Document pmr9kVG8KV4rQdweO5G644vmE

4 Client. Ilutl Interactions, I tt (1977) `2-17 2f I *i KlM'vji'r/Norlli liullHixi Scientific Putilishcrs, LH 217 'OCT 3 1 STIMULATION OF HEPATIC DRUG HYUROXYLATION AND CONJUGATION BY A CUTANEOUSLY APPLIED PCB-MIXTURE (CLOPIIEN A 50) JUKKA MARNIKMI*, MARKKU NOKKALAb, HARRI VAINIO', nd KAAHLO J.W. IIAHTIALA1'. "77ir Itehahillloliioi t:\nmitwlions Centre of The Soeial Insitranrr Institution. 20720 Tltrhu 72. hllr[nirlinrnt of Thysiulogy, llniuersily of Turhu. 2Oh 20 Tnrlm 02 unit r Itriiortnient of hutlistnut Hygiene and Toxicology, Institute of Orcu/jalinnal Health 00290 llelsinlti 29 If inland) (Hereiveil .InmiJtry .'11 si, 1977) (itevisinn received May 1 Hitt, 1977) (Arrrplrrl May 1977) introduction The polychlorinated biphenyls (PCBs) are widely used compounds |1,2|. Because of Iheir widespread use and long half-life, the environment has liecome contaminated with PCBs |3). They have been found in tissues of many birds and fish (1,5), human milk 16] and fat 17 ]. PCBs ure known to bo strong inducers oT~both the hepatic mTcrosomnl monooxygonase 18--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 phcnohnrhitaJ and 3-mcthylcholanLhrenc 112) exhibiting properties of both of them. If is also known that components of PCB-mixtures like those of Aroelor 1254 are rapidly absorbed through the skin, leading to enhancement of hepatic cytochrome P-450 and drug hydroxylation reactions after cutaneous administration (13). 'Phe present study was carried out to investigate the effects of small amounts of a commercial PCB-mixturc (Clophcn A 50) administered via the skin, on epoxide hydrase and glucuronidc and glutathione conjugation of the liver, compared to effects of monooxygenasc. Materials and methods Adult mule random-bred Wistar rats (weight about 250 g) were used. Clophen A 50 (50% chlorinated, Oy Suomen Bayer, Helsinki, obtained as a Ahhveviiilinus: PCM, polyt'lilonmilrri biphenyls. MONS 001156 248 . gift from the State College of Veterinary Medicine, Finland) in acetone (200 or 400 jd/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 CaCIj'ageregation method as earlier described (14J. The microsomes were treated in vitro with digitonin and trypsin as described by Hanninen [15} and Mamicmi [1GJ, respectively. Knzytnc assays. The aryl hydrocarbon hydroxylase activity in hepatic microsomes was determined fluorimctrically was 3,4-benzpyrene as sub strnte 117.3 8J and p-nitroanisole o-demethylasc was measured speetrophotometrically (19) by quantitating the formation of/r-nitrophenol (18,201. NADIMl cytochrome c reductase and the cytochrome l*-450 content were determined Recording to Phillips and Langdon (21] and Omurn and Sato 122], respectively. U1)P Olucuronosyltransferase activity was assayed with p-nitrophenol as the aglyconc by following spcctrophotometricaily its disappearance 115,23 j The supernatant enzyme, glutathione S-transferase, measured from CaCl,supernatant (24| and microsomal epoxide hydrase [25,26] were both determined radiochcmically using tritium-labeled styrene oxide as substrate. Hcsulls and discussion Cutaneously applied small doses of Clophen A 50 ap)>cared to enhance drug hytiroxylnlion enzymes of the liver. Both the component enzymes (NADPH cytochrome c reductase and cytochrome P-450) and total hydroxyhition reactions (p-nilroanisole o-demcthylase. 