Download pdf - Brain asymmetry as a potential biomarker for developmental TCDD intoxication: a dose-response study

Brain Asymmetry as a Potential Biomarker for Developmental TCDDIntoxication: A Dose-Response StudyDiane S. Henshel, J. William Martin, and Jamie C. DeWittSchool of Public and Environmental Affairs, Indiana University, Bloomington, Indiana 47405 USA

Previous studies have indicated tit in evo exposure to 2,3,78 ra ddiio:(TCDD) and related compounds is correlated with the development of o mmetic braThis asmmetry is m Isted as a d ce betwn the two hals of the foreb an ditetecta. Previosy, onlildli s on,co eagle) hadbe s*.this response_ nh id offerences ha bee creaewihteldsopoyhoiatddbnopdontoceqvAlecfacors (fs ine im h s nes (hen . We dt of in +erposure to TCDD on the brai toout delm et in a sensitive t mod (chickr-en). Embryos from chickn egg (Gan galw) injected with one of sevral doses ofTCDD orvehicle control wre sacrificed afer 9, 11, 13, 15, 17, or 20 days of incion, or cubaed toha and d either 24 hr or att;3 we pos h M of botihi;

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..atin deveoigcikn; 2) this rin symer aiiaota obseve in axml eaoein the wild to .a mue ofTCDD t d cont n s there was a do d in sboth the and severity of brain aymmetr ob d at all agesmeasured; and 4) theasymmetry was easuble in embryonic brains at an age when the braincase was a thin, flexibleLayer (embry-oida9), implying -tht the effect ofTCDD was direct on .the developing brainand not i diect Case. Key aymet, brindvpment di

, embryo, T Rssn 105:718-725 (1997)

Polychlorinated dibenzo-p-dioxins (PCDDs),polychlorinated dibenzofurans (PCDFs) andpolychlorinated biphenyls (PCBs) are virtu-ally ubiquitous environmental pollutantsthat are known to bioconcentrate in animaltissue, biomagnify up the food chain, crossthe placenta into mammalian embryos, andbe deposited into the eggs of egg-laying ani-mals (1-3). These compounds are part ofthe broader class of compounds known aspolyhalogenated polycyclic aromatic hydro-carbons (PHAHs), the most acutely toxic ofwhich is 2,3,7,8-tetrachlorodibenzo-p-diox-in (TCDD) (3). TCDD in particular, andPCDDs, PCDFs, and PCBs in general, areknown to be embryotoxic and teratogenic,

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causing abnormalities in the palate or beak,heart, and kidney, as well as causing generaldepression of embryonic growth (1,4,5). Inaddition, these compounds are linked toabnormalities in the development of thenervous system. PCBs, for example, havebeen linked to changes in the biochemicaldevelopment of parts of the nervous systemand nervous system-related tissue (6).PCBs, PCDFs, and PCDDs, as well asother organochlorines, have all been linkedto behavioral changes in a wide variety ofanimals (2,6-8). Indications from the bio-chemical studies, however, are that some ofthe nervous system effects of these com-pounds may not be mediated by the laterally

substituted, coplanar compounds prototypi-cally represented byTCDD (6).

Previous studies from our laboratory haveindicated that in ovo exposure to TCDD andrelated compounds is also correlated with thedevelopment of brains that are grossly asym-metric (9-12. This asymmetry is manifestedas differences in several parameters betweenthe left and right halves of the forebrain andis also evident in the tecta, again as aleft-right difference. Previously, only wildlifespecies (heron, cormorant, eagle) have beenshown to manifest this response (9-12). Inthe wildlife studies, the frequency and degreeof left-right interhemispheric differences hadbeen correlated with the levels of PCDDsand/or TCDD toxic equivalents in eggs fromthe same nest (heron, cormorant) or in theblood (eagle). As environmentally exposedwildlife species are virtually always exposed toa mixture of compounds, the questionremained whether the asymmetry was beinginduced by the TCDD-like compounds orby some other compounds that coexisted

Address correspondence to D.S. Henshel, School ofPublic and Environmental Affairs, 10,h and FeeLane, Room 340 SPEA Building, IndianaUniversity, Bloomington, IN 47405 USA.This work was supported by the WildlifeToxicology Fund (World Wildlife Fund, Canada),the Sustainable Development Research Initiative ofBritish Columbia, NSERC (Canada), the BritishColumbia Ministry (Environment), the ArdeBulova Foundation, and a Biological ResearchSupport Grant. J.W.M. and J.C.D. were supportedby research assistantships from the School of Publicand Environmental Affairs, Indiana University.Received 16 October 1996; accepted 3 March 1997.

