Document nky53zak3BXzykKzvpE66yO5m

\ !' .** - V ,* >A /'i . ^Vv*i* "'"' *,> ';< \j>."**':*'i^rll^`.^AYv.'.pmVp'-.'-.- * , *,. ` * ' `V- V v ^ ' .V-v-/ I .J'KA.'i V >* V-V-V/M- &<%/!.?&?& :mmM mmI iiHipll|: mm mm r%*>fAq)7oy m&i r*r `j\ v{t* $HmSmf liYs'fr *''; :'-'-* ' jyv'Tv/PZt 'it*-1 : '; . -h'>.i :T i\ TOXICOLOGY AND AITl.lft) PHARMACOLOGY 28, 66- 8J (1974) The Influence of Pure Polychlorinated Biphenyl Compounds on Hepatic Function in the Rat1 G. J. Johnstone, D. J. Hcohiciion and O. Hutzinger2 Deportment ofPharmacology, Daihonsie University, Halifax, Nova Scoria, Canada, and Atlantic Regional Laboratory, National Research Council, Halifax, Nora Scotia, Canada Received June 19, 1973; accepted September 5, 1973 The Influence of Pure Polychlorinated Biphenyl Compounds on Hepatic Function in the Rat. Johnstone, G. J., EcomctioN, D. J. and Hut/.ingek, O. (1974). Toxicol. Appl Pharmacol 28, 66-81. Pure bi phenyl and isomerically pure mono-, di-, tetra-, hexa-, and octachlorobi- phenyls of known chemical composition were injected ip (50 mg/kg/day) into young male NVistar rats for J days; the animals were killed 96 hr after the last injection. The potency of the pure l'CBs was compared to that of 0,/Z-DDT and p,p'-DDT and commercial Aroclors (1254, 1260) admini stered at the same concentrations. Hepatic function was assessed by pento barbital sleeping times and in vitro assays of hepatic microsomal Odcmcthylasc, N demethyiase, aniline hydroxylase, nitroreductase, carboxylcsterase and the cytoplasmic bromosulfophthalcin-glutathionc con jugating enzyme. Biphenyl and 4-monochlorobiphenyl did not cause induc tion of hepatic drug-metabolizing enzymes. Microsomal mono-oxygenases were induced by hexa- and octachlorobiphenyls and by di- and tetrachlorobiphenyls with chlorines substituted at the 3 and 4 positions. Nitroreductase and carboxylcstcrasc activities were affected only by the highly chlorinated compounds whereas all agents, including biphenyl, caused a marked induction of the bromosulfophthalein-conjugating system. The ability of the commercially available, polychlorinated biphenyls (Aroclors, Phenoclors, Clophcns, Kancchlors, etc.) to influence hepatic function has been well documented (Lincer and Peakall, 1970; Fujita el al., 1971; Villcncuve cl al, 1971; Bickers el al, 1972; Litterst et al, 1972). Toxicologic assessment of these mixtures has been complicated by the heterogeneity of the PCB isomers, by marked differences in physical and chemical properties influencing the rates of absorption, distribution, metabolism and excretion and by the possible presence -T such toxic impurities as chlorinated dibenzofurans and dibenzo-p-dioxins (Vos el a!,, 1970; Sissons and Welti, 1971; Zilko and Choi, 1971; Grcig, 1972; Hutzinger et al, 1972; Webb and McCall, 1972). A statement by Tucker and Crabtree (1970) suggested that sublelhal effects were directly correlated with the chlorine content, while lethal effects were inversely corre lated. Because of the uncertainty surrounding the interpretation of toxicologic results and the inability to determine which of the many isomers were toxic, it was decided to 1 Presented in part at the Twelfth Annual Meeting, Society of Toxicology, New York, New York, Match 18-22, 1973. 1 Atlantic Regional Laboratory, National Research Council, Halifax, Nova Scotia, Canada. Copyright * (974 by Academic Press. Inc. All n^hK of reproduction In nny form reserved. Printed in Great Britain 66 0$w 025728 STLCOPCB4009683 V -V- . 'Jtijir $&hb*iAtkiAW. ,-f; PCI! AND HEPATIC FUNCTION 67 study the influence of a series of isomerically pure chlorobiphcnyl compounds on a number of functionally diverse, hepatic, drug-metabolizing enzymes of the rat. With this series of pure chlorobiphcnyl compounds of varying degree and known position of chlorination, several questions could possibly be answered. Are (he alterations in hep atic function due to the biphenyl nucleus itself, to the position(s) occupied by individual chlorine atoms, or to the number of chlorines present on the biphenyl nucleus? METHODS Synthesis of chhnohiphenyls. The various chlorobiphenyl compounds were syn- (II utzingcr or <7/., 197J). The synthesis of2,4'-dichlorobiphcnyl was accomplished by the reduction of 2,4'-dinitrobipheny! (Columbia Organic Chemicals) with zinc and acetic acid followed by conversion of the crude 2,4'-diaminobiphcnyl to 2,4'-dichlorobiphenyI via a diu/.otization reaction and treatment with cuprous cMoridc (Sandmeyer reaction) (Hulzinger et al., 1971). TABLE 1 Physical Proper iij s or Some Isomi ricau.y Pure. Chi.orobipiienyi.s Compound Melting point (CC) Rf x 100" Water solubility (/<g/ml) 4-Chloro 77-78 43 1.18 2,2-Dichloro 59-60 38 1.50 2,4-Dicltloro 24-25 50 1.87 4,4'-Dichloro 148-149 41 0.075 2,5,2',5'-Tc(rachloro 86.5-87 656 0.046 2,4,2',4'-Tetrachloro 41-42 58 0.0675 3,4..V,4'-Tcirachloro 172-173 44 0.175 2,4.5.2',4',5'-Hexacliloro 262-263 74 0.0088 2t3,5,2',3',5'-llcxachloro J28-129 70b -- 2,4.6,2',4',6'-Hcxachloro 112-113 69 .-- 2,3,4,5,2',3',4',5'-Oclachloro 156-157 82" 0.0070 2,3,4,5,6,2',3',4',5',6'-Dccachloro 305-306 76 0.015 In heptane 011 commercially prepared thin-layer plates (Merck silica gc! I--254). ' In hexane on preparative (20 x 100 cm) thin-layer plates (Merck silica gel F-254). The agents were purified by preparative thin-layer chromatography on 20 x 100 cm silica gel plates coated with Merck Silica gel F-254 (layer thickness of 0.9mm) using 77Iiexatic or n-hcp(anc as the developing solvent. T he chlorobiphcnyls were rccrystallized from aqueous methanol or ethanol (Hutzingerc/c/., 1971). All the agents prepared were homogeneous on thin-layer chromatograms and gave single peaks when analyzed by gas-liquid chromatography using a 4.0% SE-30 column (Zitko et a!., 1971). Mass spectra, obtained for all compounds, confirmed the molecular composition (Safe and Hulzinger, 1972). Tabic 1 presents some physical characteristics of the isomerically pure chlorobiphcnyls investigated. '; >' w'j^y':; ;V). T ' .