (mouth and digestive system)– Dermal (skin)– Inhalation (nose and respiratory system)Inhalation (nose and respiratory system)
• Pathways– Food and water– Residential and non-occupationalResidential and non occupational
• External to Internal– ADME modifiers
• Aggregate• Aggregate– 1996 Food Quality Protection Act (FQPA)– multiple pathways and routes of exposure– http://www epa gov/opp00001/trac/science/aggregate pdf
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http://www.epa.gov/opp00001/trac/science/aggregate.pdf
Pesticide RegulationPesticide Regulation• Federal Insecticide, Fungicide, and Rodenticide Act g
(FIFRA, 1947) administered by USDA• Federal Food, Drug, and Cosmetic Act (FFDCA, 1954)
established pesticide tolerances on foodestablished pesticide tolerances on food– Delaney Clause, forbade the use of carcinogens as food
additives• Food Quality Protection Act (FQPA 1996) reauthorizedFood Quality Protection Act (FQPA, 1996) reauthorized
FFIFRA provisions– Tolerances reassessed as part of reregistrations
single health based standard– single, health-based standard– aggregate risk from all routes of non-occupational exposure– evaluating endocrine effects
extra tenfold uncertainty factor for children/in utero
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– extra tenfold uncertainty factor for children/in utero
Vulnerability of ChildrenVulnerability of Children
Greater exposureGreater exposure• On a caloric consumption:body-weight ratio
Children are 2.5x adults. Diet less varied (fruit and milk)
• Hand to mouth activitySki f b d i ht i d bl • Skin surface area per body weight is double that of an adult
• Rate of respiration Rate of respiration
19
Vulnerability of ChildrenVulnerability of Children
Greater physiological susceptibilityGreater physiological susceptibility• Period of rapid development of nerve cells• Loss of organ function can be permanently Loss of organ function can be permanently
imprinted• Absorption and elimination of pesticides• Metabolizing enzymes not fully developed
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Pesticide Testing- US EPAPesticide Testing US EPAHarmonized Test Guidelines
810 - Product Performance Test Guidelines830 - Product Properties Test Guidelines835 - Fate, Transport and Transformation Test Guidelines, p840 - Spray Drift Test Guidelines 850 - Ecological Effects Test Guidelines860 - Residue Chemistry Test Guidelines870 Health Effects Test Guidelines870 - Health Effects Test Guidelines
Test Guidelines/Acute Toxicity - Acute Oral Toxicity Up-And-Down-Procedure875 - Occupational and Residential Exposure Test Guidelines880 - Biochemicals Test Guidelines885 - Microbial Pesticide Test Guidelines890 - Endocrine Distruptor Screening Program Test Guidelines
http://www.epa.gov/opptsfrs/home/guidelin.htm
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http://www.epa.gov/opptsfrs/home/guidelin.htm
Pesticide Testing-H lth Eff t T t G id liHealth Effects Test Guidelines
Group A – Acute Toxicity Test Guidelines870.1000 - Acute Toxicity Testing--Background (December 2002) (PDF) (9 pp, 23K)870.1000 Acute Toxicity Testing Background (December 2002) (PDF) (9 pp, 23K)
