Document 3Zo7EyBJj906gqORajXDnB9x

Beitrage zur Okologischen Chemie LXXXJIP In Vitro Metabolism of Polychlorinated Biphenyls~>4C by V. CHE*', W. Klein,* F. Coulstoi*. L Colberc, and F. Konrc1 Inuiiui* of Experimental Pathology and Tosteology Albany Medical College, Albony,N.Y. 11208 `Chomitehot Inttitut dor UnivtnitMt Bonn S3 Bonn, W. Germany *Inttitut filr dkologueht Chemie dor CetelUchoft fUr Strahlon- ttnd Vmweltfortehung mbH, Miinchmn D3KS St. Augutlin J, w. Germany INTRODUCTION Commercial polychlorinated biphenyls (PCBs) are un suitable for metabolic studies as they are mixtures consisting of many Individual ingredients resulting from the method of manufacture. Toxicological testing of all the various chlorinated biphenyls is a formi dable task, and Initially we can only investigate cer tain representative compounds. At naturally occurring levels of exposure, the toxicity of PCBs to mammals is generally very low. Lower chlorinated biphenyls, however, are of special interest because their biolo gical degradability might lead to metabolites of in creased toxicity. Lower chlorinated biphenyls are ex creted very rapidly by the Rhesus monkey (GREB et al. 1973) and are converted to hydroxylated products (BLOCK and CORNISH 1959, HUTZINGER et al. 1972, YOSHIMURA and YAMAMOTO 1973) by animals. In chronic feeding studies the main changes were found in the li vers: hepatic porphyria in chickens and rabbits (VOS 1972) and proliferation of hepatic smooth endoplasmic reticulum in rats (NORBACK and ALLEN 1970), mice and monkeys (NISHIZUMI 1970, ALLEN and NORBACK 1973). Enzyme Induction has been reported for PCBs with high and low chlorine contents (LITTERST et al. 1972, BENTHE et al. 1972, BICKERS et al. 1972). As the hydroxylating enzymes are located in the smooth endoplasmic reticulum their metabolic activity might be indirectly responsible for the gross pathological changes observed. Therefore, we Investigated the effect of these enzymes on the PCBs by incubating lower chlorinated blphenyla with the microsomal fraction* of rat liver homogenate. Identifi cation of metabolites is an essential step in the evalua tion of safety of these compounds. LXXXII. Communication: GAB, S. et al.: Chemosphere 2* in press (1974). 424 ItillMli of CMOnl.illn I TulMlitr, vi. ia, n*. ms hr s,n.,.r.VwU n- Y**k MQNS 082052 EXPERIMENTAL The compounds studied were 2,2'-dichlorobiphenyl, 2,4' "dlchlorobipheny1, and 2,5,2'-trichlorobiphenyl. They were labelled with 4C and synthesized in the Ger man laboratory, except the last one, which was bou9ht from Mallinckrodt Chem. Corp. (Lot Nr. 4449a). Trying to gat a maximum conversion rate, adult female rats (Swiss Webster strain) weighing 200 - 300 g were trea ted with phenobarbltal daily,for 3 days (50 mg/kg i.p.) to increase mixed function oxidase activity. The animals were killed on the fourth day, the livers were removed, washed and homogenised, and centrifuged at 10,000 g. The supernatant was centrifuged at 100,000 g for 1 hour and the pellet resuspended in buffer (0.25 M sucrose in 0.01 M Tris-HCl, pH 7.4). The NADPH regenerating system and incubation medium consisted of 30 raM MgCl,, 0.1 ml IN nicotinamide, 0.05 ml 30 mM TPN, 150 mM glOcoee-6phosphate, 100 units glucose-6-phosphatedehydrogenase, and 1 ml 50 mM Tris-HCl (pH * S.6) per ml microsomes. biphenyl hydroxylase activity was measured by the method of CRAVEN et al. (1965) and protein content determined using bovine serum albumin as a standard (SUTHERLAND et al. 1949). The low degree of aqueous solubility of the PCBs