-a ,f. * . ; ! pf ..'v-i DSW 025729 68 JOHNSTONE, liCOHICMON AND MUTZINGLK Agents studied. Analytical grade o,//-DDT and /;,//-DDT (Gcigy Chemical Corp., Ardsley, Mew York) and Aroclor 1254 and 1260 (Monsanto Chemical Company, St. Louis, Missouri)3 were used as "control'' inducing agents of hepatic drug-metaboliz ing enzymes for comparison with biphenyl (Eastman Organic Chemicals, Rochester, New York) and the scries of isomcrically pure chlorobiphenyls shown in Tabic 1. The agents were dissolved in warm peanut oil by continual stirring until solution was effected. 7 he solution was adjusted to a final volume oflO ml with additional oil, yielding a final concentration of 17.5 mg/ml. Solubility problems were encountered with 2 compounds. Decachlorobiphenyl was insoluble in peanut oil and could not be included in the investigation. The 3,4,3',4',-tetrachlorobipheny! was soluble in peanut oil when heated slightly above 40'C but, on cool ing to room temperature, a portion of the material precipitated from solution. In order that this isomer could be studied, the mixture was heated until a solution was formed, and it was injected as a warm solution (approximately 37CC). Animal treatment. The animals used were young male Wisiar rats (Woodlyn strain) weighing 50-65 g at the beginning of the experiment. Each treatment group, composed of 7 animals, w as housed in w'ire-floored, metabolic cages (2-3 rats per cage) during and after drug administration. The animals were provided food and water ad libitum. For comparison purposes, the "control" inducing agents, biphenyl and the pure chlorobiphcnyls were administered at a dosage of 50 mg/kg ip in a volume of 0.15 0.20 ml of peanut oil solution. Control animals ( -- 19) W'ere given peanut oil on the same volume basis as were the experimental animals. Two groups (n ~ 5 per group) of vehicle-treated controls were tested during the investigations with the various agents to ensure repro ducibility of lire assay procedures. The dosage schedule used by Schwark and Ecobichon (1967) was used, the animals being injected once daily for 3 days and killed 96 hr after the last injection. Prior to killing the rats, a pentobarbital sleeping time w'as determined by injecting 40mg/kg of sodium pentobarbital (0.4 0.6 ml jp) and placing them in a thermally neu tral environment under a heating lamp at 32-34c'C. The animals were continually handled and turned during the period of drug effect. The sleeping time w as measured from the loss of the righting reflex until it was regained. On recovery, they were killed by C11C13 anesthesia, quickly opened and the livers and kidneys were excised, weighed and placed in chilled 50-ml beakers in crushed icc. Tissue preparation. All subsequent steps of tissue homogenization and fractionation were carried out at 0~4C. The entire liver or a 5.0-g sample was minced finely with scissors and W'as washed twice with cold 1.15% KC1 to remove residual blood. The minced tissue was completely homogenized W'ith 20 ml of 1.15% KC1 solution by 10 strokes of a loose-fitting, motor-driven Teflon pestle in a 50-ml Potter-EIvchjem glass homogenizer. The homogenate (25 ml) w'as centrifuged at 4aCfor 242,000 g-min (10,000 rpm for 20 min) in a Sorvall RC2-J3 centrifuge. The postmitochondrial supernatant was rcccnlrifuged at 4,621,500 g-min (34,000 rpm for 50 min) on an International B-60 centrifuge. The supernatant w'as carefully removed and dialyzed against buffer in * Abbreviations used: o.p'-DDT, ],I,1-trichloro-2-(<>-chlorophenyl)-2-(p-chIorophcnyl)ethane; p,p'-T)DT, l,l,l-trichloro-2,2-bis(/?-chloiophenyl)elhane. The nomenclature of the chlorobiphenyl compounds follows the accepted form, the position of chlorines on one phenyl being indicated by num bers from 2 to 6 while chlorine positions on the second ring are designated by a number with a super script prime, i.c., 2,2'-dichlorobiphenyI. DSW 025730 '.'.Wcjrvy ' STLCOPCB4009685 ) *t ,/.. '*.`VC V' -, .' ^ A- V'i'.^v'-if'vVt! *: i;:'"'i yf. ',)*; } *1 S' . vsi'v<!t '.< r>'^: "']; i?1*-. '!vr^*>''^ ^^ffV.'; t'y* ?!$> i,: Vc. *'$}''*!?'* 'i "* m..................................................... *tylv-. .f-<'?'< v l*/?&/-.*/ >''' : , `A /''. ^ 7>V ^'y.*: Jjj pen and m:PATic function 69 picpuration for a subsequent enzyme assay (sec below). The combined pellets (micro semes) were resuspended and washed in cold 1.15% KCI, recentrifuged for 2,295,200#min (34,000 rpm for 30 min), and the pellet was resuspended by homogenization in 1.15",; KCI sufficient to yield a suspension in w hich 1.0 ml contained the microsomcs of 500 mg liver. This crude microsomal fraction was used in the following enzymatic assays, being stored not longer than 2 hr in crushed ice. TIME fmioutes) CONTROL p.p'DDl T-> O.p'DDT AR0C10R 12H IB-1 AR0CL0R I26D fjL BIPHENYL 4-MON0-Cl 4I0 2,2'Di Cl 7,4'Oi Cl SO 120 _J_ _L_ 3-^1I 33% -41. % 4.4' 01 Cl 3 2,5.2,'5' 1ETRA Cl 7,4.7:4' TURA Cl ?,4,s.2!:y hexa cih ?,. s. 7:j.'S' hexa cig-1 2.4.6,2.'4:6' HEXA Cl _JI SLEEPING TIME (PinlotarD 40 roj/Kjl i 2,3,4, S, ?;3,'4:5' OCTA 004 ` Tig. I. The effect of prctieatmcnt of young male rats with DDT (o,p'~ and p,/>'-isomcrs), Aroclor 1254 mid 1240. biphenyl and u ser ies of isomerically pure chtorobiplicnyls on (tic sleeping time produced by an injection of 40 mg/kg sodium pentobarbital. Animals were treated by ip injection for 3 consecutive days, and the sleeping time was determined 9((hr after the last injection. The values represent the mean sleeping lime (min)d SD for J9 control nnimals and 7 animals per treated group. Assays. The nucleic acid (DNA and RNA) content of the hepatic homogenate prior to centrifugation was determined on an aliquot of homogenate from each animal by the method described by Schneider (1957). Suitable blanks were prepared with the TCA solution and standard curves were prepared for the nucleic acids using known concen trations of purified calf thymus DNA type I and yeast type XI RNA (Sigma Chemical Company, St. Louis, Missouri). Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin for the preparation of a standard curve. The mixed function oxidases (A'-dcmcthylasc, (9-dcmetliylase and aniline hydroxy lase) were assayed under the same conditions, incubating for 30 min in an air environ- $$$&A/c '/s.'TV' A.V' * ' f*: ?* '> '*'j ' f txc.- w:`:- v -> ; ft < ^ r* y.:x : v-. r <* ;.7fvS V**-- *'r/H ft'*.: \>v;v' ji.O.AT.r-..,;-: S.,a.j4 > .'Sfv.'VV,,.2 V'/'V'ivI.:' f.'v tvtV;^ '.-. 't :r.'< : .r>.-j-V' T::.v.,'.; 4. 7-i ; ". z. -- .* #*z- .T:y ^,s W` [ V' V-; 5v .. i . A - - T\ v f,i J 4"'-.' . s>V% h .>; )r.` > r ' > - l-.r 'c,.; r j. . >l* ` `."iV 1 '* . ' * 'j-n* -i' 4 ' { '.;<. ' ; .. '< , 1, ,s;' 9 ! 2; OSW 025731 )' V-'* V'* STLCOPCB4009686 70 JOHNSTON):', IICOH1CHON AND HUTZINGLR mcnt at 31CC and using 25-ml Erlcnmeycr flasks in a PubnofT metabolic shaker (115 rpm). The 'reaction mixtures for each assay consisted of the following: (1) 0.5 ml of nticrosomcs equivalent to 250 mg liver; (2) 50 pmol of glucose 6-phosphate; (3)0.5 unit of glucose 6-phosphalc dehydrogenase (Torula yeast, type XII, Sigma Chemical Com pany) (4) 100 pmol of nicotinamide; (5) 1.0 pmol of NADP; (6) a variable volume of 0.05 M TrisHCl buffer, pH 7.4; (7) substrate solution. The volume of the incubation mixture was 5.0 ml. For the A'-demethylase and aniline hydroxylase assays, 25 pmol of hMtXE$/ng fflouiff nM0US/mg PR0HW ftVOUSfraj ftoifib JTg. 2, The effects or preucatmcut of young male rats with DDT (o,p'~ anti/>,//-isomers) Arodor) 254 and 1260, biphenyl and a series of isomerically pure cblorobiplieoyls on hepatic microsomal aniline hydroxylase (A), p-nitroanisole O-demethylase (B) and aminopyrine A'-clemelhylasc (C'> activities. Animals were treated by ip injection for 3 consecutive days and w ere killed and assayed 96 hr a Tier the last injection. Activities arc expressed as nmol of product formed, nig of microsomal protcin/30 min incubation. The values (bars) represent the mean enzymatic activities, +SD of the means of 19 control animals and 7 animals per treated group. MgCf, were added. The assay of microsomal nitroreductase activity was done in the same manner except that the incubation period was 60 min and thcrcaction was carried out under a nitrogen atmosphere- (Pouts and Brodie, 1957). The dcmethylation of p-nitroanisole(5 /unol) top-nitrophenol (O-demethylase) was assayed by the method of Netter and Seidel (1964). The dealkylation of aminopyrine (15 /ttuol) (A'-demethylase) was measured colorimctrically using the Hansch reaction to detect the formaldehyde released (Cochin and Axelrod, 1959). Thehydroxylation of aniline (15 pmol) was meas ured by the method of Kato and Gillette (1965). The reduction ofp-nitrobenzoic acid (20 /unol) to p-aminophenol was measured by the method of Kato and Gillette (1965). PCI? AND HIU'ATIC PUNCT10N 71 T he activities were calculated in terms of the nmol of substrate changcd/mg of protein/ 30 mill (mixed function oxidases) or 60 min (nitroreductase). Nonspecific carboxylcstcrasc activity was determined on aliquots of the postmitochondrial fraction by a spectropbotometric technique, continuously monitoring the formation of free a-naphthol from 1 x I0~3 M a-nnphlhyl acetate at pH 7.4 and 25C (Ecobichon, 1970). A Pyc Unicam SP500 recording spectrophotometer equipped with a thci moslatted cell compartment and a Perkin Elmer model 165 recorder were used. Activity was presented as nmol of substrate hydrolyzed/mg of protein/min. CONTROL p.p'OOT o.p'DDI AROCIOR 12M AROCtOR W BIPHtNYl Z34-M0N0-CI Z3< nMOUS/mg PROTON 16 }4 _t_ _1_ X _l NIIRO MDUCTASt ?,7'Dl Cl ?,4'di a Z3 4,4' 01 Cl 3^ 7.5,^' 1URACI i.4,2:4* nmz\ 3< 2,4,5,2;*:v HtXA a wxa ci 2,4(6,?:W HtJCA C! 3-> r,v,ty OCTA Cl Fig. .1.1 hccfTcet of presentment of young male rats with DDT(o,//- and/j,/>-isomeis), Aroclor 1254 and 1260, biphenyl nnd a series of isomcrically pure chlorobiphcnyls on the activity oT hepatic micro somal p-niitobcn/oic acid nitroreductase. Activities arc expressed as nmol of product forined/ing of microsomal protein,60 min. For other details, see Fig. 2. The conjugation of bromosulfophthalein (BSP) with reduced glutathione was measured using the postmicrosomal supernatant (cytoplasmic fraction) which had been dialyzed overnight at 4:C against 0.1 m sodium pyrophosphate buffer, pH 8.2. The assay (Goldstein and Combes, 1966), was based on the observation that the conjugated form of BSP has a greater absorbance at 330 nm than does free BSP. An aliquot (0.1 ml) of dialyzed cytoplasmic fraction was added to 3.0 ml 0.1 M sodium pyrophosphate buffer pH 8.2 in a 25-ml Erlenmcycr flask. One milliliter of BSP solution (1.0 pmol/ml) was added and the thoroughly mixed contents were poured into a second 25-ml flask con taining 0.3 ml of reduced glutathione solution (310 Atmol/ml). After a 30-sec interval of mixing, the reaction mixture was poured into a cuvette and the enzymatic activity was measured spectrophotomctrically, continuously monitoring the rale of conjugate `'5, : > l ih -* ' *vnn-4-,.'v m j , - . v"e* c>-*,; i\. * ; IT Ilk i yv*v - fytfZYWMhtgr:',- ' V.!vv, i <rl;Si' .'