Document mpQnakdQ06gq0OpN1amR9Er6d

PATHOBIOLOGICAL RESPONSES OF PRIMATES TO POLYCHLORINATED BIPHENYL EXPOSURE J. R. Allen, D.V.M., Ph.D., and D. H. Norback* Abstract Mela and female rhesus monkeys received varying keels of polychlorinated biphenyls (PCB's) end were evaluated for toxic effects, reproductive dysfunctions, and metabolism of the compounds. Female rhesus monkeys exposed to dietary levels as tow as 2.S and S.0 ppm of FCB (Arocfor 1248) developed facial acne, erythema, subcutaneous edema, conlunctlvit/e, and tots of eyelashes. Reproductive dysfunctions were manifatted by Irregular menstrual cycles, early abortions, and stillbirths. At a result of transplacental migration of the compounds, all Infants bom of PCB-exposed animals contained PCB's to their tissues at birth. The infants, which continued to be exposed to PCB's by Ingestion of milk from thefr lactating mothers, developed skin lesions and 60percent expired within 4 months. Metabolic studies demonstrated 90 percent absorp tion of the PCB's from the gastrointestinal tract and distribution to organs of high lipid content Hydroxytoted metabolites were formed to the liver and excreted through the biliary and urinary routes. Lower chlori nated congeners were more rapidly metabolised and excreted, while concentrations of the highly chlorinated biphenyls persisted to the adipose tissue in excess of 2-1/2 years. The detection of the urinary metabolite trans-3,4-dihydro-3,4-dihydroxy'tetrachlorobiphenyl suggests diet the mechanism ofmetabolism is through an arena oxide Intermediate, to vivo and to vitro studies demonstrated binding of PCB's with macrxrmolecufet. INTRODUCTION human exposure to these compounds only recently het become of widegaread eoneern. The scientific communi ty was alerted to the potential environmental health problem by Jensen in 1906 (ref'. 1) after PCB's were identified in tissue extracts of birds experiencing repro ductive difficulties in Sweden. The magnitude of the problem was brought to the forefront by the "Yusho" incident when over 1,000 Japanese suffered prolonged ill effects from exposure to PCB-contaminated rice oil (ref. 2). Further concern about tha potential danger of PCB's on human haalth followed disclosure of increasing levels of these compounds in various foods. The contamination has bean attributed to incorporation of these com pounds within the food chain or from packaging of food products in PCB-impragnated paper containers (raf. 3). Increasing levels of PCB's in human tissue samples attest to the magnitude of the human exposure (refs. 4,5). Within this laboratory various animal models, including the nonhuman primate and rodent, have been evaluated following exposure to PCB's for the develop ment of lesions which parallel those recorded'in man. Emphasis has bean placed on determining pathophysi ological alterations that arise in animal models as a result of exposure to several levels of PCB's for variable periods of time. Tha absorption, tissue distribution, and rata of excretion of PCB's have been determined. The inter action of d>e PCB's or their metabolites with cellular macromolecules has also been a major area of investi gation. The following report, which includes previously unpublished observations, presents a summary of tht progress that has been made in this laboratory on the above mentioned areas of PCS research. Evan though the polychlorlnatad biphenyls (PCB's) have been used extensively for various industrial pur poses for the past 40 years, the health significance of 'Daparttivm of Pathology end Regional Primate Research Center, University of Wisconsin, Madison. Wisconsin. This inves tigation was supportad In part by U.S. Public Health Service grants 8$-00*72. ES-009SS end RR-00167 from the National Institutes of Health, end the University of Wisconsin Set Grant Program. The majority of th* date prasented In this report were obtained through the efforts of our collssguss In tht Experi mental Pathology Laboratory: D. Person). K. Blomqultt. L. Certtsns. I. C. Hsu, R. Msrisr, L. Moors. D. Petarson, J. Sey mour. J. Van Miller. A portion of this research was conducted in the University of Wlsoontin-Madison Biotron, a controlled environmental research facility