Document pp2yXvbM8de9GdJn3gnGnaY37

('hem. /Unt Interactions, 1 8 (1 977 ) 7 2f> I (o Klsrvicr/North-Hollaiul Scientific Publishers, L('J 247 STIMULATION OF HEPATIC DRUG HYDROXYLATION AND CONJUGATION by a cutaneously applied PCB-MIXTURE (CLOPIIEN A 50) JUKKA MAKN1KMI*, MARKKU NOKKALAb, HARRI VAIN10C, and KAARLO J.W. UAR'JIAUA1'. *The Rehabilitation examinations Centre of The Social Insurance Institution, 20720 Turku 72. hItepartment of Thystology, Vniuersity of Turku. 20520 Turku 52 and c Department of Industrial Hygiene and 'Toxicology. Institute of Occupational Health. 00200 Helsinki 20 (Tmland) < Krrcivrd .January 81s(, 1977) (itevision r. coived May 18th, 1977) (Arrrpl.rrl May 7filh, 1977 ) Introduction The polychlorinated biphenyls (PCBs) arc widely used compounds |1,2|. Because of their widespread use and long half-life, the environment has become contaminated with PCBs [3], They have been found in tissues of many birds and fish 14,5), human milk |6J and fat |7). PCBs are known to be strong inducers of both the hepatic microsomal monooxygenase |8--10) and UDP glucuronosyltransferase [11J. PCBcompounds have been reported to differ catalytically as inducers of drug hydroxylation from the two earlier known inducer types, namely those of phenobnrbital and 3-methylcholanthrenc |12J exhibiting properties of both of them. It. \s also known that components of PCB-mixturcs like those of Aroelor 1254 are rapidly absorbed through the skin, leading to enhancement of hepatic cytochrome P-4 50 and drug hydroxylation reactions after cutaneous administration [13). The present study was carried out to investigate the effects of small amounts of a commercial PCB-mixture (Clophen A 50) administered via the skin, on epoxide hydra.se and glucuronide and glutathione conjugation of the liver, compared to effects of monooxygenase. Materials and methods Adult male random-bred Wistar rats (weight about 250 g) were used. Clophen A 50 (507<, chlorinated, Oy Ruomen Bayer, Helsinki, obtained as a Abhvcvialions: I'UR. polychlorinated biphenyls. OCT 3 1 * 0SW 031564 STLCOPCB4015526 gift from the State College of Veterinary Medicine, Finland) in acetone (200 or 400 gl/kg) was applied on the shaved nuchal region of the rat skin (4 or 8 mg/kg) once daily for 3 or 6 consecutive days. Corn oil and dimethyl sulfoxide were also tested as solvents but the results were similar to those obtained with acetone. The rats were killed 24 h after the last administration and the microsomes of the liver were prepared by the CaCl^aggregation method as earlier described [14]. The microsomes were treated in vitro witli digitonin and trypsin as described by Hanninen [15] and Mamiemi [16], respectively. Enzyme assays. The aryl hydrocarbon hydroxylase activity in hepatic microsomes was determined fluorimetrically was 3,4-benzpyrcne as sub strate 117,18] and p-nitroanisole o-demethylase was measured spcctrophotomotrically |19] by quantitating the formation of p-nitrophenol [18,20]. NADP11 cytochrome c reductase and the cytochrome P-450 content were determined according to Phillips and Langdon [21] and Omura and Sato [ 22], respectively. UDP Glucuronosyitransferase activity was assayed with p-nitrophenol as the aglycone by following spectrophotometrically its disappearance (1 5,23]. The supernatant enzyme, glutathione S-transferase, measured from CaCl2supernatant. (24] and microsomal epoxide hydrase [25,20] were both determined radiochemically using tritium-labeled styrene oxide as substrate. Results and discussion Cutaneously applied small doses of Clophen A 50 appeared to enhance drug hydroxylation enzymes of the liver. Both the component enzymes (NAI)PH cytochrome e reductase and cytochrome P-450) and total hydroxylation reactions (p-nitroanisole o-demethylase, aryl hydrocarbon hyd roxylase) were increased (50--80%) already after 3 days application. Six days administration still increased the enzyme activities, the highest, enhancements (dose 8 mg/kg) being obtained in p-nitroanisole o-demethylase activity and cytochrome P-450 content (220 and 170%, respectively). These results are in accordance with those produced with Aroclor 1254 (13) when a dose of 25 mg/kg was given cutaneously during 6 days. According to the present study as small a dose as 4 