^v. A' &-$'}>/ -XT * !: t? (' . f ?.>*`fe{&; '<. ft ..V * h.-'.v'.- Y'. - Fr* ,S.y-s> <; ic' Vki.1 v-. y< fib' V1". *V P* > W*.'yVs1'.*,'. > * iyy' C .' r; L'[ tvVV-'V'fV1' .Vvs>vVVvv 1 STLCOPCB4009688 %<Xi>K:^>^X< r /t^.-;^:-' ,;V ;# '' ' ' ^' :>.Tr:f;:>v *m VV:,'' v z;^>V- 72 JOHNSTONE, UCOfilCHON AND HU JZ1NGEK formation for 10 min at 25C. The changes in absorbance were related to a standard curve prepared with the conjugated form of BSP. Since this compound was not com mercially available, it was synthesized and purified in our laboratory by the method described by Whelan et aJ. (1970). coujroi p.p'coi 0,p'PDT AROCIOR 1ZM AROCLOR 1260 BIPHENYL 4-MONO-Ct 2.2'DI Cl ?.4't Cl M' Di Cl ?ty?;y turau 2.4,2:4' UTRA Cl n Aioiw H/tf'olyjf/ng Prolfin ICLO, 2CO I300 ___ ___ 400 3- 23-i CARBOXVUSTlRASt '' -- MAW HE.XA Cl 2,3,5,W HEXA Cl 2.4,6,2:W HEXA Cl 2.3,4,5, ?;W OCTA Cl =aZEH ~>--j-H Fig. 4. The effect of prctrealmeiit of young male rats with DDT (o,/>'-andp,p'-i$omeis), Aroclor 1254 and 1260, biphenyl and a series of isomerically pure chlorobiphenyls on the activity of hepatic carboxylcstcrasc activity. Activities are expressed as nmol of *-naphthyl acetate hydrolyzcd/mg of pro tein min. The fraction used in the assay was a postmhochondrial supernatant containing both micro somal and soluble cai lioxylesterases. For other details, see Fig. 2. Statistical analysis of significance of the various parameters measured in treated and control groups was assessed by a Student's / test for 2 sample means based on indepen dent samples. Statistical significance was assessed at the 5% level of probability (.p < 0.05). RESULTS Figure 1 shows the influence of the DDT isomers, commercial Aroclors, biphenyl and the series of isomerically pure chlorobiphenyl compounds on pcntobarbilal-induced sleeping time. This histogram, as well as the subsequent diagrams, is arranged to show the results obtained for control, vehicle-treated animals followed by those treated with "control" inducing agents (o,//-DDT,p,;/-DDT, Aroclor 1254, Aroclor 1260) and for the animals treated with the individual pure compounds, arranged from low to high < DSW 025734 PCD AND lJbl'ATIC FUNCTION 73 chlorination. The mean sleeping time observed for control animals was 87 min. Highly significant reductions in sleeping time were observed with the commercial Ai odors and DDT isomers, though o,//-DDT was not as effective. Biphenyl and 4-chlorobiphcnyI caused no significant (p > 0.05) changes in sleeping time. Of the dichloro-isomers, a significant reduction was observed only with the 4,4'-isomcr. Both tetrachiorobiphenyls caused a significant reduction in sleeping time though the 2,5,2',5'-jsomcr was less effective. All the hexa- and octachlorobiphenyls tested, caused statistically significant (/> < 0.05) reductions in sleeping time, being comparable to p,//-DDT, Aroclor 1254 and 1260 in effect. pj COSJUCAH/mj PROTEIN I*iti, 5. The died ofpiclrcalment ofyoung male rats with DDT (o,//-and/M/-isomers), Aroclor 1254 anti 1260, biphenyl und a series of isomcrically purechtorobiphcnyls on the hepatic cytoplasmicenzyme which conjugates BSP with reduced glutathione. Activities arc expressed as pg ofconjugate forined/mg of protcin/inm. Tor other details, sec Fig. 2. The influence of each agent on (1) liver weight, (2) DNA and RNA content of whole homogenate (25% w/v) and (3) microsomal protein extracted per g of liver arc pre sented with the results for control vehicle-treated animals in Table 2. Statistically signi ficant (p < 0.05) increases in liver weight were observed only following treatment with ;>,/>'-DDT, Aroclor 1254 and Aroclor 1260. The quantity of DNA extracted from the whole homogenates oflrcatcd animals was rclaiivcly constant, no significant differences being observed from the control values. Jn contrast, many of the agents tested caused significant increases in hepatic RNA. No increases in hepatic RNA were observed with p,/)'-DDT, Aroclor 1260, biphenyl, 4-chlorobiphenyl while 3 agents, 4,4'-di-, 3,4,3',4'- Group treatment Control. p,p'-DDT o.p'-DDT Liver weight (g) 4.77 1.14 5.59 0.89* 4.91 + 0.46 DNA (mg/g) 3.08 0.11 2.84 0.83 3.34 0.26 RNA (mg/g) 8.02 0.40 8.28 0.94 13.37 0.74* Microsomal protein (mgis) 6.76 1.40 9.36 2.32* 7.70 0.98 o ovzzH; It; Ptj:/"V: ^ v, _ _ - S^S? Aroclor 1254 Aroclor 1260 6.23 0.30* 6.15 0.49* 2.69 0.14 2.94 0.44 10.17 0.79* 9.04+1.43 8.42 1.54* 9.52+ 1.02* no Biphenyl 4-Chloro 2,2-DichIoro 2,4/-Diehloro 4.87 + 0.11 4.76 0.69 4.85 0.96 4.69 0.34 3.34 0.17 3.17 0.43 3.60 0.49 3.39 0.12 8.08 0.51 7.63 + 0.69 13.50 1.98* 13.89 0.30* 6.22 0.60 5.5S 1.26 6.56+ 1.78 7.73 + 0.94* oz > z fetfgl -v' '' %? 4,4'-Dich!oro 4.21 0.44 3.40 0.36 6.44 0.36 5.54 1.92 2,5,2',5'-Tetrachtoro 2,4,2',4'-TetrachIoro 3,4.3',4'-Tetrachioro O Co at 2,4,5,2'A'5'-Hexachloro 2.4,6,2\4', 6 '-Hexach loro 2,3,5,2'.3',5'-Hcxachloro 4.68 0.69 5.03 0.48 4.73 0.47 4.18 + 0.91 3.82 1.29 5.80 0.78* 3.43 0.39 2.90 0.21 2.86 0.12 3.64 0.70 3.80 0.85 3.31 0.26 6.70 0.93 11.86 1.07* 6.78 0.92 11.44 0.71* 11.47 1.07* 11.83 + 1.08* 6.06 1.44 9.14 1.30* 5.70 + 0.93 12.06 1.10* 7.10 0.92 11.76+ 2.32* c H N z o f* i: ' > ^ w -^ v y . *. \ >- vft.. STLCOPCB4009691 o 2,3,4,5,2\3 'A', 5 -Oclach 1 oro 4.53 1.46 3.49 0.64 11.51 0.93* S.34 + 0.96* Nj Ul J Values presented are the mean SD of 14 control and 7 treated animals per group. Values are based on wet weight of .issue. OJ Values are significantly different from control values, p < 0.05. a 1>C AND HITATIO FUNCTION 75 tclra- and 2l5,2',5'-letrachlorobiphcny!s, caused slight reduction in RNA content. Significant increases (p < 0.05) in hepatocellular RNA were observed with 2,2'-di-, 2,4'-di-, 2,4,2',4'-tclra-, the hexa- and octachlorobiphcnyls tested. Significant increases (/?