oipported by the National Science Foundation end the University of Wisconsin. GENERAL EFFECTS OF PCB'S ON NONHUMAN PRIMATES The investigation of toxicity produced by PCB's in various animal species demonstrated tha limitations of rodents as animal models. Male Sprague-Dawiey rats wara able to survive for 1 year on diets containing 100 ppm PCB (Aroclor 1248, 1254, or 1262) without show ing-signs of illness (raf. 6). These observations substan tiated those of Kaplinger at el. (ref. 7). increasing the PCB content of the rat diets to 1,000 ppm did not product skin lesions; however, death occurred within 6 to 8 weeks due to widespread hepatic degeneration (raf. 8). Mala rhesus monkeys developed many of the signs 43 experlsnced by humens that had bean inadvertantly exposed to PCB's. Monkeys fad diets containing 100 and 900 ppm PCB (Aroclor 1248) (table 1) developed facial edema, erythema, acne, and alopecia within 3 weeks. The lesions became progressively more severe with Increased length of exposure (refs. 8,10). In addition, the animals developed anorexia, toss in weight, hypo* protefnemie, hypolipidemic, and anemia. Within 3 months the majority of the monkeys had died or warn moribund. Nacropsias of these animals revealed decided mucosal gastric hyperplasia with penetration of the glandular epithelium into the underlying submucosa (ref. 10). Numerous ulcerations of the hyperplastic gastric muoosa ware also present (ref. 11). In addition, there was decided hypertrophy of the liver. Female monkeys fed 25 ppm PCB (Aroclor 1240) (table 1) In the diet developed faciei lesions similar to those observed in the animals receiving higher levels of PCB'i (ref. 12). After 2 months on the PCB diet It was naoessary to discontinue the exposure due to the severi ty of Intoxication. One of the six experimental animals died 4 months after the Initial exposure to PCB's. Necropsy evaluation demonstrated severe gastric hyper plasia and ulceration. The surviving adult female monkeys continued to be devoid of eyelashes end dis played fecial acneform lesions 2 years following expowre to the PCB'i. Infants born to these females were nail (360 vs. 460 g) and contained PCB'i in their tissues at birth. Pamela monkeys given 2.5 and 6.0 ppm PCB (Arc elor 1248) (table 2) in their diets developed facial edema, swollen eyelids, erytheme, lots of hair, and acna within 2 months (rtf. 13). By tha fourth month, irregu larities in the menstrual cycles end an increased level of urinary ketotteroldt were recorded (ref. 14). Following 6 months of PCB exposure the female monkeys ware bred to control males. Six of eight animals on tha 5.0 ppm diet conceived (table 2). The remaining two were bred on five separate occasions without eonoalving. Four of the six females experienced abortion early In gesta tion. Eight of eight of die 2.6 ppm PCB fed animals oonatlved; however, only five were able to carry their Infants to term. As was the case with infants of animals given the higher levels of PCS's. all the infants were nail and at birth thairskln contained detectable (avals of PCB'i. Tha Infants ware permitted to nurse their mothers for 4 months. Within 2 months focal areas of hyperpig mentation, swollen lips end eyelids, loss of eyelashes, and acneform lesions of the face developed. The skin of tfwse infants showed a decided increase in the PCB level over this period. Within 4 months, 3 of the 6 infants died due to PCB intoxication. After weening, the remaining three have shown improvement of the skin lesions during the 4-month period. * Four edult male rhesus monkeys were also exposed to a diet containing 5.0 ppm PCB's (Aroclor 1248) (table 1) for 17 months (average total intake of PCB'i 460 mg). They began to develop a slight periorbital edema after 6 months of exposure; however, it was much less severe than in the female monkeys receiving a similar level of PCB. The morphological features and viability of the spermatozoa as well as the ability to fertilize control female rhesus monkeys was unaffected during the initial 12 months of PCB exposure. Subse quently on# of the four males lost weight and developed alopecia, acne, periorbital edema and decraasad libido. A testicular biopsy of this animal showed a