Document 12bEGBk5Jw2e9mQKL7pGbDbK

'.^..v i -;-\-i ^j '<'^:xii':.'-iZf':.#h >*.JC^Vvnli*!.:.j2^<'3>.dciV.*? .*, -i*`y.<-* u*.V'.;i*.,;> pen AND LIVER MORPHOLOGY 427 Ml'i/inoir, 0., Saif, S. and Zuko, V. (1971). Polychlorinated biphenyls: synthesis of sonte individual chlorobiphcnyls. Bull. Environ, Contain. Toxicol. 6, 209-219. Hui/.ingih, 0., Nash, I). M., Sam-, S., Di:Fri;iio$, A. S. W., Nokstrom, R. J., Wildish, D. J. and Zuko, V. (1972). Polychlorinated biphenyls: Metabolic behavior of pure isomers in pigeons, rats and brook trout. Science 178, 312-314. Joknmoni., G. J., Ecoimcmon, D. J. and HuTziNom, O. (1974). The influence of pure poly chlorinated biphenyl compounds on hepatic function in the rat. Toxicol. Appl. Pharmacol 1ft, 66-61. KiMimouoM, R. D.,) iNora, K. I:. and Gainfs, T. B. (1972). Morphological changes in livers of ruts fed polychlorinated biphenyls. Arch. Environ. Health 25. 354-364. Koi.uk, L. D. and Zjnkl, J. G. (1973). Pathology of polychlorinated biphenyls in rabbits. Amor. J. Puihol. 70, 363- 373. Mu.U K, J. W. (1944). Pathologic changes in animals exposed to a commercial chlorinated biphenyl. V.S. Public Health Hep. 59, 1085-1093. Nisik/umi, M. (1970). Light and electron microscope study of chlorobiphenyl poisoning in mouse and monkey liver. Auh. Environ. Health 21, 620-632. Nomhack, r>. J. and Ai.i.i.n, J. R. (1972). Chlorinated aromatic hydrocarbon induced modifi cation of hepatic endoplasmic reticulum: Concentric membrane arrays. Environmental Health Perspectives 1, 137-143. Pi am , D. C. 0964). Histological Techniques for Electron Microscopy. Academic Press, New York. Kihiud, I). M., Bradlly, R L. and Sunde, M. L. (1972). Toxicity study of polychlorinated biphenyls in the chick. 2. BioehcmicHl effects and accumulation. Poultry Sci. 51.488-493. Sisson*, 13. and Wr.i.n, D. (1971). Structural identification of polychlorinated biphenyls in commercial mixtures by gas-liquid chromatography, nuclear magnetic resonance and mass spectrometry. J Chionwtagr. 60, 15-32. Tucxir, It. K. and Charirii., 13. G. (1970). In: Handbook ofToxicity ofPesticides to Wildlife. U.S. Depl. of Interior, Bureau of Sport Fisheries and Wildlife. Pub. 84. Vu i.iNtuvr, IX C, Grant, D. L., PHiu.irs, W. U. J,, Clark, M. L. and Clegg, D. J. (1971). Lfleets of PCI) administration on microsomal enzyme activity in pregnant rabbits. Bull. Environ. Contnni. Toxical. 6, 120-128. Vos, J. G. anu K<mman, i. H. (1970). Comparative toxicologic study with polychlorinaied bipltenyls in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue reuduev Toxirnl. Appi. Pharmacol. 17, 656-668. Vos, J. G. and Nounroom-Ram, li. (1972). Comparative toxicity study of 2,4,5.2',4',5'- hexachhnohlphenyl and a polychlorinated biphenyl mixture in rabbits. Toxicol. Appl, Pharmacol. 23, 563-577. Vok, 3. G., Km man, i. 11., Van Dt: Mas*. H. L., ten Notvttt de Braow, M. G. and de Vos, R. II. (1970). Identification and toxicological evaluation of chlorinated dibenzofuratt and chlorinated naphthalene in two commercial polychlorinated biphenvls. Food Cos/net. Toxicol. I, 625-655. * Wihii, K. G. and McCall, A. C. (1972). Identities of polychlorinated biphenyl isomers in Aroclors. J Art. Offie. Anal. ( hem. 55, 746-752. von Wi.di i., H., Hoi.i.a, W. A. and Denton, J. (1943). Observations on the toxic effects result ing from exposure to chlorinated naphthalene and chlorinated phenyls with suggestions for prevention. Rubber Ape 53, 419-425. Ziiku, V. and Choi, P. M. K. (1971). PCI) and other industrial halogenated hydrocarbons in the environment. Fish. Hes. Bd. Can. Tech. Rep. 272, 1-64. C* lAv?!.- .................. WSSSM 1; Vr>.w b jL'ii.?*-. < . r /*'?" !<V '*/ >%'$* ft-2 n ' * / C -V;;, ' Wi V'-r h f*'.? A,,.