< 0.05) in microsomal protein were observed in animals treated with />,/;'-DDT, Aroelor 1254, Aroclor 1260,2,4,2',4'-tclra-, 2,4,5,2',4,,5'-hcxa-, 2,3,5,2',3',5'-hexa- and the octachlorobiphcnyls. The other agents caused no changes in microsomal protein concentration from that observed for the control animals. J'igurc 2 summarizes the influence of the various agents tested on the hepatic mixed function oxidases aniline hydroxylase (A), 0-dcmelhylasc (B) and //-demclhylasc (C). nwaisMflj worm so ,it.l --I 270 160 TOO 00 NO mmmma-- 0 C0VTR0L M.V,*. - 1S0MR AH mesa* Msum for >.4.);*: -niRA' g, CHtOftOPlfHtNYl NR I CAMOXYUSHRAM j ^ | | ]|ti| COTJUGATUitypROlLlK SSSS3--< UP sfassasesa-' |B MIHUnS -j SUIPtNG !i.< l ie. 6. Hie effect of prclicatmcnt of young male rats with 3,4,.V,4'-tcli'achlorobiphcnyl on the hepatic <?-dcnicthyl;tse (OD), A'-dcmcthylasc (ND), aniline hydroxylase (All), nitroreductase (NR), caiboxylesterasc, RSI' conjugating enzyme and pentobai bilal sleeping time. The animals were treated as described in the text. The values presented are the means SD for 7 control, vehicle-treated and 7 agent-treated animals. l or aniline hydroxylase, significant increases (p < 0.05) in activity were observed for all agents with the exception of 4-chloro- and 2,3,5,3',5'-hcxachIorobipbenyl. Marked increases were observed following treatment with 4,4'-di- and 2,4,6,2',4',6'-hcxachIorobiphenyl. Compared to control activity, significant increases (/; < 0.05) in (2-dcmethylase activity were observed for all agents tested with the exception of biphenyl, 4-chloro-, 2,2'- and 2,4'-dichloro- and 2,5,2',5'-tetrachlorobiphenyls. The influence of the various agents on hepatic A'-demcthylasc were similar to those observed for 0-demelhylase, marked induction being observed with 4,4'-di-, 2,4,2',4'-tctra-, the hexa- and oclachlorobiphcnyls. Aroclor 1260 had a more pronounced effect on A-demelhylase than did Aroclor 1254, the reverse being observed for O-demethylasc activity. The effects of the various agents on hepatic microsomal nitroreductase activity are presented in Tig, 3, the results being somewhat different compared to those observed I ffW V `-Ate, tr, vi; V- i. : r*. V . -X:,. : r r.'\ \*'i ' %V y-X -'J* . ' > 'r',.'^,V,T...v ; ,;riV t?1Vi v'*-V / .'t;X j > V^-Af-VV . ' t V. X -vV,-.Z - DSW 025737 STLCOPCB4009692 r';'/0---,^v>;trrv:->::& V" :-r-.-f.. -: ' - . . vV* .*> , v. :iSts''TV 'd TV-Tv' ' fvT'v ;v 'Jr r ,:>\/y? \ v : vX; v-7 " ~';K '.- '' . .y^jT.V; ') ^VY^^vv'xrp' v- V;f &}<:* frS v - ' V' J'j. W. ''>/ ; ,;y,\** - ./. ' * .v . . ' ' v, !'... X. T V-v , ; v r;v, - 'V-TyX.': .< ^X*s,,xr ;y'X3-'X : '\> :' '' TXXX'X XX'. X'V:;;vX \.v ./X. X'X -.Ty v .' v '.}' .;, ` . X ,;V,>d^'-V-yCVLT,XVW/* VVT>V`^,;xyVfS<':^X^ZrdwSZX:y:Xrf;SE\rii/,.'^^kk^V'^Xt,2^ 76 . JOHNSTONE, liCOniCHON AND HUTZINGER. for (he mixed function oxidases (Fig. 2). Marked increases were observed only in those animals treated with />,//-DDT, Aroclor 1254, the 3 bcxachloro-isomers and the oclachlorobiphcnyl. Statistically significant (p < 0.05) increases were observed following treatment with Aroclor 1260 and 4,4'-dichIorobiphenyl while the remainder of the agents (o,g'-DDT, biphenyl, 4-chloro-, 2,2'- and 2,4'-dichioro and 2,4,2',4'-te(rachlorobiphcnyl) caused reductions of the nitroreductase activity. Figure 4 is a summary of the influence of the various agents tested on the hepatic carboxylesterase activity in young male rats. The fraction used for this assay was the 242,000g-min poslmitochondrial supernatant since carboxylesterases have been found in both particulate (microsomal) and cytoplasmic (soluble) fractions (Schwark and Ecobichon, 1967). Of the agents tested, only p,p'-DDT, Aroclor 1254 and 2,4,2',4'tetrachlorobiphenyl caused increases in activity which were significantly different from control values (p < 0.05). The other agents either caused nonsignificant (/; > 0.05) in creases in activity or reduced the activity below that observed in control animals. Figure 5 presents the results observed for the cytoplasmic enzyme system involved in conjugating BSP with reduced glutathione following exposure to DDT, Aroclor 1254 and 1260, biphenyl and various pure chlorobiphenyl compounds. Significant (p < 0.05) increases in activity were observed with all the agents tested. Even agenls such as bi phenyl and 4-chiorobiphenyl, which characteristically had little or no influence on the other enzymatic activities, markedly increased the activity of this hepatic enzyme. The inductive effects of the (etracblorobiphenyls on hepatic enzymes was of particular interest since lipid solubility appeared to be a problem with one, the 3,4,3',4'-isomci. It should also be noted that this particular compound exhibited greater water-solubility than the other two tctrachloro-isomcrs examined (Tabic 1). In this study, the agent was injected as a warm (37"C) solution. It is not known to what extent the agent was ab sorbed from the peritoneal cavity though there were no visible residual depots of material when the animals were killed 96 hr after the last injection. The effects of this isomer on the hepatic functions are presented separately in Fig. 6, since the results observed were much different than those obtained with the other tetrachloro-isomcrs. Only 1 of the enzymatic functions, Odemethylase, was significantly (p < 0.05) increased. Slight, though nonsigr Taint increases in activity were