Figure 1. Drawings of the chicken brain from the (A) dorsal and (B) lateral aspects illustrating the major brain regions and the individual measurements made.Abbreviations: A, angle, D, depth; H, height; TD, tectal depth (tectal width not shown); C, cerebrum (telencephalon); T, tectum; Cb, cerebellum; SC, spinal cord; B,brain stem; OB, olfactory bulb; W, width; Hy, hypothalanus.

Volume 105, Number 7, July 1997 * Environmental Health Perspectives718

Articles * Brain asymmetry as a biomarker of TCDD exposure

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Environmental Health Perspectives * Volume 105, Number 7, July 1997 71

Articles * Henshel et al.

1 Wldth.

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Figure 3. Proportion of asymmetric brains of chicken embryos graphed by age and dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. E, embryonic day or incubation day.Measurements include width, angle, depth, and height of the forebrain and width and depth of the tectum (see Fig.1 ).



with the TCDD-like compounds in theenvironment. In addition, there was a ques-tion ofwhen during embryonic developmentthe asymmetry began to develop andwhether, once developed, the asymmetryremained evident. Finally, it was not clearwhether TCDD affected the brain directly orwhether the gross brain malformation wasinduced indirectly via an effect on the brain-case. Therefore, we initiated this develop-mental, dose-response, controlled laboratorystudy to determine whether TCDD alonecould induce brain asymmetries similar tothose observed in wildlife species contami-nated in the wild with TCDD and TCDD-like compounds.

MethodsFertile white leghorn chicken (Gallus gallus)eggs were injected before the start of incu-bation with one of several doses ofTCDD[10, 100, 300, or 1000 pg TCDD/g egg(= parts per trillion or ppt) for the embryossacrificed before hatching and 10, 30, 60,100, 300, or 1000 ppt for the embryosallowed to hatch] or with vehicle control

_ 0 pptm lOppt= 30 ppt= 60 ppt 1

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04

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(safflower oil). TCDD was obtained fromUltra Scientific (Providence, RI) predissolvedto a known concentration in safflower oil.Injection volumes were normalized to theweight of the individual eggs: 0.1 p1 ofTCDD or oil per gram of egg was injectedthrough a hole in the shell above the air sacusing a Hamilton syringe. The hole in theshell was subsequently sealed with paraffin.The eggs were incubated at 37.5°C drybulb and 300C wet bulb (approximately56% humidity) until sacrifice or hatch.Embryos were sacrificed at embryonic days(E; incubation days) E9, E11, E13, E15,E17, and E20. All eggs were removed fromthe incubator at the same time of day thatthey had initially been placed in the incuba-tor. All embryos were sacrificed in randomorder. Other injected eggs were incubateduntil hatching, and the hatchlings were sac-rificed either within 24 hr of hatching or at3 weeks post-hatch. Hatchlings raised to 3weeks were raised in groups including oneof each dose, housed by group in 1 1/2 ft(W) x 2 ft (D) x 1 1/2 ft (H) plastic cages,warmed with a 100 (week 1) or 60 watt

0.35

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(weeks 2-3) light bulb. Food and waterwere provided ad libitum. The birds wereindividually coded with permanent markeron their feathers.