870.1100 - Acute Oral Toxicity (December 2002) (PDF) (10 pp, 27K)870.1200 - Acute Dermal Toxicity (August 1998) (PDF) (10 pp, 24K)870.1300 - Acute Inhalation Toxicity (August 1998) (PDF) (11 pp, 21K)870.2400 - Acute Eye Irritation (August 1998) (PDF) (8 pp, 22K)870.2500 - Acute Dermal Irritation (August 1998) (PDF) (8 pp, 23K)870.2600 - Skin Sensitization (March 2003) (PDF) (9 pp, 29K)
Group B – Subchronic Toxicity Test Guidelines870.3050 - Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000) (PDF) (17 pp, 29K)
870.3100 - 90-Day Oral Toxicity in Rodents (August 1998) (PDF) (13 pp, 33K)870.3150 - 90-Day Oral Toxicity in Nonrodents (August 1998) (PDF) (12 pp, 30K)870.3200 - 21/28-Day Dermal Toxicity (August 1998) (PDF) (15 pp, 36K)870 3250 90 Day Dermal Toxicity (August 1998) (PDF) (14 pp 34K)870.3250 - 90-Day Dermal Toxicity (August 1998) (PDF) (14 pp, 34K)870.3465 - 90-Day Inhalation Toxicity (August 1998) (PDF) (17 pp, 40K)870.3550 - Reproduction/Developmental Toxicity Screening Test (July 2000) (PDF) (13 pp, 89K)870.3650 - Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000) (PDF) (17 pp, 89K)870.3700 - Prenatal Developmental Toxicity Study (August 1998) (PDF) (11 pp, 126K)870.3800 - Reproduction and Fertility Effects (August 1998) (PDF) (14 pp, 35K)
Group C – Chronic Toxicity Test Guidelines870.4100 - Chronic Toxicity (August 1998) (PDF) (18 pp, 44K)
870.4200 - Carcinogenicity (August 1998) (PDF) (17 pp, 41K)870.4300 - Combined Chronic Toxicity/Carcinogenicity (August 1998) (PDF) (20 pp, 49K)
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Pesticide Testing-Health Effects Test Guidelines (cont.)
Group D – Genetic Toxicity Test Guidelines870.5100 - Bacterial Reverse Mutation Test (August 1998) (PDF) (13 pp, 36K)
870.5140 - Gene Mutation in Aspergillus nidulans (August 1998) (PDF) (7 pp, 18K)870.5195 - Mouse Biochemical Specific Locus Test (August 1998) (PDF) (7 pp, 19K)870.5200 - Mouse Visible Specific Locus Test (August 1998) (PDF) (6 pp, 16K)
G ( ) ( ) ( )870.5250 - Gene Mutation in Neurospora crassa (August 1998) (PDF) (6 pp, 17K)870.5275 - Sex-linked Recessive Lethal Test in Drosophila melanogaster (August 1998) (PDF) (6 pp, 16K)870.5300 - In vitro Mammalian Cell Gene Mutation Test (August 1998) (PDF) (14 pp, 37K)870.5375 - In vitro Mammalian Chromosome Aberration Test (August 1998) (PDF) (13 pp, 33K)870.5380 - Mammalian Spermatogonial Chromosomal Aberration Test (August 1998) (PDF) (11 pp, 28K)870.5385 - Mammalian Bone Marrow Chromosomal Aberration Test (August 1998) (PDF) (11 pp, 28K)870.5395 - Mammalian Erythrocyte Micronucleus Test (August 1998) (PDF) (12 pp, 31K)870.5395 Mammalian Erythrocyte Micronucleus Test (August 1998) (PDF) (12 pp, 31K)870.5450 - Rodent Dominant Lethal Assay (August 1998) (PDF) (6 pp, 15K)870.5460 - Rodent Heritable Translocation Assays (August 1998) (PDF) (7 pp, 17K)870.5500 - Bacterial DNA Damage or Repair Tests (August 1998) (PDF) (7 pp, 18K)870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture (August 1998) (PDF) (7 pp, 18K)870.5575 - Mitotic Gene Conversion in Saccharomyces cerevisiae (August 1998) (PDF) (6 pp, 16K)870.5900 - In vitro Sister Chromatid Exchange Assay (August 1998) (PDF) (7 pp, 18K)870 5915 In vivo Sister Chromatid Exchange Assay (August 1998) (PDF) (6 pp 15K)870.5915 - In vivo Sister Chromatid Exchange Assay (August 1998) (PDF) (6 pp, 15K)