observed for A'-dcmethy]ase, aniline hydroxylase and BSP-conjugaling enzyme activities. Both nitroreductase and carboxylesterase activities were lower than control values and there was a statistically significant (/; < 0.05) prolongation of the pentobarbital sleeping time. DISCUSSION Commercial PCB mixtures have been shown to cause a marked reduction in barbitu rate-induced sleeping time (Komatsu and Tanaka, 1971; Bickers el al., 1972). Most of the enzyme inductive effects have been demonstrated using enzymatic reactions coupled to the mixed function oxidase system including D-demclhylase, TV-dcmethylase and aniline hydroxylase, all of which showed marked increases in activity (Fujita el a!., 1971; Villeneuve cl a!., 1971; Bickers el al., 1972; Liltcrst cl ah, 1972). Increases in hepatic cytochrome P-450 in rats have been observed following the administration of Aroclor 1016, 1242, 3248, 1254 and 1260 (Bickers cl al, 1972; Liltcrst el al., 1972). Hepatic microsomal nitroreductase activity was increased in rats as was carboxylesterase activity PCI) AND HEPATIC {-UNCTION in rabbits (Yillcneuvc el a!., 1971; Litterst cl al., 1972). The present results confirmed those reported in the litcralurc, the commercial Aroclors (1254 and 1260) causing marked induction of these enzymatic functions. Data have not been reported to date on the induction of hepatic, synthetic (conjugating), enzymatic reactions by PCBs, but the present results indicate that, for at Jcastonc conjugative enzyme system (Fig. 5), marked induction occurred following the administration of Aroclor 1254 and 1260. Few studies have been published concerning the effects of isomerically pure chlorobiphcnyl compounds on hepatic enzyme activities. Fujita el al. (1971), studying the effects of oral administration of4,4'-di-, 3,4,3',4'- and 3,4,2',4'-tetra-, 2,4,5,3',4'-pcnta- GROUP Biphenyl 4-Mono-CI J.V 2,4' O.D. N.D. A. H. N.R. BSP Sip. Time --f t 4b ---- -- *-- --1 t t-- ---- --t i 4.4' Z.3.2'5' 2,4.2'4' 4^ t * * -- t 4 t 4&> t 4- 4 4 2,4.5.2'4'V 4b ** j.j.s.rj'y 2,4,6, Z'4'6' && ** 4b 4b 4 4b 4b * Zi314,5,r3,4,5` 4 > -4 4 4b * ------- NO DIFFERENCE . SUCHt MARKED I-to. 7. A summary ofthccfiecls of pretreatment of young male rats with biphenyl and a scries ofisoniciically pure chlorobiphcnyls on hepatic microsomal O-demclhylasc (O.D.) A'-dcmcthylasc (N.D.), aniline, hydroxylase (A,IF), nitroicducta.se (N.R.), the BSP conjugating system and the pentobarbital sleeping time. The data of the previous figures arc simplified (o show ihat there were (1) no differences, (2) slight differences, or (.1) marked differences from control values. and 3,4,5,3')4',5'-hcxachiorobiphenyls to rats, observed increased liver weights, in creased microsomal protein, decreased barbiturate-induced sleeping times and marked increases in hepatic O-dcmethylasc and aniline hydroxylase activities. The greatest changes were observed with the penta- and hcxachlorobiphenyls, the former being the most active. Of the 2 tetrachloro-isomcrs studied, the 3,4,3',4'-isomer exerted a more marked influence than did the 3,4,2',4'-tclrachIorobiphenyl. Fujita el al. (1971) con cluded that the positions of the chlorine atoms on the rings was a factor in determining the intensity of the inductive effects. In contrast to the results of Fujita et al. (1971), who found that orally administered 4,4'-dichlorobiphenyl scarcely altered the activity of hepatic enzymes, the present study employing ip injections, revealed that, with the U fv-Jv",: 3/tv `X-V- 1 -.i t . v 2',: >- Y``Si --JT -.,-f -V. Ipifff 'b\-Aw-V y:,!'/: f W:r. ft--.' S|g|: ittto&eJP.W.c ` <-,v wpPP a Cii'- .y.- >1 ; h"- ii v-?f SU: i Vf r. v.-4 cf ;r V .. . .' Ov ; b f \ --V /*. . vi-Y/ ,,:.V j, :1-"I:'X<~ V. '-.j-. < :.b.: * ,;;.v .^.u; S DSW 025739 ja'.jv .jS CV,V-,?;F b ' ; -Vi', '' STLCOPCB40C ,' V V- :'.. V .\ ,, . 1 ,,'~t ,; .\V> J. . >> I- J-V V. r '; "V. ,vvv , . ..-.V.v:-;; = *:"V^ v *'' \;i., ';/ - .V: 'vV jp*/;'vc.V1 ;F' :, r VV^; V' *v ,*.#:.-/. 'V,;'. ,P.V: - >- ' i5*;'-' ^ '* . 'S, >i * - --J' v<7-.-. ; . -V /-'.i .^V/- > >v V'-- : 'A'w- Yi :-tt|VFlpZV.-.tl'Sl Mil Sr W&fWMn Mp iSfcMte ',: V .. /-n* / ....,* ..- ^}h-;. .\ r 78 JOHNSTONI:, CCOWCIJON AND 1IUTZINGIR exception of carboxylcstcrase and nitroreductase activities, the 4,4'-dichIoro-isomcr was as potent an inducing agent as the commercial mixtures or the isomers of high chlor ine content when given ip. Vos and Nolcnboom-Ram (1972), using 2,4,5,2',4',5'-hcxachlorobiphenyl in a chronic study, showed that hepatic injury was more severe with this isomer than that produced by Aroclor 1260, a marked proliferation of hypertrophic, hypoaclivc smooth endoplasmic reticulum being observed. In the present study, 2,4,5,2',4',5'-hcxachlorobiphcnyl caused marked induction of all enzymatic functions tested, Bcnlhcc/n/. (1972) reported that equimolar doses of tctrachlorobiphenyls were more effective than dichlorobiphenylsin inducing rat hepatic enzymes, but it must be pointed out that Aroclor 1248 and Aroclor 1232 were used as the source of tetra- and dichlorobiphcnyls, respec tively. These commercial compounds contain a number of other biphenyls of different degree of chlorination (Webb and McCall, 1972). Recently, Chen et at. (1973) reported the effects of the 2,3,4,5,- and 3,5,3',5'-tetrachlorobiphenyl isomers on hepatic micro somal enzymes following short-term oral administration to rats. Ethylmorphine Ndcmethylase, aniline hydroxylase and p-nitroanisole (7-dcniethy!asc activities were all markedly increased, especially the 0-demethylase, which appeared to be extremely sensitive to these agents (Chen el a!., 1973). Our investigations demonstrated