- . . V . `'f : t. i nuN$ 018?4 _________________ k*;*;..H'Hewii'*wwtyi)w:iaxeyreii)'W4*1 , . .X- ^ . zipiUg*?.'" ^ '' >-c*^'-r ' '' *v' ` ' -" PUttl? PCB AND HEPATIC FUNCTION 95 METHODS Agents studied. The rioiucrically pure chlorobipltcnyls were purchased from Analabs Inc., a division of New England Nuclear, Boston, Mass., and were used without further purification. Since it was important to study only the effects of position and degree of chlorination, biphenyl (Eastman Organic Chemicals, Rochester, New York) was used as the "control'' agent. The biphenyl was purified by preparative thin-layer chromato graphy on 20 x 100 cm silica gel plates coated with Merck silica gel F-254. n-Heptane was employed as the developing solvent. The material was eluted and recrystallized from aqueous methanol (Hutzingcr et al., 1971). The agents were dissolved in warm peanut oil by continuous stirring until solution was elTcctcd. The solution was adjusted with additional oil to a final concentration of 17,5 mg/ml. No solubility problems were encountered with the exception of 3,4,3',4'tclrachlorohiphcnyl, as was previously noted (Johnstone ttai, 1974). This tetrachlorobiphenyt was administered as a warm (approx. 37C) solution. Animal treatment. The animals used were young male Wistar rats (BioBreeding Laboratories) weighing 65- 85 g at the beginning of the experiment. Each treatment group, composed of six animals, was housed three to a cage on hardwood shavings, and was provided food and water ad libitum. The agents were administered at a dosage of50 mg/kg ip in a volume of 0.15-0.25 ml of peanut oil solution. Control animals (n 18) weie given purified biphenyl in peanut oil at the same concentration and in the same volume of solvent. Groups of biphenyl-treated animals were tested at intervals during the investigations to ensure reproducibility of the assay procedures. The animals were given daily doses for 3 consecutive days and killed 96 hr after the last injection (Schwark and Ecobichon, 1967; Johnstone et al.y 1974). Tissue preparation. The animals were killed by cervical dislocation, quickly opened, and the livers were excised, weighed, and placed in chilled 50-ml beakers in crushed ice. All subsequent steps of tissue homogenization and centrifugation were carried out at 0-4*C. A 5.0-8 sample of liver was minced finely with scissors and waswashed twice with cold 1.15% KCI to remove trapped blood. The minced tissue was completely homo genized with 20 ml ofl. 15 % KCI solution by 10 strokes of a loose-fitting, motor-driven Teflon pestle in a 50-ml Pottcr-EIvchjem glass homogenizer. The homogenate (25 ml) was centrifuged at 4'C for 242,OOOg-min (I2,000g for 20 min) in a Sorvall RC2-B centrifuge. The posimitochondrial supernate was carefully removed, stored in crushed icc, and used within 2 hr of preparation. 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 using the method of Schneider (1957) as described previously (Johnstone et al., 1974). Protein concentrations of the 242,000g-min supernate were determined by the method of I lartrec (1972), using bovine serum albumin for the preparation of a standard curve. 1 he mixed function oxidases(p-nitroanisole O-demethylase and aniline hydroxylase) were assayed by the methods of Netter and Seidel (1964) and Kaloand Gillette (1965), respectively. The reaction mixtures contained 0.5 ml or homogenate, 50/rmol of glucose-6-phosphatc, 1.0 unit of glucose-6-phosphate dehydrogenase, 100/imol of nicotinamide, 1.0/imol NADP, substrate (5.0 /imol of p-nitroanisole or 15/imol of aniline) ami sufficient 0.05 m Tris buffer pH 7.4 to yield a final volume of4.0 ml. For the '*** iWk'-. y rtf ' : ' mm i` ' ` a . f-'-r-y )-: i*-* "V .CV1 r ,J ' Wf sv..->*i2r- mm mm |Ti-* ,.-v- | mm MUNS 0818 76 96 U OiltCHON AND COMLAU aniline hydroxylase assay, 25 /iniol cf MgC.