Group E – Neurotoxicity Test Guidelines870.6100 - Acute and 28-Day Delayed Neurotoxicity of Organophosphorus Substances (August 1998) (PDF) (10 pp, 27K)
870.6200 - Neurotoxicity Screening Battery (August 1998) (PDF) (13 pp, 32K)870.6300 - Developmental Neurotoxicity Study (August 1998) (PDF) (14 pp, 35K)870.6500 - Schedule-Controlled Operant Behavior (August 1998) (PDF) (8 pp, 20K)870 6850 - Peripheral Nerve Function (August 1998) (PDF) (9 pp 22K)870.6850 Peripheral Nerve Function (August 1998) (PDF) (9 pp, 22K)870.6855 - Neurophysiology Sensory Evoked Potentials (August 1998) (PDF) (14 pp, 35K)
Group F – Special Studies Test Guidelines870.7200 - Companion Animal Safety (August 1998) (PDF) (10 pp, 23K)
870.7485 - Metabolism and Pharmacokinetics (August 1998) (PDF) (14 pp, 34K)870.7600 - Dermal Penetration (August 1998) (PDF) (14 pp, 37K)870.7800 - Immunotoxicity (August 1998) (PDF) (13 pp, 34K)
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y ( g ) ( ) ( pp, )Group G – Health Effects Chemical-Specific Test Guidelines870.8355 - Combined Chronic Toxicity/Carcinogenicity Testing of Respirable Fibrous Particles (July 2001) (PDF) (17 pp, 181K)
http://www.epa.gov/opp00001/reregistration/status.htm
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What is the acute and chronic Point-of-D t f ti id t i it ?Departure for pesticide toxicity?
Reference Dose (RfD) = NOAEL x UF
25
26ToxRefDB website: http://www.epa.gov/ncct/toxrefdb/
In Vivo Toxicity Data in ToxRefDB
Chronic/CancerMultigenationMultigenationDevelopmental
mic
als
Che
m
Martin et al 2009a,bKnudsen et al 2009
2730 years and more than $2B worth of data
Chronic/Cancer Toxicity Profiling
Liver & ThyroidToxicants
Spleen & KidneyToxicantsToxicants
28
http://www.ehponline.org/members/2008/0800074/0800074.pdf
Chronic Rat & Mouse Endpoints
29
Reproductive Toxicity Profiling
Systemic Toxicity&&
Delayed SexualMaturation
DecreasedReproductiveP fPerformance
30
http://toxsci.oxfordjournals.org/cgi/reprint/kfp080
R d tiReproductiveEndpoints
31
Profiling developmental toxicity
in vivo endpoints (target, description)www.epa.gov/ncct/toxrefdb
toxicity
ToxRefDB 387 chemicals, 751 prenatal studies,988 effects annotated (enhanced DevTox.org)
283 chemicals x 293 effects 19 targetsystems from rat ( ) and rabbit ( ) studies
32
SOURCE: Knudsen et al. (2009) Reproductive Toxicology (in press) DOI 10.1016/j.reprotox.2009.03.016Also see Abstract #16
Is a pesticide carcinogenic?Is a pesticide carcinogenic?
• Genotoxic?Genotoxic?• Non-genotoxic?
H l ?• Human relevance?
33
Rat Liver Histopathologyfrom Chronic Bioassaysfrom Chronic Bioassays
37
21
No PathologyProliferative LesionsPre-neoplastic Lesions
68
Neoplastic Lesions
N = 248 ChemicalsN 248 Chemicals
34
Chemicals Evaluated for Carcinogenic P t ti l b US EPAPotential by US EPA
35http://www.epa.gov/pesticides/carlist/
Is a pesticide an endocrine di ?disruptor?
• EstrogenicEstrogenic• (anti) Androgenic
Th t i• Thyrotoxic• Other
36
P i t l 2005
37
Panzica et al 2005
http://www epa gov/endo/pubs/regaspects/testorders htmhttp://www.epa.gov/endo/pubs/regaspects/testorders.htm
38
Reprod cti eReproductive and
EndocrineEndocrine Organ
Toxicity yEndpoints
from T R fDBToxRefDB
39
What is a hit?
