Document MpyJYyxN5ZrKe9ZDx3Rp3avV

y COMPARATIVE EFFECTS OF POLYCHLORINATED BIPHENYLS AND ORGANOCHLORINE PESTICIDES IN INDUCTION OF HEPATIC MICROSOMAL ENZYMES1 Joseph C. Street, Francis M. Urry, D. Jes9e Wagstaff and Adrian D. Blau Department of Animal Science Utah State University Logan, Utah 84321 Chlorinated biphenyls (PCB) , triphenyls (PCT) and other related compounds hove important Industrial uses and have been produced in large vulitmes for many years. Evidently some amounts reach the general environ ment. Pesticide residue cheroist9 became interested in such materials following a report from Sweden of their presence in wildlife tissues.* This ha3 been shown to interfere with the routine residue analyses of many organochlorine pesticides since complex patterns are produced in EC-GLC. * Moreover, routine cleanup procedures fail to eliminate such interferences unless special precautions are taken. These materials un doubtedly contributed to some of the "apparent" DDT found in biological specimens predating usage of DDT in agriculture or public health and constitute part of the present background of lipoidal organic chlorine in the biota. Polychlorinated biphenyls produce acute toxic effects in mammals similar to those of chlorinated naphthalenes and other chlorinated aro matics. The special vulnerability of the liver to such agents is well documented.^ Many lipid-soluble chemicals induce various enzymes of the hepatic endoplasmic reticulum (microsomal enzymes) including the drugmetabolizing hydroxylative system. Several organochlorine pesticides are potent inducers of such enzymes, with the result that treated animals metabolize drugB, insecticides and other foreign compounds more rapidly.^ 1 Presented at the 158th meeting, American Chemical Society, Pesticide OhemlStry Division, September 8, 1969. DSW 026707 STLCOPCB4010668 I) In some situations such stimulation is beneficial as, for example, in causing a more rapid detoxication of dieldrin and a lower residue stor age in the body. The induction could likewise be detrimental because certain toxifying reactions, such as activation of organothiophosphate insecticides, are also stimulated. Enhanced steroid degradation also occurs which raises possibilities of disturbing certain physiological processes due to altered endocrine relationships. The latter has been suggested as the basts for the thin egg shell phenomenon and poor repro duction in certain birds chronically exposed to DDT, dieldrin and some Other organorhlorlne 1nnecticides.^ Some polychlorinated biphenyl mater ial n hflvo already been reported to induce microsomal enzymes.^ The pre sent investigation was undertaken to compare the induction potencies of several type9 of polychlorinated biphenyls and triphenyls to those of various organochlorine pesticides, and to obtain preliminary information about tissue storage trends for such materials. The results contribute to our understanding of molecular characteristics required for microsomal enzyme induction and also allow some speculation about the consequences of various types of chlorinated compounds found in biota, METHODS Groups of male and female rats were individually fed Purina Labora tory Chow diets containing 25, 50, and 100 ppm concentrations of one of the several PCB and PCT materials tested. The test materials, Aroclors provided by the Monsanto Co., consist of crude products obtained by chlorination of biphenyl, or triphenyl, to specified limits; each is therefore a complex