% were added. Incubations were curried out fur 10 hikI 20 min in 37"C with continuous shaking in a Pubnoff metabolic shaker; the reactions were stopped by the addition of 2.0 ml of 20% TCA. Initial rates of product formation were calculated from the rectangular hyperbola plotted for the (no incubation intervals. Nonspecific carboxylcstcrasc activity was determined on .aliquots of the postmitochondriai supernatant fraction by a spccirophotomelric technique winch consisted of a continuous monitor at 322 nm of the formation of free alpba-naphthol from I x 10 ' m alpha-naphthyl acetate at pH 7.4 and .37C (Ecobichon, 1970). The conjugation of sulfobromophthalein (DSP) with reduced glutathione (GSU) was measured using an aliquot of the supernatant fraction which had been dialyzed overnight at 4`C against 0.1 m sodium pyrophosphate buffer pH 8.2. The method of Goldstein and Combes(l966) was used; the rate of conjugate formation was monitored for ] min at 37C rind pH 8.2 as described previously (Johnstone ct a!., 1974). Enzymatic activities were presented as nanomoles (0-demcthylasc and hydroxylase) of product formed or as micromoles (carboxylcstcrase) of substrate changed per minute in (he total weight of fresh liver per JOO g of body wt, The activity of the USP-GSH conjugating enzyme was expressed in the same way but in terms of micrograms of conjugate formed per minute. Statistical significance of the various parameters measured in treated and control groups was analyzed by Student's / test for two sample means based on independent samples. Significance was assessed at the 5 % level, p < 0.05. RESULTS U was essential, before studying the effects of chlorination, to determine what effect the biphenyl nucleus had on hepatic enzymes. Table 1 shows the results of the ip TAHLE 1 Tm. Enters or InruAi'EtUTONEALLY Adminisiehed Pf.anui Oil, Commercial Hii'mfnyl, a no EumriW) Diphenyl Dissolved in Peanut Oil on Enzyme Activities or K.u Livtu* Activates (in total wl of fresh livcr/100 g body wt)* Enzyme Vehicle Commercial biphenyl Purified biphenyl p-Nitroamsole O-demethyinse Aniline hydroxylase Curboxyleslcrasc HSP-GSH conjugating enzyme 168.0 + 23.9 102.7 + 26.7 234.4 53.5 5.00.9 211.5 + 38.7 352.5 102.8' 336.7 + 55.3'' 14.4 1.9e 195 61 19. J 97.3 1 15.1 236 : + 24.7 0.9* 1 he animats received 50 mg of biphenyl kp 10.15-0.25 ml of solution) for 3 consccu1 dii)1* and were killed 96 hr after the last injection. Vehicle-.'.fated animals received peanut .vl on thc&jniv illume basis Twelve eniinnls "ere treated with vehicle. II "idi commercial biplwnjl, and 18 with purified l>iplicny|. TJic fictivilius of 0-(tenicihyl;**e ami aniline hyUrtnjIflsc arc expressed as nanomoles of product funwdiuimute. Catlxwylcslemc activity is expressed as micromoles of substrate hydrolyzcd/minutc. while (he DSI'-CiSM conjugating enzyme twtivuy it expressed as nviciograms of conjugate forme.)/ minute. Activities arc presented in terms of ihf total livcr lOOg of hotly wt. Values arc stalisticnlly different from vehicle-treated control values at p -t 0 05 HONS 081.877 rURL PCB AND HliPATIC ruNCTION 97 administration of vehicle (peanut oil), commercially available biphenyl and purified biphenjl on the activities of hepatic 0-dcntcthylase, aniline hydroxylase, carboxylcstcrasc, and BSP-GSH conjugating enzyme. Treatment with unpurified biphenyl resulted in statistically significant (p < 0.05) increases in activities of three of the en* zymes overvalues observed in vehicle-treated controls. In contrast, chromatographicaliy purified biphenyl caused a significant increase only in the BSP GSH conjugating enzyme activity. Since relatively pure compounds were to be investigated, the values for the purified biphenyl administered in peanut oil were used as control data. TABLE 2 CtiANuis in Ltvru Wuom, DNA, and RNA Following Intraperitoneal Administration or Purl Cmlorobipmesvls to Rats' Agent Li'er /I00 g body Wt. DNA (mg/g) RNA (mg/mg DNA) Biphenyl Monochloro: Dichloro: Trichloro: Tetrachloro: Higher coitgenera: 5.33 0.25 3.95 0.49 3.99 0,11 2- 5.10 0.12 3.26 0.26 6.76 0.19* 3- 5 29 O.Jg 3.62 0.07 5.90 0.19* 4- 5.52 0.46 3.73 0.24 5.52 0.13* 2,2'2,4'3.3'4,4'- 4.90 0.20 5.58 0.38 5.39 0.37 5.27 0.18 4.26 0.36 3.39 0.20 3.1910.33 3.36 0.23 4.480.14* 4.22 0.17* 6.50 0.34* 4.58 0.24* 2.4,4'2.5.2'- 2.5.3'2,5.4'- 3.4.2'- 5.74 0.46 5.29 + 0.30 5.20 + 0.18 6 28 0.62* 5.19 + 0.29 2.94 0.21* 3 31 0.29 3.18 0.22 2.78 0.30* 3.53 0.23 6.64 0.28* 5.90 0.52* 5.60 0.24* 5.96 0 34* 5.80 0.29* 2.4,2',4'2,6.2',6'2,3,2\3'2.5,2'.J'3,4.3',4'- 5.94 0.38 5.37 0.15 5.23 0.62 5.19 0.34 6.59 0.36* 3.29 0.12 3.67 0.18 3.53 0.57 3.58 0.21 3.27 0.27 4.76 0.19* 