40
HISTORY OF DDT1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane, , , (p p y )
DDT was discovered to be an insecticide in 1939 by Paul Muller. He was a scientist working for Geigy, a Swiss firm that was focused on the chemical Swiss firm that was focused on the chemical development of agricultural insecticides. Products with DDT entered the Swiss market in 1941. Seven years later, in 1948, Muller received the Nobel Prize for medicine and physiology in recognition for the lives
WWII DDT d b th lli t
medicine and physiology in recognition for the lives DDT saved.
• WWII – DDT was used by the allies to suppress a typhus epidemic in Naples
• 1943-1944 DDT was applied directly to the head pp yof humans to control lice
• Success with DDT hastened the development of aldrin, dieldrin, endrin, chlordane, benzene
41
aldrin, dieldrin, endrin, chlordane, benzene hexachloride etc.
CURRENT STATUS:• No US registration, most uses cancelled in 1972, all uses by 1989
N US d ti i t t
DDT• No US production, import, or export• DDE (metabolite of DDT) is regulated as a hazardous air pollutant
(Clear Air Act)• Priority toxic pollutant (Clean Water Act) DDT• DDT can take more than 15 years to break down• Found in animals far from where they were it is used
y p ( )
y• Bio-accumulates in fish and marine mammals. Found concentrations in
these animals are many thousands of times higher than levels in water • DDT can be absorbed by some plants and by animals and humans who
eat those plants eat those plants • DDT is fat-soluble and is stored in adipose tissues of humans and
animals
HUMAN EXPOSURE FROM:• Eating contaminated fish and shellfish • Eating imported food exposed to DDT g p p• Infant exposed through breast milk • Eating products from crops grown in contaminated soil
Insecticide advantages of DDT• Low volatilityy• Chemical stability• Lipid solubility• Slow rate of biotransformation and degradationg
Disadvantages of DDT• Persistence in the environmentPersistence in the environment• Bioconcentration• Biomagnification in food chain• Profound effects on wild life (“Silent Spring”)Profound effects on wild life ( Silent Spring )
Health Effects of DDT• Paresthesia of tongue lips and • Hypertrophy of hepatocytes• Paresthesia of tongue, lips, and
face• Irritability, dizziness, vertigo,
tremor, and convulsions
• Hypertrophy of hepatocytes• Hepatic tumors• No epidemiological evidence linking DDT
to carcinogenicity in humans
43
• Hypersusceptibility to external stimuli (light, touch, and sound)
• Low rate of absorption through the skin• Human health effects minor
44Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001
Sites of DDT poisoning
45Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001
Is a pesticide a developmental i ?neurotoxicant?
46
Inhibition of choline esterase or action potentialp
• Organochlorine Insecticides
Organophosphate • Organophosphate Insecticides
• CarbamatesCarbamates
• Pyrethroid insecticides
• Botanical Insecticides
47Klaassen, CD. CASARETT AND DOULL's Toxicology: The Basic Science of Poisons. McGraw-Hill 2001
• Most chemical insecticides act by
i i th poisoning the nervous system of the target organisms
• CNS of insects are highly developed and similar to that of the mammal