mixture. The ten products ranged from 21 to 68 per cent chlorine. Except for the negative controls, the diets were also treated to contain 1 ppm dieldrin. One group of rats in each experiment was fed diet containing 50 ppm DDT and 1 ppm dieldrin. The diets were -2 DSW 026708 STLCOPCB4010669 fed for 15 days. During that period tests of drug and insecticide metab olism _in vivo were conducted. Later, tests of microsomal enzyme activity were carried out with liver homogenates, Hexobarbital sleeping time measurements were made on the tenth day of treatment. Hexobarbital sodium was administered at the rate of 100 mg/kg i.p. in aqueous solution. The duration of deep sleep ending with the return of the animal's righting reflex was recorded as the sleeping time. The oxidative detoxification of EPN (0-ethyl-0-(p-nitrophenyl)-phenylphosphonotlifoate) and the O-demethylation of p-nitroanisole (PNA) were measured by incubation of the respective substrates with the 10,000 X g supernatant of liver homogenates following the method of a Neal and DuBois as modified by Kinoshita et_ al. The ring hydroxylation of aniline was measured by the incubation of aniline hydrochloride with the 10,000 X g supernatant of liver homogenates following the method of Kato and Gillette. Residual dieldrln in adipose tissue was determined by electron capture gas chromatography on hexane extracts of the tissue. The adipose tissue extracts required special cleanup to eliminate PCB interference in the detection of dieldrln. This was accomplished by passing the extract over a 2-stage chromatography column consisting of 1:1 MgO-Celite (5V) on the bottom and Florisil containing 47. moisture (4") on the top. After eluting with hexane (200 ml) to bring out the PCB components, dieldrln was recovered by eluting with 400 ml of a 1 + 4 dichloromethane + hexane solution. Qualitative and semi-quantitative evaluation of PCB storage in adipose tissue was possible after careful evaluation of GLC data comparing parent material and that recovered from tissue. DSW 026709 -3 STLCOPCB4010670 )~ RESULTS Short term feeding of polychlorinated biphenyls and triphenyls to rats at rates up to 100 ppm in the diet had essentially no effects on food consumption, weight gains or efficiency of food utilization (Table 1). Liver enlargement was observed with all products tested, increasing in dogree with the percentage chlorine in the product. Products with 60 percent chlorine or more caused greater liver enlargement than equivalent doses of the Insecticide DDT. Evidence of PCB induction of liver hydroxylating enzyme activity was obtained with all enzyme tests employed. Hexobarbital sleep times were markedly reduced by the PCB materials at both 50 and 100 ppm in the diet for 10 days (Fig. 1). The rate of decrease was reasonably linear with increasing degree of PCB chlorination. Enzymatic aniline hydroxylation, EPN degradation and demethylation of p-nitroanisole, as determined with liver homogenates, were increased with preparations from PCB-treated rats (Fig. 2 and Table 2). In general, higher chlorination of the biphenyl resulted in greater enzymatic activ ities. These enzyme activities were similarly affected in both sexes by the treatment materials, although the male appeared to be more responsive (Table 3). Dieldrin metabolism in vivo, judging from its residue storage in adipose tissue, was stimulated by the treatment materials, especially those with high degrees of chlorination (Fig. 3). The products