4.32 0.33 3.87 0.40 4.33 0.26 4.50 0.52 2.3,4.2',5'2,4,S,2',4'.5`2,4,6,2'.4',6'2,3,4,5,2',3',4',5'- 5.48 + 0.21 6.17 0.14* 5.47 0.45 5.60 0.35 3.51 0,21 3.65 0.21 3.21 0.29 2.56 0.10* 5.94 0.27* 4.33 0.14 6.20 0.25* 7.33 0.27* * Values preurnted are the mean SD of II biphenyl-treated (control) and 6' animals per group. Values are besed on wet wi of tissue. * Values ate significantly different from control values, p < 0.05. Tlte influences of each chlorobiphenyl on (1) liver weight in terms of 100 g body wt, (2) DNA and, (3) RNA content of whole homogenates (20 % w/v) arc presented with (he results for control, biphenyl-treated animals in Table 2. Statistically significant (p < K- V. '' ; / ' ;s .. iY ;YY ; :vt: : ' . S*-; i 'l.' ' ,; v. ;:.V 'rV MONS Q81878 V '' <- 'p' 98 I COmCHON AND COMI;AU 0.0.*') increases in liver weight were observed for only a very few agcnl$(2,5,4'-trithloi o-, 3,413',4Vlctrachloi(>-> ami 2,4,5,2',4',5'-hcxacllorobiplcnyls). All agents tested caused a reduction in DNA content, which suggested increased cellular size, though these were only significnnily dilTcrcni from biphenyl-treated controls in a few instances (Table 2). Increased RNA concentrations were obseivcd with all of the chlorobiphcny) agents tested though significant increases were observed only for some of the agents. The influence of monoclilorobiphcnyls on the hepatic 0-dcmethylase, hydroxylase, curboxylcslernsc, and BSP-GSH conjugating enzyme activities are compared in Table 3 with (he effects following treatment with purified biphenyl. No significant changes in microsomal G-dcmclhylasc were noted, (hough microsomal aniline hydroxylase activities were significantly increased over control values. C'nrboxylestcrusc activity was significantly (/ < 0.05) increased following treatment with the monochlorobiphcnyls. No significant {/> > 0.05) differences were observed for BSP-GSH conjugating enzyme activity between biphenyl- and the chlorobiphenyl-trcalcd animals. ' TABLE 3 Einns of Acme IniHAitunONtAC AoMiNisraAiioN of Uiphinvl and Monochum'ohu'Henyi.s on Hh-atic F.n/ymf Activity Treatment Activities (in total wt of fresh livcr/100 g, body wt)* OD n (nmol/min) AH (nmol/min) CE Oimol/min) USP (//g/min) Diphenyl 2-Chlorobiphcnyl .V Chlorobiphcny) 4-Chlorobiphvnyl 18 195.6 19.1 97.3 13.7 236.224.7 8.8 + 0.9 6 184.8 17.2 188.3 30.4* 309.2+ 35.1* 7 51 1.5 6 193.6 28.1 225.8 4 55.3* 381.7 1 64.3* 9.8 1.5 6 233.4 .12.5 168,1 37.4* 306.2 24.5* 9.8 i 1.6 * Values presented are the mean iSD of the number ofanimals per group. * Values Arc s'jjniftcnmly different from values obtained from biphenyl-treated animals, /> < 0 05. ' The enzymes investigated include p-mlroonisolc O-demethylase (OH), aniline hydroxylase (AH), cnrboxyVeslcinw (Ctl), and sutfoUromophthalcin-glmailtionc conjugating enzyme (BSP). Figure I shows the influence of a series of isomcrically pure dichloiobiphcnyls on hepatic O-demethylase, aniline hydroxylase, carboxyleMcrasc, amt BSP-GSH conju gating enzyme activities in comparison with the effect produced by purified biphenyl. Statistically significant (p < 0.05) increases in Die activities of (he mixed function oxi dases (G-dcmcthylasc and hydroxylase) were observed only with 4,4'-dichlorobiphenyl, whereas carboxylcslerasc activity was markedly increased by 2,2'-, 2,4'- as well as by 4,4'-dichlorobil>hcnyl. The BSP-GSH con jtigntingenzyme was increased only following treatment with 2,2'- and 4,4'-dichlorobiphenyI. No enhancement of activily was observed following treatment with 3,3'-dtchloTobiphcnyl; the activities of all four hepatic enzymes were lower than those of control, biphenyl-treated animals. Figure 2 summarizes the influence of a group of isomcrically pure trichlorobiphenyls on (he hepatic enzymes as compared to (he activities detected in biphenyl-treated animals. On the basis of O-demelhylasc activity, significant increases were observed HONS 061879 rum- pen and m-PATic function 99 l:n.!. The cfTcclsof pictrcalment ofyoung male rats with biphenyl and a series of isomcrically pure diehlnrohiphcnylt on hepatic /i-nitroanisole O-dcmciliylasc (OD), aniline hydroxylase (AH), carboxyl* cMerac(CJ:.),anil kiilfobiomophthukin-glululhione conjugating enzyme (BSP) activities. Animals were liciiirtl by ip injection for .1 consecutive days and were killed and assayed 96 hr after the Inst injection. Activities arc expressed as nanomoles ofprodnclformcd/minute(OD and AH), micromoles ofsubstrate hydrulyrcd/minulc (CP), or ns mirrograms ofconjugate formed/minutc (BSP), based on the total weight of fresh liver/100 g body wl. The values (kirs) represent the mean enzymatic activities SD (lines) of ]9 control biphciiyMrcntcd animals and 6 animals per treated group. The asterisk () indicates values KtntiMicully diH'cicnt (p < 0.05) from else biplrcnyblrcatcd controls. only following treatment with 2,5,4'* and 2,4,4'-trichlorobiphcnyl. The response of ttiicrosomnl aniline hydroxylase activity wrs somewhat different; significant increases were observed following treatment with all but 2,5,3'*trichlorobiphcnyl. Significant increases in cnrboxylcstcrusc and BSP-GSH conjugating enzyme activities were ob served with all agents tested though the most marked response was observed for 2,5,4'attd 2,4,4'*tricl)1oiobiphcnyl. 1 he effects ofa series oftctrachlorobiphcnyls on (he various hepatic enzyme activities Acnvnr i m tail * a ima umruoi Mr i i lie. 2. The cflectj of presentment of young male rats with biphenyl ami a series ofisomci ically pure Oirhlniobiphcnyli on hepatic/Miitroarmolc 0-dcinethylase (OD), aniline hydroxylase (AH).carboxylc>.tcrusc (CF), and sulfubiomophthnlrin-glutnthionc conjugating enzyme (BSP) activities. For other details, tec Mg. I. -rV- 1o&' ^iS.yUA;/ k> mimim a&Pj*. t T- r ' "',**.* > ". 1 ..Vv ,.s io l*il#.'iii V * EC0U1CH0N ANU COMhAU iPHnm .<..< ACTIVITY (IB loUl t tffc'ih IMr/IQD| My U rtMtefxta nmMTivIn (> rnotalnln af/nta IfO <00 MO (00 p............. in no xo p too <9 uo no s io n IZ3 M r.j.r.i* ur.i' SS&-' ffiSK M AH (I IS** Fig. 3. Thccffectsofpretrcatmenl oryoung mate rats with biphenyl And k serin ofisomericully pure letraehlorobiphcnyls on bepailv p-nitroanisolc O-dcmeihylasc (OD), aniline hydroxylase (All) carboxylesierasc (CE), and sulfobroroophthalein-glulathione conjugating enzyme (BSP) activities. For other details sec Fig. 1. as compared to those or biphenyl are shown in Fig. 3. While all the agents tested caused significant (p < 0.05) increases in hepatic cnrboxylesterave activity, the most marked cflccts were observed with 2,4,2',4'- and 2,6,2',6'-tetrachlorobiphcnyl. In contrast, the mixed function oxidase, O-demethyJase, was markedly increased by 2,4,2',4'- and 3,4,3',4'-tetrachIorobiphenyl. A small though significant increase was observed in the activity of the latter enzyme following treatment with 2,<\2,,6'-tctrachlorobiphcnyl. As was observed in Fig. 2, aniline hydroxylase responded somewhat differently following treatment with Ictrachlorobiphenyls. While significant induction was observed with 2,6,2',6'- and 2,3,2',3'-tctrachlorobiphenyl, the most marked increases were observed with the 2,4,2',4'- and 3,4,3',4'*isomcrs. Significant (p < 0.05) increases in the BSPGSII conjugating enzyme were observed only with 2,4,2',4'-, 3,4,2',4'-, 3,4,3',4'-, and 2,3,2',3'-tctrachlorobiphenyl. ACTIVITY I In taUJ nt l Idlh ltwU| My M HMlIwmibV'V/ v::* 00 AH (t Sir Fio. 4. The effects of pretreatment of young male rats with biphenyl and selected penta-, hexn, ami octachlorobiphenyls on hepatic p-nilroanisolc O-demethylusv (OI>), aniline hydroxylase (Alt), caTboxytesterasc (CE), and sulfobromophthalcin-gliilathione conjugating enzyme (HSi\i Mciivmo. For other details, see Tig. I. MONS ublddl PUKE PC AND HEPATIC FUNCTION 101 ! ip.mc *1 summarizes the influence oftreatment with a pcntachloro-, two hcxachloro-, ami nit octnchlorobiphcnyl on hepatic enzymes compared to the activities detected in biphenyl-treated rats. With the exception of 2,4,6,2',4',6'-hcxachlorobiphenyl, all of the agents tested caused significant increases in the four enzymatic functions assayed. In conti a\l, 2,4,6,2',4',6'-hcxachlorobiphcnyl