• Chemicals that act on the insect nervous the insect nervous system may have similar effects on higher forms of lifehigher forms of life
Stenersen, J. Chemical pesticides: Mode of Action and Toxicology. CRC Press 2004
General Modes of Action
Pesticides acting on the axon (impulse t i i )transmission):
• Interference with transport of, Na+, K+, Ca2+, or Cl- ions
Pesticides acting on synaptic transmission:
• Inhibition of specific enzyme activities:activities:GABA-ergic (inhibitory) synapsesCholinergic synapses
• Contribution to the release or persistence of chemical transmitters at nerve endings
49Stenersen J, Chemical Pesticides Mode of Action and Toxicology, CRC Press 2004
PON1 polymorphism:PON1 – human serum paraoxonase, enzyme important for lipid metabolisms that is also involved in metabolism of organophosphate compounds
PON1R192 PON1Q192
Rapid hydrolysis Rapid hydrolysis p y yof paraoxon
p y yof sarin, soman,
diazoxon
Two-dimensional enzyme analysis to characte-i PON1 l hi i h l tirize PON1 polymorphisms in human population:
analyze hydrolysis of PON1 substrates, diazoxone vs. paraoxone (panel c)O PON1Q192/PON1Q192 (glutamine)
50From: Hulla et al. Toxc. Sci. (1999)
■ PON1Q192/PON1R192 (glu/arg) PON1R192/PON1R192 (arginine)
Pyrethroid Insecticides
• Newest class of insecticides• Newest class of insecticides• New analogs will be (hopefully):
– More stable in light and airBetter persistence
51
– Better persistence– Low mammalian toxicity
Soderlund et al. (2002)
Importance of Importance of Structure-Activity-Toxicity
Relationships
Soderlund et al. (2002)
P th id UPyrethroid Use
• Household sprays• Flea preparations for pets• Flea preparations for pets• Plant sprays for home• Plant sprays for greenhouses
• Similar to DDT
Pyrethroid Poisoning
• Not highly toxic in animals• Toxic ingredients
– Chrysanthemic acid
53
y– Pyrethric acid
Figure 1. Nine neonicotinoid insecticides and four nicotinoids.The neonicotinoids are nitromethylenes (C==CHNO2), nitroguanidines (C==NNO2), and cyanoamidines(C==NCN). Compounds with 6-chloro-( ) p3-pyridinylmethyl, 2-chloro-5-thiazolylmethyl, and 3-tetrahydro-furanmethyl moieties are referred to as chloropyridinyls (or chloronicotinyls),py y ( y ),chlorothiazolyls (or thianicotinyls), and tefuryl, respectively. The nicotinoidsare naturally occurring [(−)-nicotine and (−)-epibatidine] and synthetics ( ) p ] y(ABT-594 and desnitroimidacloprid).
Tomizawa & Casida (2004)
Tomizawa & Casida (2004)
56
Biotransformation of XenobioticsBiotransformation of XenobioticsThe M in ADME (absorption, distribution, metabolism and excretion)
• Metabolism of xenobiotics in liver is defense against lipophilic compounds accumulating in bodycompounds accumulating in body
• Nuclear Receptors coordinate body’s defense against these compounds
• Biotransformation: Phase I, II, III– Phase I: functionalization reaction
• Cytochrome P450 Monooxygenase System• Microsomal
M j ti i id ti• Major reaction is oxidative• Minor reaction is reductive• May result in metabolic activation
– Phase II: conjugation reaction• Glucuronidation, sulfation, acetylationGlucuronidation, sulfation, acetylation• Cytoplasmic• Increases water solubility
– Phase III: transporter activity• Basolateral and bile canalicular expression
TP d d
57
• ATP-dependent export pumps
Importance of BiotransformationParent/Metabolite Methoxychlor (human ER)