deckled hypo- activity of tha seminiferous tubules. There was an absence of mature spermatozoa and a predominance of Sanoli cells of the tubules. The remaining three males have remained healthy and sexustty active (ref. IS). ABSORPTION, METABOLISM, TISSUE DEPOSITION, AND EXCRETION OF PCB's Over 90 percent of a single oral dose (1.6 or 3.0 g per kg) of PCB's (Aroclor 1248) given to adult rhesus monkeys was absorbed from the gastrointestinel tract. Chromatographic analysis of the tissues 14 days after exposure revealed e predominance of higher chlorine Isomert that had a predilection for the adipose tissue and organs containing a high fat content (ref. 16). Rhus monkeys fed 25 ppm PCB (Aroclor 1248) in the diet attained levels of 127 pg/g within the adipose tissue after 2 months. Eight months after discontinua tion of exposure to PCB's the levels ware 34 jjg/g within the adipose tissue. After 33 months, the levels within the adipose tissue ranged from 3 to 14 pg/g; the residues contained greatly increased proportions of highly chlori nated congeners. Transplacental movement of the PCB's was demonstrated by the pretence of PCB's In the infants born to exposed females. The tissues of an infant, born to a female 8 months following the discon tinuation of PCB's in the diet, contained 26 pg/g of PCB in the fat and adrenal tissues at the time of birth. In an infant born to .a female 29 months following the discon tinuation of PCB's, the levels within the adipose tissue were 3.38 pg/g at 4 months of age. Female monkeys given 5.0 ppm PCB in their diets attained maximum levels of PCB's within their adipose tissue at 6 months (141 to 177 pg/g adipose tissue)., However, it required approximately 14 months on the 2.5 ppm diet for the monkeys to reach similar maximum PCB levels in their adipose tissue (126 to 144 pg/g). Males which received 5.0 ppm PCB's attained levels 44 MONS 083356 Table 1. Experiments on exposure of primates to PCB's (Aroclor 1248*1 Level of PCB In diet ' (ppm) No. of animals Length of exposure (months) Total PCB Intake 300 100 25 2.5 5.0 -- 5.0 6 males 6 males 6 females 8 females 8 females 4 males - 3 2 2 16-19 16-19 17 Monsanto Co., Inc., St. Louis, Missouri. 3.6 to 5.4 g 0.8 to 1.0 g 250 to 400 mg 243 to 303 mg ~ 460 to 614 mg 530 to 692 mg .. - Table 2. Modification in reproduction In primates that were exposed to Aroclor 1248 In the diet Control 2.5 ppm 5.0 ppm Total Impregnated (no./no. ^^yartlmalsT 12/12 . .v.'rt,*'': Resorptions or abor- . . tlons (no./ no. animals) 0/12 8/8 -'6/8 3/8 4/8 " Stillborn (no./no. animals) 0/12 0/8 - 1/8 Normal ' births (no./ '' no. animals) 12/12 5/8 1/8 ;45 I v '.v 083351 ranging from 12B to 200 Jig per g adipose tissue et 14 months. Infants born of mothers exposed to 2.5 and 5.0 ppm PCB's within the diet contained concentrations of PCB's ranging from 1.0 to 4.8 ug/g within the skin at birth. While nursing from mothers consuming PCB diets, the infants continued to accumulate the compound. At 3 months, the levels within the tissues ranged from 86 to 136 jjg/g- The concentration of PCB's within the milk ranged from 0.15 to 0.40 jig/g. The tissues of the infants which died while nursing PCB-fed mothers contained high levels of PCB's within the thymus, ovaries, brain, kidneys, adrenal glands and pancreas (20*48 pg/g tissue). Lower levels were found in the liver, lymph nodes, and bona marrow (8*16 jig/g). Monkeys and rats demonstrate species variation in the metabolic response to the PCB congener 2,5f2',5'tetrachloroblphenyt (TCB). Over 66 percent of the single dote (600 mg/kg) administered to rats was recovered from the feces, end an additional 10 percent was prasant In tfw urlnp within tha initial 72 hours (ref. 17). The materiel present in the body was concentrated within tha adipose tissue. Thera was a transient high level of TCB within tha blood at 24 hours. Other organs which contained significant quantities of TCB, however at lower concentrations, included the liver, skin, and muscle. Tha major urinary metabolite was identified as 30H*2,6,2*,6'*tetrechlorobiphenyl, Other monohydroxy TCB metabolites ware present in minor quantities (ref. 17). Over B0 percent of an oral dost (500 mQ/kg) of *H TCB administered to Infant