treatment resulted in significant (p < 0.05) increases in aniline hydroxylase and carboxylcstcrase activities whereas the 0 dcmcthylnsc and BSP GSH conjugating enzyme activities were not significantly differ ent from activities in biphenyl-treated animals. DISCUSSION The ability of commercial chlorobiphcnyl mixtures to alter hepatic function by ultrastructural changes and induction of a variety of hepatic enzymes which act on biotruiisfornuiiion, has been well documented (Nishiz.umi, 1970; Vos and Bcenis, 1971; Kujita etui., 1971; Villcncuvc eta!., 1971; Kimbroughetai, 1972; Bickersetai, 1972; Kiltcrsl et al, 1972; Bruckner ct al,, 1973; Hansel! and Ecobichon, 1974). The hetero geneity of these mixtures has hindered toxicologic evaluation and has raised several pertinent questions. Do dichlorobiphenyls produce the same effects as tetra- or hexa* chlui obiphvnyls ? Do all isomers ofthe same degree ofchlorination produce comparable toxicologic responses? Arc the pathologic and toxicologic alterations observed due to the biphenyl nucleus, to the position(s) occupied by individual chlorine atoms or to the number of chlorines present in the structure? Are hepatic enzymes other than those associated with the endoplasmic reticulum affected by these agents? It is now known that not ail chlorobiphcnyl congeners possess the same physicochemical and pharma cologic properties (Johnstone et al., 1974). A high priority has been suggested for studies using individual pure compounds (Hammond etai, 1972), though to date such studies have been limited (1-ujita et al., 1971; Vos and Notenboom-Ram, 1973; Chen etai., 1973). T he hepatic enzymes, p-nitroanisole 0-dcmcthylasc and aniline hydroxylase, that were investigated in this study were selected as representative ofendoplasmic reticulumbound enzymes showing characteristic type 1 and type II spectral changes involving substrate interaction with cytochrome P450 (Remmer et a!., 1966; Schcnkman et al., 1967). Hepatic carboxyleslcrascs were selected as examples of enzymes localized predominantly in the endoplasmic reticulum though a small quantity of cytoplasmic enzyme may be present due to a possible solubilization of the membrane-bound enzyme (Schwerk and Ecobichon, 1968; Ljungquist and Augustinsson, 1971; Chow and Ecobichon, 1973). The enzyme which catalyzes the conjugation of BSP with GSH is a soluble cytoplasmic enzyme (Combes and Stakelum, 1961). The administration of such well-known inducing agents as phenobarbit.nl and DDT results in markedly enhanced activities of each of these enzymes (Conney, 1967; Schwark and Ecobichon, 1968; Johnstone etai., 1974). As was shown (Table 1), biphenyl significantly increased the activity of BSP-GSH conjugating enzyme. Compared to the enzyme activity in biphenyl-treated animals, there were no statistically significant (p > 0.05) differences observed following treat ment with monoelilorobiphcnyls but, with the exception of 2,4'- and 3,3'-di-, 2,5,2',5'-, and 2,6,2',G'-tctra-, and 2,4,6,2',4',G'-hcxachlorobiphcnyls, all of the chlorobiphenyl I PUKE PC AND HEPATIC FUNCTION 101 ! ip.mc *1 summarizes the influence oftreatment with a pcntachloro-, two hcxachloro-, ami nit octnchlorobiphcnyl on hepatic enzymes compared to the activities detected in biphenyl-treated rats. With the exception of 2,4,6,2',4',6'-hcxachlorobiphenyl, all of the agents tested caused significant increases in the four enzymatic functions assayed. In conti a\l, 2,4,6,2',4',6'-hcxachlorobiphcnyl treatment resulted in significant (p < 0.05) increases in aniline hydroxylase and carboxylcstcrase activities whereas the 0 dcmcthylnsc and BSP GSH conjugating enzyme activities were not significantly differ ent from activities in biphenyl-treated animals. DISCUSSION The ability of commercial chlorobiphcnyl mixtures to alter hepatic function by ultrastructural changes and induction of a variety of hepatic enzymes which act on biotruiisfornuiiion, has been well documented (Nishiz.umi, 1970; Vos and Bcenis, 1971; Kujita etui., 1971; Villcncuvc eta!., 1971; Kimbroughetai, 