contain ing 607. chlorine or greater reduced dieldrin storage to levels found in untreated control animals in our laboratory. This activity toward diel drin metabolism had been predicted by analogy to the similar effects of enzyme-inducing drugs and organochlorine pesticides. OSM 026710 -A - STLCOPCB4010671 ) Higher dosage rates (100 ppm vs 50 ppm in the diet) produced greater degrees of enzyme induction. The dose-response curve (Fig. 4) obtained with 627. Cl-PCB, in the range from 5 ppm to 100 ppm in the diet, was generally similar to that of DDT or phenobarbital yet that substance was greater in potency at every dosage level. Heptachlor epoxide, by contrast, was considerably more potent than any of these compounds. The two products containing chlorinated triphenyl components (one 427. Cl-PCT and the other a mixture of PCB:PCT, 60:40, containing 657, chlorine) fronted were, in comparison to the PCB materials, less active in frerms of effect on the enzyme activities measured in vitro. However, hexobarbital sleep time was more strongly influenced by the 42% Cl-PCT material than by the equivalent PCB, and dieldrin metabolism was highly stimulated by both the PCT-containing materials (Table 4). The Arochlor products fed and the residual components recovered from adipose tissue were all examined by electron capture gas-liquid chromatography. Some degree of storage of each product was observed al though the residues had greatly modified composition in comparison to the parent materials administered. Those products and individual components having the least chlorine appeared to be stored the least in adipose tissue. That can be seen in the comparison illustrated (Fig. 5) for 62% Cl-PCB and its tissue residue. The early GLC peaks (components low in chlorine) in the residue were greatly diminished relative to most of the later peaks in the chromatogram. Peaks C and D in that residue match, in retention time, two of the three major peaks reported by Risebrough et_ al.^ in PCB residues from tissues and eggs of birds in the San Fran cisco bay area. The tissue residues for PCBs lower in chlorination were much lower in proportion to that of the highly chlorinated PCBs and -5- OSW 026711 STLCOPCB4010672 consisted chiefly of minor components of those materials. Work is in progress to quantitate these residues in terms of organic chlorine. SUMMARY To summarize all the responses evaluated, hepatic microsomal enzyme induction was minimal with the PCBs of low chlorination (21-32/i chlorine) but increased to very high values for those materials averaging over 607. chlorine. The latter exceeded DDT in potency of induction, but not heptachlor epoxide, , Mic roRomnl enzyme induction caused by PCB materials is probably r ndd<rive with that produced by organochlorine pesticides.^Hence, if i any of the toxicological aspects of organochlorine pesticides in the environment are related to their enzyme inducing action, the presence of highly chlorinated biphenyls and triphenyls would intensify such biological effects of the pesticides. The very marked influence of the highly chlorinated PCB and PCT materials on dieldrin metabolism (which, by analogy, would affect all other cyclodiene insecticides and DDT as well) could greatly confound the interpretation of exposures of animals and birds bearing mixed residues. The toxicological implications of such exposures would be equally con founded . This work extends our previous investigations of the structureactivity aspects of microBomal enzyme inducing agents (presented in part at the 156th ACS meeting, Agriculture and Food Chemistry Division) and the results fit well with our working hypotheses regarding the activities of various bridged and unbridged diphenyl compounds. DSW 026712 -6 STLCOPCB4010673 literature cited 1. Jensen, S. New Sci. 32^66(1966) 2. Reynolds, L. M. Bull. Envir. Cont. Toxicol. 