rhesus monkeys was absorbed from tha gastrointestinal tract and was highly oonosntritad in tha skin, adrenal gland, liver, and adi* poet tissue. At 72 hours, lass than 2 percent of tha dose had bean eliminated in tha urine and 1 paroant in tha facts. Monohydroxy TCB, a major metabolite present in tha rat urine, was a minor metabolite in tha urine of tha monkeys. The two major metabolites wart dihydroxy* TCB and trani-3,4-dlhydro-3,4-dfttydroxy TCB (raf. 18). A second minor metabolite was hydroxy*3,4*dihydro* 3,4-dihydroxy TCB. Following tha oral dost of *H-TCB (1 g/kg) to juvenile monkeys, a major percentage of the material was absorbed from the gastrointestinal tract and was highly concentrated In the adranal, adipose tissue, and skin. Significant quantities ware prasant within the liver, muscle, and uterus. After 4 weeks, 14.8 percent of the dose was secreted into tha bile. Approximately 75 paroant of the biliary material was reabsorbed by the gut and the nonabsorbad material was recovered from tha Moat during this period. Over 90 percent of the PCB's excreted in the bile wes In the form of weter soluble glucuronic acid conjugates. An additional 8 percent of the total dose was recovered from the urine. Administration of the higher chlorine congener *H 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) to rets or monkeys demonstrated low levels of exretion into the bile. An oral dose of HCB (1 g/kg) administered to rets resulted in excretion of 0.3-0.7 percent of the dose per day in the bile over a period of 14 days. Tha urine was free of detectable levels of radioactivity (ref. 19). At 14 days, 65 percent was recovered fro.m tha body tissues. The oompourtd was highly concentrated within the adrenal glands, adipose tissue, and skin end in the female within the ovaries and uterus. Following the administra tion of a single dose of HC8 (1 g/kg) to juvenile rhesus monkeys, less than 2 percent wes excreted vie the biliary-fecal route over e 3-week period. There wes no detectable radioactivity within the urine. The organs with the highest concentration of the material Included the edrenal, adipose tissue, and skin. Qua to tha targa mass of muscular tissue, this was a major reservoir of tha compound. INTERACTION OF METABOLITES WITH CELLULAR MACROMOLECULES ' Following tha administration of 2,5,2',5'-titrtcMorobiphenyl (TCB) to infant rhesus monkeys, inter action of TCB and macromoleeules of ceils end of serum wes evaluated (ref. 20). Separation of the serum constit uents by polyacrylamide gel electrophoresis demon strated association of the TCB primarily with serum albumin. Over 90 percent of the radioactivity of liver homogenates eluted from a Sephadex G-2B column was In the protein and nucleic acid fractions. Tha majority of the macromolecular-associated HCB apparently was bound by hydrophobic association. Extraction of liver homogenates with hexene, precipitation of the hexaneextracted homogenate with TCA, and subsequent extrac tion of the TCA precipitate with methanol resulted in extraction of the majority of the radioactivity. In the extracted residue, 1.1 percent of the radioactivity remained which may represent covalently bonded material. Recant in vitro studies employing monkey microsomes incubated with an NADPH generating system demonstrated 20 percent of the metabolised *H-TCB wes bound to microsomal protein and RNA in a nonextracteble form. Binding of *H-TCB was pravtnted by haating tha microsomas to 100C prior to incubation Iref. 21). DISCUSSION These experiments employing nonhuman primates have demonstrated development of parallel signs end 46 MONS 083358 lesions of PCB intoxication in humans and rhesus monkeys exposed to similar levels over comparable periods of time. Acne, subcutaneous edema of the face, and edema of the eyelids were observed in man (ref. 22) and lower primates (ref. 15) exposed to PCB's, The facial signs of PCB exposure appear to be a sensitive indicator of PCB intoxication. The rhesus monkeys after exposure for 2 months to dietery levels of PCB's (2.5 and 6.0 ppm) developed facial alterations after a total oonsumption of 32-50 mg of PCB's. It is noteworthy that PCB levels of 5.0 ppm are presently permitted in foods destined for humen