1972; Bickersetai, 1972; Kiltcrsl et al, 1972; Bruckner ct al,, 1973; Hansel! and Ecobichon, 1974). The hetero geneity of these mixtures has hindered toxicologic evaluation and has raised several pertinent questions. Do dichlorobiphenyls produce the same effects as tetra- or hexa* chlui obiphvnyls ? Do all isomers ofthe same degree ofchlorination produce comparable toxicologic responses? Arc the pathologic and toxicologic alterations observed due to the biphenyl nucleus, to the position(s) occupied by individual chlorine atoms or to the number of chlorines present in the structure? Are hepatic enzymes other than those associated with the endoplasmic reticulum affected by these agents? It is now known that not ail chlorobiphcnyl congeners possess the same physicochemical and pharma cologic properties (Johnstone et al., 1974). A high priority has been suggested for studies using individual pure compounds (Hammond etai, 1972), though to date such studies have been limited (1-ujita et al., 1971; Vos and Notenboom-Ram, 1973; Chen etai., 1973). T he hepatic enzymes, p-nitroanisole 0-dcmcthylasc and aniline hydroxylase, that were investigated in this study were selected as representative ofendoplasmic reticulumbound enzymes showing characteristic type 1 and type II spectral changes involving substrate interaction with cytochrome P450 (Remmer et a!., 1966; Schcnkman et al., 1967). Hepatic carboxyleslcrascs were selected as examples of enzymes localized predominantly in the endoplasmic reticulum though a small quantity of cytoplasmic enzyme may be present due to a possible solubilization of the membrane-bound enzyme (Schwerk and Ecobichon, 1968; Ljungquist and Augustinsson, 1971; Chow and Ecobichon, 1973). The enzyme which catalyzes the conjugation of BSP with GSH is a soluble cytoplasmic enzyme (Combes and Stakelum, 1961). The administration of such well-known inducing agents as phenobarbit.nl and DDT results in markedly enhanced activities of each of these enzymes (Conney, 1967; Schwark and Ecobichon, 1968; Johnstone etai., 1974). As was shown (Table 1), biphenyl significantly increased the activity of BSP-GSH conjugating enzyme. Compared to the enzyme activity in biphenyl-treated animals, there were no statistically significant (p > 0.05) differences observed following treat ment with monoelilorobiphcnyls but, with the exception of 2,4'- and 3,3'-di-, 2,5,2',5'-, and 2,6,2',G'-tctra-, and 2,4,6,2',4',G'-hcxachlorobiphcnyls, all of the chlorobiphenyl I PIIK1! ICH AND Ht-PATIC FUNCTION 103 presence of a different hydroxylation mechanism for highly chlorinated agcnls having ikolnlcd unsubstituted positions. The rate of hydroxyla( ion for the latter is much lower tlimt (hat observed for chlorobiphcnyls with unsubstituted vicinal carbons. When 2,4,5,2',4',5'*licxHchlorobiphcnyl was given to rats it was slowly converted and excreted as a inonoltydroxy derivative, substituted at either the 3- or 6-position. The exact structure awaits confirmation. The inability to rapidly hydroxylate low chlorinecontaining agents chlorinated in the 4- and/or 4'-positions and those highly chlorinated agents with isolated unsubstituted positions would contribute markedly to their persistence in animals and result in more marked induction of hepatic enzymes and other toxic effects. These pieces of evidence are supported by the hepatic enzyme induction studies herein, the most marked induction being observed with chlorobiplicnyls substituted in the 4- and/or 4'-positions. To conclude by answering the questions raised earlier, it would appear that for enzymes closely associated with the hepatic endoplasmic reticulum, the induction of enzyme activity was linked not to the biphenyl nucleus but to the position of chlorine substitution on the biphenyl structure. Isomers of the same degree of chlorination did not produce the same toxic effects, and dichlorobiphenyls may be quite similar to hexachlorobiphenyls depending on whether chlorine substitution occurs at the critical 4- and 4'-positions, thereby interfering with biolransformation. For enzymes less