At sacrifice, the older embryos (E13,E15, E17, E20), hatchlings, and 3-week-oldbirds were all anesthetized with phenobarbi-tal and either perfused through the heartwith phosphate buffered saline (PBS, pH7.4) followed by 4% paraformaldehydemade in PBS (PARA, pH 7.4) or were sacri-ficed with phenobarbital and then dissectedrapidly to remove the brains. The dissectedbrains were then immersion-fixed in 4°CPARA. Sacrifice method was by injection setacross all doses and controls, with injectionsets of three birds each per dose and by ageof sacrifice. The younger embryos were sac-rificed by immersion in cold PARA (40C).All embryos were stored in PARA at 40Cuntil shortly before necropsy, when theywere transferred to PBS (4°C) with 0.625%sodium azide added to prevent moldgrowth. All animals were handled followingprotocols approved by the University ofBritish Columbia (UBC) Animal Care

0_40.35

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Figure 4. Mean symmetry differences in brain of post-hatch chicks averaged by age and dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. P21, postnatal day 21.Measurements include width, angle, depth, and height of the forebrain and width and depth of the tectum (see Fig.1 ).

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Articles - Henshel et al.

Committee, and handling of hazardousmaterials was approved by the UBCOccupational Safety and Health office.

Six measurements were made on eachbrain, four on the forebrain region and twoon the tecta. The tectal depth and forebraindepth were measured from the lateral aspectof the brain, while the tectal width wasmeasured from the ventral aspect of thebrain. The other three measurements(height, width, angle) were all made fromthe dorsal aspect of the forebrain. The fourforebrain measurements (illustrated in Fig.1) were mediolateral width just rostral tothe pineal (width), mediolateral measure-ment from the same central point just ros-tral to the pineal and out to the lateral edgeat a 350 angle from the horizontal definedby the width measurement (angle), ros-tral-caudal length from mid-wulst (the dor-sorostral forebrain protrusion) to the cau-dal-most part of the forebrain on that ros-trocaudal axis (height), and the dorsoventraldepth of the brain at the point where thehypothalamus meets the telencephalon (atthe level of the pre-optic area, depth).

Brains were measured as described pre-viously (7,8), except that the tecta were alsomeasured for this study. Briefly, each mea-surement was made using a ruler proppedparallel to the surface of the brain. The eyewas centered over the center of the brain forthe width and angle measurements and overthe center of the area being measured forthe other measurements. All measurementswere made two times. If the measurementsdid not agree, each measurement was madean additional three times and the resultswere averaged. The measurements reportedhere represent the difference between leftand right halves of the brain and are all pre-sented as measurements from the left side ofthe brain minus measurements from theright side of the brain.We regressed each measurement on

TCDD concentration and on age using theRegression and General Linear Models pro-cedures in SAS (PROC REG, PROC GLM,SAS Institute, Cary, NC). Significance wasdetermined using a p-value of 0.05. Probitanalysis was also performed using SAS(PROC PROBIT) to estimate the ED10s(the dose at which a 10% populationresponse is elicited) and the median effectivedoses (ED50s) for the observed asymmetry.The standard deviation (SD) reported for theED50 is the standard deviation (a) reportedby SAS for the average (the p or the ED50).No such values were given for the EDlos bySAS; therefore, no SDs were reported for theED10s. Further, 95% fiducial limits are notincuded in this paper as they were inconsis-tently reported by SAS, depending on the

ResultsTCDD clearly affected both the frequencyand the degree of brain symmetry in a dose-dependent manner (Fig. 2-5). To give anindication of relative variability in the data,the sample size and standard error of themeans are listed in Table 1 for the anglemeasurement (Fig. 2 and 4). Noticeably,the TCDD-induced forebrain asymmetrywas only evident at the lowest dose (10 ppt)in the E13 and older animals. However, thetectal asymmetry was already present in thebrains exposed to the lowest dose ofTCDDat E9. It is also clear from these data thatthe asymmetry persisted to at least 3 weekspost-hatch, through a period of rapid post-hatching growth. Similarly, although theforebrain measurements were only madefrom the dorsal aspect, the brain asymmetrywas noticeable from both the dorsal and theventral aspects of the brain (Fig. 6). Table 2lists the PROBIT determined EDlos andED50s by age and measurement.