4:128-143(1969) 3. Risebrough, R. W. , P. Reiche and H. S. Olcott. Bull; Envir. Cont. Toxicol. 4:192-201(1969) 4. Von Oettingen, W. F. U. S. Dept, of Health, Education and Welfare, Public Health Service Publication No. 414, 1955. The Halogenated Aliphatic, Olefinic, Cyclic, Aromatic, and Aliphatic-Aromatic Hydrocarbons Including the Halogenated Insecticides, Their Toxicity and Potential Dangers, p. 306-307. 5. Street, J. C. Ann. New York Acad. Sci. 160:274-290(1969) 6. Peakall, D. B. Nature 216:505(1967) 7. piocbi-o.igh, R. W. , P. Reiche, D. B. Peakall, S. G. Herman, and M. N. Kirvcn. Nature 220:1098-1102(1968) 8. Kinoshita, K. F., J. P. Frawley and K. P. DuBois. Toxicol. Appl. Pharmacol. :505(1966) 9. Kato, R. and J. R. Gillette. J. Pharmacol. Exptl. Therap. 150:280(1965) 10. Street, J. C., D. J. Wagstaff, and F. Mayer. Proc. 6th Interaraerican Conf. on Toxicol, and Indu9t. Med., Univ. Miami School of Medicine, 1968, (in press) 0SW 026713 STLCOPCB4010674 Table 1. Food consurapt' h, weight gain and efficiency, p liver wergiita of female rats treated with polychlorinated bipnenyls and DDT. ^ Treatment Trial A Basal Dieldrin, 1 ppm Food Cons. g 226 t 5.6 233 3.3 + DDT, 50 ppm 224 6.0 -1- 487. Cl-PCB, 50 ppm 100 ppm + 547. Cl-PCB, 50 ppm 100 ppm 226 4 4.7 225 4 5.5 224 5.4 231 t 9.0 + 607. Cl-PCB, 50 ppm 100 ppm 226 * 7.1 225 t 5.5 + 687. Cl-PCB, 50 ppm 100 ppm f PC^ + 657. ci; + , 50 ppm \ PCB 100 ppm 224 9.6 227 6.3 228 4 8.3 232 t 7.9 + 427. Cl-PCT, 50 ppm 100 ppm 218 4 4.6 211 5.7 Weight Gain g Wt. Gain f Food g/lOOg Liver Wt g/lOOg 45 + 1.6 49 3.3* 44 2.4 20 * 0.6 21 4 1.6 20 0.9 4.1 0.44 4.3 4 0.27 4.5 4 0.44* 47 3.2 44 + 6.5 21 i 1.3 20 i 3.0 4.7 4 0.45** 4.7 4 0.46** 47 + 2.0 48 + 2.0* 21 4 0.7 21 1.7 4.7 4 0.39** 5.5 4 0.46** 50 3.1* 22 4 1.4* 4.6 0.28** 59 2.2** 26 3.0** 5.3 4 0.22** 50 t 5.0* 51 5.5* 22 4 1.8* 22 1.8* 5.1 4 0.16** 5.1 4 0.46** 43 3.0 49 1.7* 40 + 4.3 48 + 5.8* 19 4 0.9 21 t l.l 18 4 1.8 18 4 1.1 4.8 4 0.31** 5.2 4 0.37** 4.4 4 0.21 4.7 4 0.01** Trial B Basal Dieldrin, L ppm + DDT, 50 ppm + 217. Cl-PCB, 50 ppm 100 ppm + 327. Cl-PCB, 50 ppm 100 ppm + 427. Cl-PCB, 50 ppm 100 ppm + 627. Cl-PCB, 50 ppm 100 ppm 237 4 3.8 234 i 6.9 39 + 2.3 40 8.0* 16 2.5 17 4 3.0 4.0 4 0.31 4.3 4 0.32* 221 4 2.6* 228 6.6 228 6.9 32 2.3 33 + 1.3 33 5.5 15 1.6 14 4 1.7 14 4 2.5 4.5 0,21** 4.1 0.33 4.1 0.56 225 4.5* 232 4 A.3 38 + 1.7 39 + 4.7 17 4 1.9 17 4 1.3 4.4 0.52** 4.7 4 0.44** 226 7.7 39 i 2.1 211 5.0** 31 3.1 17 4 1.9 15 4 3.8 4.5 0.26** 4.6 0.28** 216 4 5.4** 35 2.5 16 1.1 4.8 4 0.22** 230 4 5.3 48 3.6** 20 1.2** 5.3 4 0.33** All groups except the basal received 1 ppm dieldrtn. Data are expressed as means 4 S.E. for groups of five rats. Asterisks, * and **, indicate values judged significantly different from the basal group by LSD analysis with P'S .05 or .01, respectively. DSW 026714 STLCOPCB4010675 r' Table 2. ,\ Enzyme respon. i and reduction in hexobarbital .leep time and dieldrin storage in female rats treated with polychlorinated biphenyls, -triphenyls or DDT. i Treatment Trial A EPN ' Detox. 