ER di li d
Parent/Metabolite
80
100
120
Methoxychlor (human ER)
HPTE (human ER)Methoxychlor (bovine ER)
HPTE (bovine ER)HPTE (hER) HPTE (bER)tio
nER radioligand binding assay
0
20
40
60 BottomTopLogIC50HillSlopeIC50
2.448= 100.0-1.349-0.91970.04476
1.725= 100.0-1.697-0.98770.02009%
of I
nhib
it
-3 -2 -1 0 1 2-20
0
Conc (log M)
Parent/Metabolite
ER cellular (HEK293)
60
80
100
BottomMethoxychlor0 3017
HPTE-3 912
MethoxychlorHPTE
trol
(HEK293) transactivation assay
0
20
40BottomTopLogEC50HillSlopeEC50
0.301741.860.57145.4663.727
3.91235.70-0.71312.2580.1936%
of C
on
58
-3 -2 -1 0 1 2-20 Conc (log M)
NR Regulation Phase I Phase II Phase IIIXenobiotics AhR XRE CYP1A1(+) UGT1A1(+) ABCG2(+)
CYP1A2(+) UGT1A6(+)
Ligand NR Response ElementTarget Gene
gof Metabolic
Enzymes
CYP1A2(+) UGT1A6(+)
CYP1B1(+)
Xenobiotics CAR DR-3, DR-4, DR-5 CYP2A6(+) UGT1A1(+) ABCC2(+)
Phenobarbital SR-6, ER-6 CYP2B1(+) ABCC3(+)
CYP2B6(+) ABCC4(+)
CYP2C9(+)
CYP2C19(+)y CYP2C19(+)
Xenobiotics SXR/PXR DR-3, DR-4, DR-5 CYP1A2(+) SULT2A1(+) ABCA1(+)
Steroids ER-6, ER-8 CYP2B6(+) UGT1A1(+) ABCB1(+)
CYP2C9(+) UGT1A3(+) ABCB11(+)
CYP2C19(+) UGT1A4(+) ABCC1(+)
CYP3A4(+) ABCC2(+)
CYP3A7(+) ABCC3(+)
Phase I Phase II Phase IIIoxidation conjugation transportersreduction glucuronic acid CYP3A7(+) ABCC3(+)
CYP7A1(-) ABCG2(+)
CYP3A(+)
Bile Acids FXR IR-1 CYP7A1(-) UGT2B4(+) ABCB4(+)
DR-1 CYP8B1(-) SULT2A1(+) ABCB11(+)
ABCC2(+)
Oxysterols LXR DR-4 CYP2B6(-) ABCA1(+)
reduction glucuronic acidhydrolysis sulfationcyclization glutathione
decyclization
Oxysterols LXR DR-4 CYP2B6(-) ABCA1(+)
CYP3A4(-) ABCG1(+)
ABCG4(+)
ABCG5(+)
ABCG8(+)
Fatty acids PPAR DR-1 CYP4A1(+) UGT1A9(+) ABCA1(+)
Fibrates CYP4A3(+) UGT2B4(+) ABCC2(+)Fibrates CYP4A3(+) UGT2B4(+) ABCC2(+)
CYP7A ABCD2(+)
ABCD3(+)
Fatty acids PPAR DR-1 CYP4A(+) UGT1A(+) ABCA1(+)
Carboprostacyclin
Eicosanoids PPAR DR-1 CYP4AB(+) UGT1A(+) ABCA1(+)
Thiazolidinediones ABCG2(+)
59
Thiazolidinediones ABCG2(+)
1,25(OH)- VDR DR-3 CYP2B6(+) SULT2A1(+) ABCC2(+)?vitamin D3 ER-6 CYP2C9(+)
IR-0 CYP3A4(+)
Glucocorticoids GR GRE CYP2C9(+)
CYP2B6(+)
Cytochrome P450/CYPCytochrome P450/CYP
• Heme-containing proteins
• RH+O2+H++NADPH → ROH+H2O+NADP+
60Medscape
Phase II Metabolism• Glutathione S-transferases • Mercapturic acid biosynthesis • UDP Glucuron(os)yltransferases • UDP-Glucuron(os)yltransferases • N-Acetyltransferases • Amino acid N-acyl transferases Amino acid N acyl transferases • Sulfotransferases
61
Phase III: TransportersPhase III: Transporters
Bile Canaliculus
62
PXR LigandsPXR Ligands
• Estimated that 50% mof drugs are PXR ligandsM i t l • Many environmental chemicals are PXR ligandsligands
• Most promiscuous of the NRs
• Large, flexible binding pocket
63
Focus On a Mode of Action …
NR activators stimulate intracellular processes that lead to hyperplasiaChronic stimulation increases the risk of neoplasms
EnvironmentalCh i l
Molecular Cellular TissueChemicals response response response
Cell fate AdverseOutcome
Chemicals
Pesticides NR-sig Gene-reg. Transcription
Molecular Response (Early)
Proliferation
DeathApoptosisNecrosis
Hyperplasia
Tumor
Cancer
PesticidesConazolesPyrethroids
ToxicsDE-71PCBs
NR sig Gene reg. Transcription
CARPXRPPAR
cis-reg.trans-reg.
Xen. Met.Phase I
Phase II
Phase III
64
PhthalatesPFOA/PFOS
Phase III