Of the four forebrain measurements,width, angle, depth and height, the firstthree had the highest R2 values whenregressed against age and TCDD concen-tration (see equations below). The heightmeasurements had the lowest R2 values of

all of the measurements, in general. The R2values for the two tectal measurementstended to be intermediate between thewidth, angle, and depth measurements andthe height measurement, although the R2for tectal depth was similar to the R2- forthe width, angle, and depth measurements.When evaluated by age and TCDD con-centration, all six measurements were sig-nificant at the 0.0001 level. The regressionequations (Eq. 1-6) in the shaded boxdescribe the relationships between eachmeasurement, TCDD, and age; p-valuesfor the individual parameters within eachequation are listed below each parameter.

DiscussionTCDD injected at the start of incubationclearly induces brain asymmetry in chickenembryos in a dose-dependent manner. Theasymmetry is manifested as a consistentleft-right hemispheric difference in both theforebrain and the tectum and is present afterthe initial formation of these brain regions.

Chickens have a 21-day incubationperiod. Chicken brains (and avian brainsin general) do not develop evenly through-out development (Fig. 7). During earlyincubation (organogenesis), the chicken

Table 1. Standard error of the mean and sample size for angle difference measurementsa

Dose (ppt)Age 0 10 30 60 100 300 1000

E9 0 (9) 0 (8) ND ND 0.13 (8) 0.11(3) 0.11(5)Eli 0.07 (14) 0 (10) ND ND 0.13 (10) 0 (3) 0.14 (4)E13 0 (13) 0.1 (12) ND ND 0.12 (8) 0.12 (10) 0.14 (4)E15 0(15) 0.1 (11) ND ND 0.18(8) 0.2(9) 0.14(4)E17 0 (13) 0.13 (6) ND ND 0.18 (9) 0.35 (7) 0.18 (2)E20 0 (8) 0.1 (6) ND ND 0.2 (7) NS (1)P0 0 (22) 0.07 (22) 0.13 (7) 0.13 (8) 0.13 (18) 0.11 (5) NSP21 0 (5) 0 (5) 0.24 (4) 0.06 (4) 0.13 (7) 0 (2) NS

Abbreviations: E, embryonic day or incubation day; P, postnatal day; ND, not done; NS, no survivors.aValues in parentheses are sample size.

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sample sizes for each age and dose.


Articles - Brain asymmetry as a biomarker of TCDD exposure

brain first segments into the prosencephal-ic, diencephalic, mesencephalic, andrhombencephalic regions (listed from ros-tral to caudal). By E3-E4, the two forebrainhemispheres (the telencephalon) bubbleout in the prosencephalic region of theneural tube. By E4, the mesencephalonbegins to develop as an enlarging centralstructure. The partitioning of the mesen-cephalon into two (left and right) tectalprominances begins about E5. By E7 orE8, the dominant outgrowths of the brainare the tecta, caudal to the telencephalicoutgrowths. The telencephalic hemispheresbegin to appear more prominent aroundE9 or E10 so that, by E12, the forebrainhemispheres are almost equal in size to thetectal outgrowths. By E16 or E18, the fore-brain hemispheres have begun to dwarf themore caudal tecta (13,14).

Given the difficulty of dissecting outearly embryonic brains, we only made asym-metry measurements on brains from E9embryos and older. By this age, even thoughthe telencephalic hemispheres are still verysmall and undeveloped, there is clear fore-brain asymmetry induced by the higherdoses of TCDD used and tectal asymmetryat all doses of TCDD used. Once the fore-brain hemispheres begin to develop again, inthe latter half of the 21-day incubation peri-od, the brain asymmetry is also manifestedat even the lower doses ofTCDD. Once thebrain has started to develop asymmetrically,evidence from the brains of the hatchlingand 3-week-old birds indicates that thebrains remain asymmetrical. Thus, theasymmetry endpoint appears to be increasedin sensitivity following and during periodsof rapid development of the brain regionsaffected (forebrain and tecta). Once theasymmetry is present, the brain appears tocontinue to grow such that the asymmetryremains. This implies that TCDD may beaffecting the brain during periods of highmitogenic activity and may be differentiallyinducing increased mitogenesis on the twosides of the brain. The ED1Os (Table 2) con-firm this increase in sensitivity in the fore-brain measurements at the time of the fore-brain expansion and indicate that the sensi-tivity of the tectal measurements are alreadymaximal by E9 (posttectal expansion).