presen ted a problem since many organic solvents are known to decrease microsomal enzyme activity. After testing the inhibitory effect of a series of solvents, dimethylsulfoxide (DMSO) was selected together with an emulsifier as vehicle. Although DMSO stimulates aniline metabo lism in rats in vivo (STOCK et al. 1969), it partly in hibits enzyme activity in vitro. The PCBs were admi nistered in 0.1 ml DMSO, 20 mg Tween 60, and 0.2 ml H-0 and incubated for 1 hour at 37C. The reaction was stopped by adding 2 N HC1. The acidified mixture was extracted with 2 x 20 ml hexane and 3 x 10 ml chloro form/methanol (3:1) so tha.t 99 % of the radioactivity could be recovered. After concentration, the compounds were purified by preparative layer chromatography on silica gel (Merck Nr. 5765, 5766, solvent: dlchloromethane). Radioactivity was determined by the "scraping technique" and liquid scintillation counting (Packard Model 3380) or autoradiography (Kodak X-ray film) (CECIL et al. 1966). Compounds were methylated by dlazonethane In ether overnight, GC carried out on Packard model 873 (EC-detector, 2 m. It OV-1) and fi nal analysis performed with a GC-MS- combination (LXB 9000 A). Experimental details are provided in Table 1. 42S 'i i HONS 082053 TABLE 1 Experimental Data on PCB Application to Rat Liver Homo genate dose tyi moles) dose <yuCl) g liver/ml homogenate mg proteln/ml enzyme activity (% 4-OH-BP formed from 1.2 ^Mmoles biphenyl /ml) vol. of mlcrosome sus pension (ml) level of PCB used, dose (^moles/x&l) conversion in % 2,2*-Di 2,4 '-Di- 2,5,2' -Tri- chlorobi chlorobl -chlorobl- phenyl phenyl phenyl 3.97 1.02 1.25 14.8 16.3 1.51 7.72 1,12 13.4 12.6 1.43 14.25 1.12 13.4 12.6 6.0 0.66 10.0 12.0 0.12 35.8 6.0 0.23 31.0 RESULTS AND DISCUSSION The rate of conversion of the PCBs varied between 10 and 95.8 I on the basis of their concentrations in^molae per ml incubation medium. Smaller concentrations increa sed the yield of metabolites. The difference in yield between doses of 0.23 and 0.12/Mmoles/inl, however, was very small (31 t and 35.8 %, respectively). Determining the enzyme activity, 16.3 % 4-hydroxybiphenyl were for med from 1.2/tmolee/ml biphenyl, while only 10 % of 0.66y,moles/Tnl 2, 2*-dichlorobiphenyl were converted. Although less concentrated, the PCB was mstabollzed more slowly than the biphenyl. Radioactive extracts of the incubation mixtures showed thin layer chromatograms similar to each other: a main peak comsponding to un changed PCB, two smaller peaks corresponding to monoand dihydroxymetabolitea, and a small fraction at the origin which was not identified. 426 MONS 08*05*. Table 2 shows GLC - and MS-data of 2.2 *-dlchloroblphenyl and Its methylated metabolites. Four monohydroxy derivatives (R.-R.) were detected and characterized by GLC/MS. Although four metabolites were detected on TLC, matching dihydroxy derivatives (R.-Rg), only one of them (R) occurred in amounts sufficient for methy- lation, GLC and MS. Table 3 gives GLC - and MS-data of 2,4*-dlchloroblphenvl and Its metabolites. Two monohydroxy derivatives - U5) and two dihydroxy derivatives (U, - 01) were isolated and characterized by GLC/MS. Zn Table 4, figures are listed related to GLC and MS of 2,5,2*-trlchloroblphenyl and its metabolites, three mo- noSydroxy derivatives (L^ and two dihydroxy deri- vatIves All identified metabolites contained one or two hydroxyl groups In the PCB-molecule. Primarily monohydroxy pro ducts were formed. While we found all possible monohydroxy-dichloroiaomers of the 2,2'-dichloroblphenyl, only