consumption. The most debilitating lesions in the monkeys were the severe hyperplasia and ulceration of the stomach. Whether similar changes occur in the stomach of man exposed to PCB's remains to be clarified. Nausea and anorexia described by the human subjects suggest poten tial gastric alterations. Liver hypertrophy, proliferation of tht endoplasmic reticulum, end increased hepatic microsomal enzyme activities were observed in man (raf. 23) and in lower primates (ref. 11). Menstrual irregularities, decreased libido, occurrence of stillborns, reduced birth weights, end transplacental movement of PCB's in humans end rhesus monkeys have been recorded (refs. 13,24). The reproductive failures of monkeys exposed to PC8't were due to inability to maintain a pregnant state. The majority of the animals did not experience appreciable difficulty in conception; however, a large percentage of the animals aborted during the first 45 days of pregnancy. These observa tions suggest an Inability of Implantation or inability to maintain the implanted embryo during the early stages of pregnancy. Although the mechanism of reproductive dysfunc tion has not been clarified, there is some indication of hormonal modifications. Alterations in the urinary kttosteroids were reported in humans exposed to PCB's (ref. 22). Increased levels of urinary ketosteroids have been observed in the nonhuman primates that experienced reproductive failures (ref. 14). One mechanism of altered steroid metabolism may be secondary to the increase in the hepatic mixed function oxidases that ere present in the hypertrophic livers of exposed animals. It has also been a consistent observation that the organs associated with steroid production, tht adrenals and ovaries {par ticularly the corpora lutea), have contained relatively high concentrations of PCB's in exposed animals. Thus the oompounds `may possibly hava a direct effect on these organs. The presence of PCB's in the milk of other species has been previously reported (ref. 3). This avenue of infant exposure and the potential morbidity and mor tality was vividly demonstrated in the infant monkeys who nursed from mothers exposed to 2.5 and 5.0 ppm in the maternal diets. The presence of relatively low levels of PCB's in the diets of iactating females repre sents a potential source of PCB intoxication to nursing infants. Metabolic studies of the rhesus monkey demon strate over 90 percent absorption of tht PCB's from tha gastrointestinal tract following oral administration. The material is concentrated in organs with high lipid content. Including the adipose tissue, skin, edrenel, corpora lutea of the ovaries, and brain. Within tha liver the greatest portion of the materiel Is associated with the membranes of the endoplasmic reticulum. Studies with the single congener TCB demonstrate the metabolism of the compound to hydroxyiated forms which are conjugated with glucuronic acid and excreted into the bile. Enterohepatic circulation of tha PCB's undoubtedly occurs as only 20 percent of the material sacrated into the bila was recovered from the feces. The greatest portion of the metabolized TCB was excreted through the urinary system. The more highly chlorinated biphenyl HCB was more slowly eliminated from the body via the biliary-fecal route; HCB or metabolites ware not detected within the urine. The facilitated metabolism and excretion of the lower chlorine con geners was also indicated by the relative decreased stor age of these compounds, and conversely the accumu lation of higher chlorinated congeners, within the adipose tissue. Metabolic studies that hava been conducted on non human primates suggest mechanisms of interaction of PCB's with tissues and, more importantly, indicate potential mutagenic and carcinoganic effects of the PCB's. Metabolites have been isolated from rhesus monkeys exposed to the PCB congener 2.5.2#,5'-tetrechlorobiphenyl that era formed through an arena oxkL intermediate (ref. 18). Similar metabolites of the PCB's and tha potential for arena oxide formation has been demonstrated in rabbits (refs. 25,26). Arena oxides formed by the metabolism of other aromatic hydro carbons have been shown to covalently bind with macro* molecules and