These results also suggest that TCDDaffects primarily the brain and not primarilythe braincase, with a secondary effect on thebrain. At E9, the earliest that we haveattempted to measure the brain asymmetry,the braincase is a very thin, almost mem-brane-like covering over the brain. At thispoint in development, the braincase is veryflexible and appears to present little or noresistance to the brain developing under-neath. Thus, while there may also be a

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Figure 5. Proportion of asymmetric brains in post-hatch chicks graphed by age and dose of 2,3,7,8-tetra-chlorodibenzo-p-dioxin. P21, postnatal day 21. Measurements include width, angle, depth, and height ofthe forebrain and width and depth of the tectum (see Fig.1 ).

direct effect of TCDD on braincase devel- previously made on several wildlife speciesopment (which we have not in any way (great blue herons, double-crested cor-attempted to assess), our results indicate morants, and bald eagles) exposed in ovo inthat there is a direct effect ofTCDD on the the wild to a mixture of TCDD-relatedbrain as it undergoes rapid development, chemicals, including (depending on the

These results in TCDD-injected chick- study) PCDDs, PCDFs, and PCBsens are consistent with observations we have (10-12). In each of these studies, the brain

Environmental Health Perspectives * Volume 105, Number 7, July 1997 723

Articles * Henshel et al.

asymmetry was highly and significantly cor-related to either TCDD levels, TCDD-toxic equivalents (TEQs), or some othermeasure that generally indicates exposure toTCDD-like compounds ethoxyresorufin 0-deethylase, an enzyme induced by TCDDexposure and a variety of other environ-mental contaminants) (15). The present

results in chickens confirm that althoughother non-TCDD-like congeners may havehad an effect on the measured brain asym-metry in the wildlife studies, TCDD alonecan induce a developmentally linked grossbrain asymmetry in avian brains.

Ibis important to correlate this grossbrain asymmetry with functional changes in

~B

Figure 6. Photographs of embryonic day 13 chicken brains from the ventral (A, B) and dorsal (C, D)aspects illustrating the flattened arc on the left side of the brain (B. D; indicated by arrows) due to in ovo(TCDD) exposure. The TCDD injected chicken brain (300 ppt) is shown in B and D. The noninjected controlbrain is shown in A and C.

Table 2. Probit (log10) determined ED10s and ED50s by age and measurement

Height Width Angle Depth Tectal Width Tectal DepthAge ED10a ED b ED10 EDs ED10 EDs ED10 EDs ED10 EDs ED10 ED50E9 832.52 1046.20 ± 1.2 22.77 107.09 ± 3.35 24.85 89.32 ± 2.71 21.00 168.68 ± 5.08 8.75 127.32 ± 8.08 8.75 127.32 ± 8.08Eli 803.38 1000.00 ± 1.19 85.36 103.98 ± 1.17 77.56 95.89 ± 1.18 53.96 164.73 ± 2.39 11.64 58.26 ± 3.51 14.62 87.53 ± 4.04E13 5.35 477.26 ± 33.23 4.97 82.07 ± 8.92 4.83 56.38 ± 6.8 32.27 131.63 ± 2.99 11.91 93.13 ± 4.98 12.39 79 ± 4.24E15 806.74 1000.00 ± 1.18 6.12 67.83 ± 6.54 4.72 67.77 ± 8.0 2.71 85.15 ± 14.73 10.02 90.93 ± 5.59 6.41 110.25 ± 9.21E17 44.07 496.50 ± 6.62 0.98 34.91 ± 16.3 1.20 47.93 ± 17.78 15.34 106.38 ± 4.53 0.58 62.51 ± 38.52 6.37 58.9 ± 7.12E20 7.21 241.29 ±15.47 6.44 42.3 ± 4.34 6.44 42.3 ± 4.34 6.37 111.17 ± 9.31 0.58 90.65 ± 51.3 4.76 111.17 ± 9.31P0 22.70 247.17 ± 6.44 9.67 86.83 ± 5.54 9.12 78.78 ± 5.38 6.85 79.03 ± 6.74 4.93 58.71 ± 6.91 7.16 62.28 ± 5.41P21 15.80 196.33 ± 7.14 0.029 33.75 ± 250.16 16.05 70.69 ± 3.18 2.16 206.97 ± 35.14 18.35 199.72 ± 6.44 1.23 195.93 ± 52.18