that, administered ip, 2,4,2',4'-tctrachlorobiphenyl was a more potent inducer of enzyme activities than w'as the 2,5,2',5'-isomer. The 3,4,3',4'-tclrachloro-isomcr caused marked induction only of p-nitroanisole O-demethylating activity, though the unusual solu bility problems of this compound should not be disregarded. Despite the available information regarding the eff ects of the PCBs, there still remains the problem of interpreting and relating effects with chemical structure. Is the biphenyl nucleus essential for induction ? Is it the position of chlorine atoms on the molecule, or is it the number of chlorines, which determines the nature and the intensity of effect? Figure 7 presents, in summary fashion, the results found in Figs. 1-3 and 5 for the various isomcrically pure chlorobiphcnyls, indicating wdielher there wras no effect, a slight (possibly significant or nonsignificant) effect or a marked effect on the enzymatic func tions and sleeping times. The biphenyl nucleus alone played little role in inducing hepatic drug-metabolizing enzymes. While the slight increases in A'-demethylase and aniline hydroxylase were statistically significant from control values, pure biphenyl and 4-monochlorobiphcnyl were the least effective inducing agents when compared w ith DDT, commercial Aroclors or the other chlorobiphcnyls tested. For the two DDT isomers, the position of the chlorine on the phenyl rings was important since greater inductive effects were observed when the 4- (para) positions were both occupied (/?,//-DDT) than when the 2- (ortho) and 4- (para) positions were occupied (o,y/-DDT). This quantitative difference w'as also observed for the PCB isomers. Considering the dichlorobiphcnyls, when the 4- and 4'- positions were occu pied, there was a much greater inductive effect for all cnz.yniatic activities than was observed with 2,2'- and 2,4'-dichlorobiphenyls. The same positional phenomenon was observed for the tctrachlorobiphenyls studied, the induction caused by 2,4,2',4'-ieiracliJorobipheoyl being much greater than that observed for the 2,5,2',5'-telrach!oroisomer. Fujita el al. (1971) found that orally administered 3,4,3',4'-tctrachlorobiphcnyl caused a more marked induction of D-dcmethylase, Ar-dcnielhylase and aniline hydroxy- DSW 025740 STLCOPCB4009695 rCl> AND HEPATIC I'UNCTION 79 jasc Ilian did the 3,4,2',4'-isomcr. While the rate of administration was different in the present study, 2,4,2',4'-lelrachlorobiphcnyl caused a 2-fold increase in (9-demelhyIase activity (Fig. 2) whereas treatment with the 3,4,3',4'-isomcr resulted in a 7-fold increase (Fig. 6). The results for the analogs containing more than 4 chlorines suggested that, with these higher chlorinated compounds, the positions of the chlorine atoms were not as impor tant. I'ujita e! al. (1971) found that the penta-, hexa-, and octa chlorobiphenyls had greater enzyme-inducing potential than did the tetrachlorobiphcnyl isomers, a result which, with the exception of4,4'-diehlorobiphenyl, was confirmed in the present study. Jt has been demonstrated that more marked induction was obtained with commercial Aroclors wit ha greater proportion ofhighly chlorinated biphenyls (Aroclors 1254,1260) than with those having predominantly low chlorine content (Aroclors 1016, 1221) (Yillcncuvcrf aL, 1971; Bickers el al., 1972). It would appear that positional specificity disappears when more than 4 chlorine atoms are incorporated on the rings since the 4aixl 4'-positions arc usually occupied. An important factor governing the ability of any foreign chemical to cause induction of hepatic enzymes is the rate at which it is degraded in the body. Several investigators have shown that, after (he administration of Aroclor mixtures, the low chlorine analogs were eliminated more rapidly from tissues than were the highly chlorinated biphenyls (Grant el al., 1971; Bailey and Bunyan, 1972; Bickers el a}., 1972; Rehfeld el aL, 1972). Evidence has been presented (hat biphenyl and 4-clilorobiphenyl are hydroxylated and conjugated into readily excreted, water-soluble products (Block and Cornish, 1959), More recently, Ilutzingcr el al. (1972) showed (hat, in (he rat, biphenyl and 4-chlorobiphenyl were, mono- and dihydroxylated and excreted rapidly in the urine. MonoJtydroxylatcd derivatives were formed from 4,4'-dichIorobiplienyI, from 2,4,2',4'- and 2,5,2',5'-tc(rach!orobiplienyl, but not from the 3,4,3',4'-isomer. Chlorobiphenyls with more than 4 chlorine atoms were excreted only in the feces, no trace of the agents being delected in the urine. On the basis of these observations, biphenyl and 4-chlorobiphenyJ mighl be less effective inducing agents than the highly chlorinated analogs because of rapid biotransformation and elimination from the body whereas the more lipid-soluble, highly chlorinated biphenyls persist in vivo. ACKNOWLEDGMENTS This study was supported by funds from (he Medical Research Council of Canada (Grant MA-36IJ). The gifts of samples of Aroclor 1254 and 1260 from ihc Monsanto Chemical Company arc gratefully acknowledged. REFERENCES Hailey, S. and Bunyan, P. J. (1972). Interpretation of persistence and effects of poly chlorinated biphenyls in birds. Nature (London) 236, 34-36. Br.NTHr, H. F., Schmoi.dt, A. and Schmidt, H. (1972). Induktion mikrosomalerLeberenzyme much cinmaligcr Gabc von polychlorierlcn Biphcnylcn (PCH) und anschliessender StressSituation. Arch. Toxikol. 29, 97-106. Bicxiiiis, D. R., Harder, L. C, Kaim*as, A. and Ai.vares, A. P. (1972). Polychlorinated bi phenyls: comparative effects of high and low chlorine containing Aroclors on hepatic mixed function oxidase. Ret. Comowtt. Client. Pathol. Pharmacol. 