discretely localized in the hepatocyte (i.e., carboxylesterases and the BSP-GSH conjugating enzyme), the position of the chlorine atoms appears of less importance; enzyme induction was stimulated by biphenyl though enhanced effects were observed when one or more chlorine atoms were present. ACKNOWLEDGMENTS This tludy was supported by National Health Grant 602-7-162 made by the Department of National Health and Welfare of Canada. The technical assistance of Miss P. II. Cameron ia giatefully acknowledged. REFERENCES Baiu v, S. and Runyan, P. J. (1972). Interpretation of persistence and effects of polychlori nated biphenyls in birds Nature (London) 236,34 36. lliCKMts, D. R., Harm*, L. C., Kappas, A., and Alvares A. P. (1972). Polychlorinated biphenyls: comparative effects of high and low chlorine containing Arodore on hepatic mixed function oxidase Res, Common. Chan. Pathol. Pharmacol. 3,505-512. llutcx, W. D. and Cornish, II. H. (1959) Metabolism of biphenyl and 4-chlorobiphenyl in tlsc rabbit. J Biol. Clwm. 234, 3301-3302. UnwKNPtt, J. V., Kiianna, K. L. and Cornish, H. H. (1973). Biological responses of the rat to polychlorinated biphenyls. Toxicol. Appt. Pharmacol. 24,434-448. Him*, )i., Tumasoms, (.'. J'. and Uakfr, F. D. (1974). Toxicity and persistence of PCB homo logs and isomers in iIk avian system. Arch. Environ. Contant. Toxicol. 2,195-212. C`m:N, P, R., Mi HI.KDAI.P, H. M., ANt> FisunuN L. (1973). Effect of two isomeric letrachlorobiphcnyls on ruts and tltcir hepatic enzymes. Arch. Environ. Contain. Toxicol. 1, 36-47. C'uow, A. Y. K. and lIcomcnON, D. J. (1973). Characterization of the esterases of guinea pig livci and kidney. Biochcni Phurtnocol. 22, 689-701. Comill's, It. and Siakii.um, G. S. (1961). A liver cn/.ynK that conjugates sulfobromophthalcin sodium withglutmhionc. J. Clin. Inoexi. 40,981-988. PIIK1! ICH AND Ht-PATIC FUNCTION 103 presence of a different hydroxylation mechanism for highly chlorinated agcnls having ikolnlcd unsubstituted positions. The rate of hydroxyla( ion for the latter is much lower tlimt (hat observed for chlorobiphcnyls with unsubstituted vicinal carbons. When 2,4,5,2',4',5'*licxHchlorobiphcnyl was given to rats it was slowly converted and excreted as a inonoltydroxy derivative, substituted at either the 3- or 6-position. The exact structure awaits confirmation. The inability to rapidly hydroxylate low chlorinecontaining agents chlorinated in the 4- and/or 4'-positions and those highly chlorinated agents with isolated unsubstituted positions would contribute markedly to their persistence in animals and result in more marked induction of hepatic enzymes and other toxic effects. These pieces of evidence are supported by the hepatic enzyme induction studies herein, the most marked induction being observed with chlorobiplicnyls substituted in the 4- and/or 4'-positions. To conclude by answering the questions raised earlier, it would appear that for enzymes closely associated with the hepatic endoplasmic reticulum, the induction of enzyme activity was linked not to the biphenyl nucleus but to the position of chlorine substitution on the biphenyl structure. Isomers of the same degree of chlorination did not produce the same toxic effects, and dichlorobiphenyls may be quite similar to hexachlorobiphenyls depending on whether chlorine substitution occurs at the critical 4- and 4'-positions, thereby interfering with biolransformation. For enzymes less discretely localized in the hepatocyte (i.e., carboxylesterases and the BSP-GSH conjugating enzyme), the position of the chlorine atoms appears of less importance; enzyme induction was stimulated by biphenyl though enhanced effects were observed when one or more chlorine atoms were present. 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Comparative toxicity study of 2,4,5,2\4',5'- hcxachloiobiplvcnyl and a polychlorinated biphenyl mixture in rabbits. Toxicol. Appl. Pharnmcol. 23, 563- 578. Wins, R. G. and McCall, A. C. (1972). Identities of polychlorinated biphenyl isomers in Aroclors. J. An. OJJic. Anal. Chem. 55, 746-752.