7. Basal 94 + Dieldrin, 1 ppm 100 + DDT, 50 ppm 431 + 487. Cl-PCR, 50 ppm 100 ppm 225 362 + 547. Cl-PCB, 50 ppm 100 ppm 444 475 + 607. Cl-PCB, 50 ppm 100 ppm 475 544 + 687. Cl-PCB, 50 ppm 100 ppm . 600 525 PNA Demeth, 7. 98 100 228 408 568 555 672 291 368 272 242 Aniline Oxidn. 7. Hexobarb. Sleep Time -7. Dieldrin Storace -7. 104 100 205 193 295 309 346 231 273 266 240 0 75.0 34.6 57.7 65.4 80.0 78.8 80.0 83.7 80.0 0 93.4 60,7 77.5 85.8 91.6 93.4 96.5 96.5 96.0 Trial B Basal + Dieldrin, 1 ppm + DDT, 50 ppm + 217. Cl-PCB, 50 ppm 100 ppm + 327. Cl-PCB, 50 ppm 100 ppm + 427. Cl-PCB, 50 ppm 100 ppm + 627. Cl-PCB, 50 ppm 100 ppm 92 100 450 117 117 183 233 200 275 625 717 114 100 300 148 128 257 411 386 474 403 474 114 100 248 138 . 154 194 225 250 305 293 312 0 78.6 11.3 31.0 48.8 55.4 37.5. 65.0 85.7 87.5 0 92.2 7.8 10.9 29.7 45.3 37.5 64.7 94.5 96.5 Each value is the mean percentage increase (or decrease) in the measured response for groups of five rats compared to the groups that received the basal diet plus only 1 ppm dieldrin. DSW 026715 STLCOPCB4010676 Table 3. Sex coi.. {rlsoriB in selected response ol Ats to various polychlorinated biphenyl materials. PCB Treatment 487. Cl, 50 ppm 25 ppm 547. Cl, 50 ppm 25 ppm 607. Cl, 50 ppm 25 ppm 627. Cl, 50 ppm 25 ppm 687. Cl, 50 ppm 25 ppm EPN Sex Detox. 7. F 193 M 155 F 293 M 225 F 413 M 340 F 433 M 408 F 467 M 430 PNA Demeth. 7. 416 265 497 341 316 331 286 363 254 359 Dieldrin Storage -7. 46.0 46.7 74.7 76.6 91.8 85.0 91.5 90,0 92.7 88.7 Data from rats fed diets containing 1 ppm dieldrin plus each treatment at the indicated level. Each value is the mean percentage increase (or decrease) in the measured re sponse of groups of five rats compared to groups that receiv ed the basal diet containing only 1 ppm dieldrin. Table 4. Comparisons of responses from polychlorinated biphenyls to poly chlorinated triphenyl materials equivalent in chlorination. Treatment EPN Detox. 7. PNA Demeth. 7. Aniline Oxidn. 7. Hexobarb. Sleep Time -7. Dieldrin Storage -7. 427. Cl-PCB 427. Cl-PCT 200 386 250 238 208 145 37.5 46.2 37.5 73.6 687. Cl-PCB 627. Cl-PCB 657. Cl-/ PCB' [+ . pct 600 272 266 625 403 293 519 249 231 85.6 85.7 66.3 96,4 95.0 95.5 Data from female rats fed diets containing 1 ppm dieldrin plus each treatment at a level of 50 ppm. Each value is the mean percentage in crease (or decrease) in the measured response of groups of five rats com pared to groups that received the basal diet containing only 1 ppm dieldrin. DSW 026716 STLCOPCB4010677 >) Figure 1. Reduction in hexobarbital sleep time in female rats after receiving PCB materials in the diet (50 ppm) for 10 days. Figure 2. Increases in activity of liver homogenates from female rats in degrading EPN and p-nitroanisole after receiving PCB materials in the diet (50 ppm) for 15 days. Data are presented as percentage increase In activity reLative to rats receiving the basal diet containing only 1 ppm dieldrin. OSM 026717 STLCOPCB4010678 Figure 3. Reduction in dieldrin storage in adipose tissue of female rats fed diets containing 1 ppm dieldrin and various PCB materials (50 ppm) for 15 days. Figure 4. Dose response curves for various microsomal enzyme inducing agents in terms of stimulation of the iji vitro degradation of EPN by liver mitochondrial supernatant preparations. The inducing agents were ad ministered in the diets of female rats for 15 days. SW 026718 STLCOPCB4010679 Figure 5. Electron capture gas chromatograms of 627. Cl-PCB material and its residue recovered frcxn rat adipose tissue. DSW 026719 STLCOPCB4010680