produce mutagenic and carcinogenic changes in mammalian cells (ref. 27). Dechlorination of tha more highly chlorinated biphenyls, demonstrated by dechlorination of 2,4,5,2',4 ,5'*haxachlorobiphenyl (ref. 26)f provides a mechanism through which metabolism of highly chlorinated biphenyls through an arane oxida intermediate would be facilitated. Evidence demonstrating the association of PCB's with liver macromolecules supports the theoretical potential of tha PCB's for covalent binding with cellular macromolecules. Thus, it appears that alkylation of macromolecules is one mechanism by which tha PCB 47 MGNS 033359 metabolites cause widespread injurious effects. Further credence for the ability of the compound to produce alterations in the macromolecules is presented in recent reports of hopetoceiluiar tumors developing in rats and mice exposed to PCB's (refs. 29-31). REFERENCES 1. S. JtnjL-r,. "Report of a New Chemical Hazard/' New Scientist. Vol. 32 (1966), p. 612. 2. M. Kuratsune, "An Epidemiologic Study on 'Yusho' or Chlorobiphenylt Poisoning/' Fukuoka Acta Mad/ca Vol. 60 (1969), p. 403. 3. A. C. Kolbye, "Food Exposures to Polychlorinated Biphenyls," Environ. Health Parsp., Vol. 1 (1972), pp. 85-86. 4. F. J. Biros. A. C. Walker, and A. Medbery, "Poly* chlorinated Biphenyls in Humen Adipose Tissue," Bull. Environ. Conam. Toxicol., Vol. 5 (1970), pp. 317-323. 6. J. Finktea, L. E. Priester, j. P. Creators, T. Hauaer, T. Hinners, and D. I. Hammer, "Polychlorinated Biphenyl Residues In Human Plasma Expose e Major Urban Pollution Problem," Amir. J. Pub. Health, Vol. 62 (1972), PP. 646-661. 6. J. R. Allen, L. A. Carstens, and L. J. Abrahamson, "Responses of Rats Exposed to Polychlorinated Biphenyls for Fifty-Two Weeks. I. Comparison of Tissue Levels of PCB and Biological Changes," Arch. Environ. Contarn. Toxicol., Vol. 4, in press. 7. M. L. Ktpilnger, 0. E. Fancher, J. C. Catandra, and E. P, Wheeler, "Toxicological Studies with Poly chlorinated Biphenyls," paper presented at tha NIEHS Polychlorinated Biphenyl Conference, Research Triangle Park, North Carolina, Oacambar 20-21,1971. ,, 8. J. R. Allan and L. J. Abrahamson, "Morphological and Biochemical Changes in the Uvar of Rats fad Polychlorinated Biphenyls," Arch. Environ. Conmm. Toxicol., Vol. 1 (1973), pp. 265-272. 9. J. R. Allen, L. A. Carstens, and D. H. Norbeck, "Biological Effects of the Polychlorinated Biphenyls In Nonhuman Primates," paper presented at Inter national Symposium on Recant Advances in the Assessment of tha Health Effects of Environmental Pollution, Parti, June 24-26,1974. 10. J. R. Allan and 0. H. Norbeck, "Polychlorinated Biphenyl and Triphtnyl Induced Gastric Mucosal Hyperplasia in Primates," Science, Vol, 179 (19731, pp. 498-499. 11. J. R. Allan, L. J. Abrahamson, and D. H. Norbeck, "Biological Eflacts of Polychlorinated Biphenyls and Triphenyls on Subhuman Primates/' Environ. Re*, Vol. 6 (1973), pp. 344-354. 12. J. R. Allen, L. A. Carstens, and D. A. Bariotti, "Residual Effects of Shert-Term, Low-Level Exposure of Nonhuman Primates to Polychlorinated Biphenyls/' Toxicol. Appl. Phwmacol., Vol. 30 (19741, pp. 440-451. 13. D. A. Barsotti, R. J. Marlar, and J. R. Allen, "Reproductive Dysfunctions In Rhesus Monkays Exposed to Low Levels of Polychlorinated Bi phenyls (Aroclor 124BI," FoodCosmat. Toxicol., in press. 14. 0. A. Barsotti and J. R. Allen, "Effects of Poly chlorinated Biphenyls on Reproduction In the Primate," Fed. Proc.. Vol. 34 (1975), p. 338. 15. J. R. Allen, "Response of Primates to Polychlori nated Biphenyl Exposure," Fed. Proc., Vol. 34 (1975), pp. 1676-1679. 16. J. R. Allen, D. H. Norbeck, and I, C. Hsu, "Tissue Modifications in Monkays as Related to Absorption, Distribution and Excretion of Polychlorinated Biphenyls/' Arch. Environ. Contam. Toxicol., Vol. 2 (1974), pp. 86-94. 17. J. P. Van Miller, I. C. Hsu, and J. R. Allan, "Distri bution and Metabolism of *H-2,5,2*.5'-tetrachlorobiphenyl in Rats/' Proc. Soc Exp. Biol. Med., Vol. 148(1975), pp. 682-687. IB. I. C. Hsu, J. P. Van Miller, J. L. Seymour, and J. R. Allan, "Urinary Mttaboiltas of 2,5,2',S'-tetrschiorobiphenyl in the Nonhuman Primate," Proc. Soc Exp. Biol. Med.. Vol. 15013975), pp. 185-188. 