two of the maximum six 2,4'-dichlorobiphenyl-monohydroxy-metabolites and three of seven of the 2,5,2*-trichloroblpheny1-monohydroxy-metabolites were formed. Although the first experiment showed that all different positions in the biphenyl-molecule could be hydroxylated, the chlorine substituent might heve a di recting Influence on the position hydroxylated by the entymee. BOme isomers were more likely to be formed than others. Biotransformation of the PCBs in vitro proceeded to metabolites containing up to two hydroxyl groups per molecule. Fig. 1 gives a survey of all metabolites Identified in this study. The high yields end variety of products formed in these experiments demonstrate that lower chlorinated biphenyls ere readily metabolized by hepatic enzymes, probably mixed function oxidases, of rats. This Investigation of the metabolism of three individual PCBs also provides some indication of their general bio degradability. Thus it is likely that at least one com mercial mixture of lower chlorinated biphenyls, Aroclor 1221, contains mostly ingredients of low persiatence. On the basis of the composition of Aroclor 1221 reported by WILLIS end ADDISON 1972, e flowsheet of metabolic con versions (Fig. 2) has been drawn up to summarize our present Information. TABLE 2 6LC - and MS-Data of 2,2'-Dichlorobiphenyl and its Methylated Metabolites Data glc Rfc (nin. ) HS-fragment H* M+-CH3 " M+-C1 2,2'-t>CB .0/185C 222 s - 187 s OH-Metabolite "i 7.3/I85C OH-Metabollta R2 OH-Metabolite R3 8.8/185C 10.8/185C OH-Metabollte R4 (OH)5-Meta bolite R5 7.3/188C 13.5/200C for 8 min for 8 min., then temp then temp, raised raised 5C/ 5C/ain. min. 252 s 252 s 252 8 252 s 282 s - 237 1 - 237 1 267 1 217 1 217 m 217 1 217 1 247 1 " metastable 158* - " m+-ch3-co - m+-ch3-ci - M+-C1j 152 1 - M+-C1-HC1 151 1 M+-CH,-COHC1 209 m 173 m 222-5 209 s 202 s 182 1 173 m 209 m 202 1 173 1 222T-5 209 s 202 m 182 m 173 239 B - Peak-Intensities: a " strong, m medium, 1 low MUNS 0 6 2 0 5 6 TABLE 3 and MS-Data of 2,4'-Dlchloroblphenyl and Its Metabolites Data 2,4'-DCB OH-Metabo- OH-Metabo- (OH) --Meta (OH)^-Meta lite 0, {not lite U, bolite U, bolite U. methylated) (methylated) (methylated) (methylated) GLC Rt (min.) MS-fracrment M+ M+-CHj M+-CO-H ' M+-C1 * M+-HC1 m+-co-ch3 3.6/185C 222 s - 203 a 202 s - 5.1/185C 238 s - 209 a 203 a 202 s - 9.5/188C for 8 min., then temp, raised 5c/ ain. 252 s - _ 217 1 _ 209 a 13.0/2COC for 8 min., then temp, raised 5C/ min. 282 s 267 1 247 1 _ - 239 m U.5/20OC for 8 min., then temp, raised 5C/ min. 282 8 - 247 1 - 209 m M+-Clj 152 s " M+-C1-HC1 151 m * H+-C12-BjO - M+-CO-CH,- - HC1 168 s 167 m 149 s - 173 1 - - _ - Peak-Intensitiest s strong, m - medium, 1 low SO?POi TABLE 4 GLC - and MS-Data of 2,5,2'-Trlchlorobiphenyl and its Metabolites Data GLC Rt (nin.) MS-fraqment M+ ' M+-CHj ' M+-C1 " m+-co-ch3 M+-CO-Cl K+-CH3-C1 M+-Clj M+-CO-CB3 M+-C13 2,5,2*-TCB OHHtetabo- OH-Metabo- OB-Metabo- (OH) ,-Meta-l (OH) ,-Me- lite l, (not lite L, lite L, bolite L. j tabof.L. methylated) (aethvfated) (aethvlated) (aethvlated) (methyl?) 5.1/18SC 8.9/18SC iI.9/162C for 9 ain., then team, raised 8C/ ain. 13.9/185C for 8 ain., then team, raised SC/ ain. 16.2/200C l.0/20OC for 8 ain.r for 8 ain., then team, then team, raised 5C/ raised 5C/ ain. ain. 256 s 272 s 286 s 286 s 316 a 316 s -- - 271 a 301 1 - 221 m - - 237 1 - 209 a - 251 1 243 a - 236 251 1 243 a - 236 1 281 a 273 a - - _ 273 a - - 186 s 202 216 1 216 1 - - - - 173 1 173 a - - 151 a - - - -- 9 5 0 7 9 0 SNOW Peak-Intensities: a- strong, a " aediua, 1 low