mg/kg of Clophen A 50 administered cutaneously for (5 (lays is enough to cause an observable enhancement of drug hydroxyla tion in the rat liver. Also the epoxide transforming enzymes, epoxide hydrase and glutathione S-transferase, were increased with these small doses of the PCB-mixture when administered via the skin (Table I). The highest enhancement (about 170%) was found in epoxide hydrase activity after 6 days application of 8 mg/kg of Clophen A 50. Glutathione S-transferase enhanced (52%) already after three days administration (8 mg/kg) and after 0 days the enhancement was of about the same magnitude. Also the lower PCB-dosc (1 mg/kg) increased the activity of both enzymes timing six days. Furthermore the enzyme catalyzing glueuronide conjugation, microsomal UDPglucuronosyllransferase was shown to he elevated (Table 1). After a DSW 031565 TABLE I EFFECT OF CUTANEOUSLY APPLIED CLOPHEN A 50 ON HEPATIC EPOXIDE I1YDRASE. GLUTATHIONE S TRANSFERASE AND UDPGLUCURONOSYLTRANSFERASE ACTIVITIES Activities were measured after 3 or 6 days administration. Results are means from 4--5 animals. The statistical significance of the differences (Student's /-test) has been denoted as follows: (X), 2P < 0.05;(XX), 2P < 0.01. Glutathione S-transferase (pmnlc/h trig prol.) Epoxide hydrase (nmole/h > nig prot.) UDP Glucuronosyltransferase Non-activated microsomes (nmole/h/mg prot. Digitonin-treated microsomes 3 days Control Clophen A 50 3.91 i 0.58 5.95 i 0.53 (X) 280 i 24 370 51 16.5 i 2.2 26.4 i 3.3 (X) 263 44 343 34 6 days Control Cluphcn A 50 (4 mg/kg) Clophen A 50 (8 mg/kg) 4.61 .- 0.35 5.80 0.51 G.5G i 0.G4 (X) 214 i 27 559 56(XX) 573 i 85(XX) 12.2 * 1.8 23.0 2.4 (X) 25.9 0.6 207 : 33 340 7-i 4 02 -. 70 (X) DSW 031566 STLCOPCB4015528 250 tliruu-day application (he increment was highest when tin' enzyme activity was measured from the native, non-aetivated microsomes (60%) hut also both in digitonin and trypsin treated microsomes slightly increased activities were' observed. After six days two- (native and digilonin treated microsomes) to threefold (trypsin treated microsomes, not shown in the table) activities were? to be seen when compared to the control level. The enhanced activities could be seen also with the smaller dose (4 mg/lcg). The data obtained indicate that even culaneously applied small doses of PCB-mixturos easily reach the liver at concentrations high enough to induce besides the monooxygation step also the subsequent epoxide hydration and glucwronide and glutathione conjugation steps of drug metabolism. In a recent study we have shown that the epoxide inactivating enzymes, gluta thione S-transferase and epoxide hydratase of the rat liver could be doubled after a single intraporitoneal dose (100 mg/kg) of Clophen A-50 |27], The present and earlier [13,28] results on the effects of culaneously applied I'CBs and those on the percutaneous penetration |2;>[ of some pesticides and herbicides stress the importance of the skin as a route to the organism of many harmful pollutants. Fortunately, this tissue has shown also a high degree of adaptation in its ability to metabolize these compounds to less harmful and more water soluble derivatives [13,28J. Supported by Grant No. Nil! AM 06018 and Juho Vainio Foundation, Finland. I J. 1'ichirallo, tweaks oT toxic substances raises issues of effects, regulation, .Science, 173 (1971 ) H(H1 Y. Masuria, It. Kagawa and M. Kuratsune, Polychlorinated biphenyls in carbonless car him paper, Nature (London), 237(1972)41. S. Jensen, Report of a now chemical hazard, New 32 (1900) Gl 2. It W. HiKchmugh, I*. Reiche, D.B. PeakaJI, S.G. Merman and M.N. Kirven, Poly chlorinated biphenyls in the global ecosystem, Nature (London), 220 (1963) 1098 J.M. Kocman, M.O.T.N. He Brauw and T.II. dr Vos, Chlorinated biphenyls in fish, mussels and birds from the river Rhine and the Netherlands coastal area, Nature (London). 221 (19G9) 1 126. L. Acker and E. Schulte, Uhrr das Vorkommen von chlorierten Biphrnylen und Hexachl0rheu7.nl neben cblorierten Insek 1 iziden in Humanmileh und menschlichem Kellgrwebc, Nalurwissenschaflen, 67 (1970) 497. F.J Biros, A.G. Walker and A. Medhery, Polychlorinated biphenyls in human adipose tissue. Bull. Environ. 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