3, 505-512. :rm .uvV .ft .K'fjfc /;*: A r ':V-Vv.Uf^ tfv j ,'t\`i V; ; ;<?V .;' ... :i .. v.-ir 51 tT ' "* :. T" :.,>. ;:r-' - 80 JOHNSTONE, r COlilOHON AND HUTZINGER Block, W. D. and Cornish, H. H. 0959). Metabolism of bipbenyl and 4-cbloiobiphcnyl in the rabbit. J. Biol. Client. 234, 3301-3302. Cm \\ P. R., Mehenimle, H. M. and Fishbein, L, (1973). Effect of (wo isomeric letrachloro- bi phenyls on rats and their hepatic enzymes. Arch. Environ. Contain. Toxicol. 1, 36-47. Cochin, J. and Axelrod, J. (1959). Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and nonnorphinc. J. Pharmacol. Exp. Ther. 125, 105-110. . Ecohiciion, D. J. (1970). Characterization of the esterases of canine scrum. Can. J. Biochcm. 48, 1359-1367. Fou is, 3. R. and Brodie, B. B. (1957). The enzymatic reduction of chloramphenicol, p-niu o- benz.oic acid and other aromatic nilro compounds in mammals. J. Pharmacol. Exp. Ther. 119, 597-207. Fujjta, S., Tsuji, FI., Kato, K., Saeki, S. and Tsukamoto, H. (1971). Effects of biphenyl chlorides on rat liver microsomes. Fukuoka Acta Med. 62, 30-34. Goldstein, J. and Combes, B. (1966). Specirophotomctric assay of the liver enzyme that cata lyzes sulfobromophthalcin-glutathione conjugation. J. Lab. Clin. Med. 67, 763-872. Grant, D. L., Phillips, W. E. J. and Villf.neove, D. C. (1971). Metabolism of a poly chlorinated biphenyl (Aroclor 1254) mixture in the rat. Bull. Environ. Contain. Toxicol 6, 102-112. Grek:, J. B. (1972). Effect of 2,3,7,8-tetrachlorodibenzo-l,4-dioxin on drug metabolism in the rat. Biochem. Pharmacol. 21, 3196-3198. Hut/.inger, O., Sate:, S. and Zitko, V. (1971). Polychlorinated biphenyls: Synthesis of some individual chlorobiphcnyls. Bull. Environ. Contain. Toxicol. 6, 209-219. Hu jzinuer, O., Nash, D. M., Sate, S., de Freitas, A. S. \V., Norstrom, R. J., Wildish, D, J. and Zitko, V. (1972). Polychlorinated biphenyls: Metabolic behavior of pure isomers in pigeons, rats and brook trout. Science 178, 312-314. Kato, R. and Gillette, J. R. (1965). Effect of starvation on NADI'H-depcndcnt enzymes in liver microsomes Of male and female rats. J. Pharmacol. Exp. Ther. 150, 279-284. Komatsu, F. and Tanaka, K. (1971). Shortening of hcxobarbital sleeping time and change of scrum triglyceride level in chlorobiphenyl-intoxicated rats. Fukuoka Acta Med. 62, 35-41. Linger, J. L, and Peak all, D. B. (1970). Metabolic effects of polychlorinated biphenyls in the American kestrel. Nature (London) 228, 783-784. Ln t erst, C. L., Fakber, T. M., Baker, A. M. and Van Loon, E. J. (1972). Effect of polychlori nated biphenyls on hepatic microsomal enzymes in the rat. Toxicol Appl Pharmacol 23, 112-122. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Client. 193, 265-275. Ni.ttek, K. J. and Seidel, G. (1964). An adaptively stimulated O-demethylating system in rat liver microsomes and its kinetic properties. J. Pharmacol Exp. Ther. 146, 61-65. Reiii eld, B. M., Bradley, R. L. and Sunde, M. L. (1972). Toxicity study of polychlorinated biphenyls in the chick. 2. Biochemical effects and accumulation. Poultry Sci. 51,488-493. Sate, S. and Hutzinger, O. (1972). The mass spectra of polychlorinated biphenyls. J. C-hem. Soc. Perkin Trans. 1, 868-691. Schneider, W. C. (1957). Methods Enzymol 3, 680. Schwark, W. S. and Ecoujchon, D. J. (1967). Subcellular localization and drug-induced changes of rat liver and kidney esterases. Can. J. Physiol Pharm,.col 46, 207-212. Sissons, D. and Welti, D. (1971). Structural identification of polychlorinated biphenyls in commercial mixtures by gas-liquid chromatography, nuclear magnetic resonance and mass spectrometry. J. Chromatogr. 60, 15-32. Tucker, R. K. and Crabtree, D. G. (1970). In: Handbook of Toxicity ofPesticides to Wildlife. U.S. Dept, of Interior, Bureau of Sport Fisheries and Wildlife. Publ. No. 84. ViLi.ENKUVE, D. C., Grant, D. L., Phillips, W. E. J., Clark, M. L. and Clegg, D. J. (1971). Effects of PCB administration on microsomal enzyme activity in pregnant rabbits. Bull. Eiwiron. Contain. Toxicol 6, 120-128. DSW 025742 STLCOPCB4009697 PCI) AND HEPATIC FUNCTION 81 Vos, J. G., Koeman, J, H., van di u Maas, N. L., ten Nof.ver de Brauw, M. C. and de Vos, R. H. (.1970). Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated naphthalene in two commercial polychlorinated biphenyls. Food Cosmet. Toxicol. 8, 625-655. Vos, J. G. and Notfndoom-Ram, 1:. (1972). Comparative toxicity study of 2,4,5,2',4',5'hcxachlorobiphcnyl and a polychlorinated biphenyl mixture in rabbits. Toxicol. Appl. Pharnituol. 23, 563 -578. Wi.hb, R. G. and McCall, A. C. (1972). Identities of polychlorinated biphenyl isonters in Aroclors. J. Ass. Offic. Anal. Client. 55, 746-752. Whelan, G., Hoch, J. and Combes, B. (1970). A direct assessment of the importance of con jugation for biliary transport of sulfobromophthalcin sodium. J. Lob. Clin. Med. 75, 542 557. Zii ko, V. and Choi, P, M. K. (1971). PCB and Other Industrial Halogenatcd Hydrocarbons in the environment. Fish. Pcs. Pd. Can. Tech. Kept. 272. Zhko, V., Hut/.inger, O. and Safe, S. (1971). Retention times and electron-capture detector response of some individual chlorobiphenyls. Pull. Environ. Contain. Toxicol. 6, 160-163. '. ,Y,: v-i :-*r, ! , y'5-t.fi-' K. '' t;. 4> >/* :l2 ,k.-A'! 'l ?S7'ti`-X1-,'-'V.aiP'' {' .* vV* , .J - i fa -J; tr > J;*.T - i &*},*. * o'- '>'*' fv**,' ' -h / - 1. -M..* ; 'C ; - '';i .r.yrwfi;- * " . i < v' > 'r v? f . T ' -' 1 . V. 2 .';---V.S4 V.Vi*.< j.S i: ' Svft'ft;-:*, i *} \ -'i' '.i ;C: * v.i*v :,v is1 'V .< j-Vi-yrvC,<A>'4T;,-.Vr;; YUV-VV`yVlAS- '-*; ,.pP X't'-iMcA 5-V V ^ /'A f < ;V. t . j y. svy OSW 025743 ; :-'v ^ ............. '"' " STLCOPCB4009698