Abbreviations: E, embryonic day or incubation day; P, postnatal day; ED10, effective dose eliciting a 10% response; ED50, median effective dose.aNo standard deviation (SD) is given by SAS (SAS Institute, Cary, NC) for the ED10s, therefore no SD is listed.bp (ED50 a(SD).

brain-controlled activity. At this time we donot know specifically which brain-mediatedfunctions (if any) may be affected by thisgross dysmorphism, although behavioralstudies are ongoing. From previous histo-logical studies we know that the pyriformcortex of the heron hatchlings from aPCDD- and PCDF-contaminated colonywas increased in both mediolateral widthand cell density (9). The pyriform cortex isassociated with the limbic system, a series ofstructures in the brain that affect emotionand instinctive behavior. In addition, thepyriform cortex is the main cortical areainvolved in olfactory discrimination and isbelieved to receive indirect input from theolfactory bulb (16). Olfaction is also gener-ally believed to affect instinctive behaviors.We did not, however, track sidedness in thebrains for the initial heron studies and,given how the brains were processed, wecan not determine sidedness after the fact.In addition, the pyriform cortex is a thinstrip on the outside of the forebrain. Therelatively large differences in the left-righthemispheres seen in this study could not betotally attributed to changes in such a thinstrip of tissue, nor could any changes in thetecta be directly attributed to changes in thethickness of the pyriform cortex. Futurestudies are needed to determine whichnuclei (an amalgamation of functionallyand/or anatomically related neurons) andbiochemical markers in the brain are specif-ically affected by TCDD.

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3. Safe S. Polychlorinated biphenyls (PCBs),dibenzo-p-dioxins (PCDDs), dibenzofurans(PCDFs), and related compounds: environmen-tal and mechanistic considerations which sup-

724 Volume 105, Number 7, July 1997 * Environmental Health Perspectives


"ig

Figure 7. Drawings of the chicken brain from the dorsal aspect between embryonic days 10 and 20, justprior to hatching. Abbreviations: C, cerebrum; T, tectum; Cb, cerebellum; SC, spinal cord. Modified fromRomanoff (14).

port the development of toxic equivalency fac-tors (TEFs). Crit Rev Toxicol 21:51-88 (1990).

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8. Moul I. Environmental contaminants, distur-bance and breeding failure at a great blue heroncolony on Vancouver Island [MSc Thesis].University of British Columbia, Vancouver,British Columbia, 1990.

9. Henshel DS, ChengKM, Norstrom R, Whitehead P,SteevesJD. Morphometric and histological changes inbrains of great blue heron hatchlings exposed toPCDDs: preliminary analyses. In: EnvironmentalToxicology and Risk Assessment, ASI-M STP 1179(Landis W, Hughes JS, Lewis MA, eds).Philadelphia:American Society for Testing andMatials 1993;262-277.

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ANNOUNCING THE

EIGHTH NORTH AMERICAN ISSX MEETINGOCTOBER 26-30 1997 A/fEilHILTON HEAD, SOUTH CAROLINA

SHORT COURSES ADDITIONAL TOPICS INCLUDE:

Four half-day short courses will be offered, two in * Susceptibility factors for diseasethe morning and two in the afternoon. * Chemistry and enzymology of biotransformation

* Drug interactionsSCIENTIFIC PROGRAM FOCUSING ON Two * Alternatives to animal modelsCENTRAL THEMES * Computational models

(1) Disposition of Biotechnology Compounds * Transgenic models* Peptides, Proteins, Oligomers * Physiological barriers* Uptake, Clearance, Metabolism * New technologies for analytical methods* Methods to Evaluate Combinational

Libraries FOR MORE INFORMATION:(2) Risk Assessment and Safety Evaluation issx

* Chemicals in the Environment P.O. BOX 3* Pharmaceuticals and Foods CABIN JOHN, MD 20818* Risk Management and Regulation FAX 301-983-5357

Environmental Health Perspectives . Volume 105, Number 7, July 1997 725