19. D. H. Norbeck, J. L. Seymour, and J. R. Allen, "Metabolic Study on * H-2,4,5,2',4',6'-b*xachlorobiphtnyl and * H-2,5,2',5'-utrachiorobiphany I in Rats," Amer. J. Path., (19701 in press. 20. I. C. Hsu, J. P. Van Millar, and J. R. Allen, "Meta bolic Fata of *H-2.5,2',5'-tatrachlorobiphtnvl in Infant Nonhuman Primates," Buff. Environ. Contam. Toxicol., Vol. 14 (1975). pp. 233-240. 21. J. L. Seymour, S. P. Schmidt, and J. R. Allen, "In vitro Generation of s Chemically Reactive Metabo lite of 2,5,2',5'-tetrachlorobiphenyl by Rhesus Monkey Liver Microsomes," Proc Soc Exp. Bio/. Med, submitted. 22. M. Kuratsune, T. Yoshimure. J. Metsuzeka, and A. Yamaguchi, "Epidemiologic Study on Yusho, a Poisoning Caused by Ingestion of Rice OH Contami nated with Commercial Brand of Polychlorinated Biphenyls," Environ. Health Penp., Vol. 1 (1972), pp. 119-128. 23. C. Hlrayema, T. Irisa, and T. Yamamoto. "Fine Structural Changes of the Liver in e Patient with 48 HONS 083360 Chlorobtphenyls Intoxication/* Fukuoka Acta Madfca, Vol. 60 (1969), p. 455. 24. M. Kikuchl and M. Hashimoto, "Hiitopathological Studies of Skin Lesions of Patients With Chloroblphenyls Poisoning," Fukuoka Acta Madlca, Vol. 60 (1969), pp. 484-488. 26. A. M. Gardner, J. R. Chen. J. A. G. Roach, and E. P. tagelis, "Polychlorinated Biphenyls: Hydroxylated Urinary Metabolites of 2,6,2',5'-ttrachloroblphcnyl Identified in Rabbits," Biocham. Biophya. Baa. Comm., Vol. 66 (1973), pp. 1377-1384. 26. 8. Safe, 0. Hutzingar, and D. Jones, "The Mech anism of Chlorobiphenyl Metabolism," J. Agric. Food Cham.. Vol. 23 (1976), pp. 851-853. 27. 0. M. Jerlna and J.W. Daly, "Arena Oxides: A New Aspect of Drug Metabolism," Sdanca, Vol. 186 (1974), pp. 673-582. 28. 0. Hutzingar, W. D. Jamieson, S. Safe, L. Petimann, and R. Ammon, "Identification of Metabolic Dechlorination of Highly Chlorinated Biphenyl in Rabbit." Nature. Vol. 252 (1974), pp. 098-699. 29. R. D. Kimbrough, R. A. Squire, R. E. Under, J. D. Strandberg, R. J. Montali, and V. W. Burse, "Induc tion of Liver Tumors In Rats by Polychlorinated Biphenyl Aroclor 1260," J. Natl. Cancer tntL, in press. 30. N. Ito, H. Nagasaki, S. Makiura, and M. Aral, "Hiitopathological Studies on Liver Tumorigenesls In Rats Treated with Polychlorinated Biphenyls," Gann, Vol. 66 (1974), pp. 546-649. 31. H. Nagasaki, S. Tomli. T. Mega, M. Msrugaml, and N. Ito, "Hepatocardnogenedty of Polychlorinated Biphenyls In Mice," Gann, Vol. 63 (1072), p. 005. DISCUSSION VOICE: I'm from the Massachusetts Society. Have you examined the samples for minute contaminants? DR. ALLEN: That is a good question. I presume your primary interests are in the dibenzofurans. The Monsanto Company has volunteered to analyze the Aroclor 1248 used in our experiments for the furans. In our discussion last week they were hope ful of having these data available for this confer ence. If Dr. Wright is in the audience perhaps he would give us a progress report on the subiect. (No answer from the audience.) J can say that we have done some preliminary work in this area end have found undetectable levels of furans in the samples. However, more detailed studies to clarify this ques tion are underway at the present time. VOICE: How can you determine that the PCB's are covalently bound to macromolecules? DR. ALLEN: Repeated extractions carefully monitored for radioactivity ere the best methods of removing any absorbed material from the protein. Standard gel chromatography methods do not differentiate between adsorption and covalently bound materials. We hope to be able to generate enough PCB bound to macromolecules to permit the determination of the exact covalent nature of the bond following macromolecolar digestion. VOICE: Which macromolecule did you use? DR. ALLEN: We were using protein end RNA from monkey microsomes. These microtomes were incubated with *H PCB in a NADPH generating system. The protein and RNA were isolated sub sequently and their radioactivity determined. VOICE: Could you tell us roughly how much PCB your animals consumed per kilogram of body weight? DR. ALLEN: The female animals on the PCB experi ments weighed between 6 and 7 kilograms. Table 1 gives the average total intake of PCB's by these ani mals during the various experiments. 49 mqns 083361