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 an* 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 6 days is enough to cause an observable enhancement of drug hydroxylalion 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-1). The highest enhancement (about 170%) was found in epoxide hydrase activity after 6 days application of 8 tng/kg of Clophen A 50. Glutathione S-transferase enhanced (52%) already after three days administration (8 nig/kg) and after 6 days the enhancement was of about the same magnitude. Also the lower PCB-dose (I mg/kg) increased the activity of both enzymes during six days. Furthermore the enzyme catalyzing glueuronide conjugation, microsomal UDPglueuronosyllransferase was shown to be elevated (Table I). After a HONS 00113? TABLE I KKKKCT OF CUTANEOUSLY APPLIED CLOPHEN A 50 ON HEPATIC EPOXIDE HYDRASE, GLUTATHIONE S TRANSFERASE AND UDPCLUCURONOSYLTRANSFERASE ACTIVITIES Activities were measured aftsr 3 or days administration. Reculta are means from 4--5 animals. T>ie statistical significance of the differences (Student's /-test) has been denoted as follows: (X), 2P < 0.05; (XX), 2P < 0.01. Glutathione S-lransferase (^mole/h rrig prol.) Epoxide hydrase (nmole/h trig prot.) UDP Glucuronosyltransferase Non-activated microtomes (nmole/h/mg prol ) Digitonin-treated microsumes 3 days Control 3.91 l 0.58 Clophen A 50 5.95 t 0.53 (X) 280 i 24 370 51 16.5 i 2.2 26.4 < 3.3 (X) 263 : 44 343 34 6 days Control Clophen A 50 (4 mK/kK) Clophen A 50 (8 mg/hii) 4.61 ' 0.36 6.80 0.61 0.56 i 0.04 (X) 214 * 27 669 66 (XX) 573 i 85(XX) 12.2 j 1.8 23.0 - 2.4 (X) 25.9 0.6 207 33 340 74 402 . 70 (X) HONS 001138 250 throo-chiy application the increment was highest when the enzyme aelivily was measured from the native, noivae(ivaUd mierosomes (60%) hut also both in digilonin and trypsin treated mierosomes slightly increased activities wen* observed. After six days two- (native and digilonin treated mierosomes) to threefold (trypsin treated mierosomes, not shown in the table) activities were to be seen when compared to the control level. The enhanced activities could he seen also with the smaller dose (4 mg/kg). The data obtained indicate that even cutancously applied small doses of PCH-mixtures easily reach the liver at concentrations high enough to induce besides the monooxygalion step also the subsequent epoxide hydration and glncuronide and glutathione conjugation steps of drug metabolism. In a recent study we have shown that the epoxide inactivating enzymes, gluta thione S lransferase and epoxide hydratasc of the rat liver could be doubled after a single intraperitoneal dose (100 mg/kg) of Clophen Af>0 |27|. The present and earlier (13,28) results on the effects of cutancously applied I'CBs and those on the percutaneous penetration |2B| of some l>cstiridcs 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 iU ability to metabolize these compounds to less harmful and more water soluble derivatives (13,28). Supported by (Irani No. NIII AM 06018 and Juho Vainio Foundation, Finland. 1 J. Pirhirallo, I/rnks of toxic substances raises issues of effects, regulation. Science, I7S (1971 )H99. 2 Y. Mnsudn, It. Kngawa and M. Kurntsunc, Polychlorinated biphenyls in carbonless rarhon paper, Nature (London), 237 (I072) 4 1. 3 S. .Jensen, Iteporl of h new chemical hazard, New Sei., 32 (1006) 01 2. 4 It W. Hisebrough, P. Reiehe, IV B. Peakall, S.O. Herman and M.N Kirv.-n, Poly chlorinated biphenyls in the global ecosystem. Nature (Immlon), 220 (190H) 109H ft .1 II. Koeman, M.C.T.N. de Brauw and T.H. de Vos. (Chlorinated biphenyls in fish, mussels anil birds from the river Rhine and the Netherlands cnnst.il arm, Nature (London), 22! (1900) 1126 0 L. Acker and B. Schulte, liber das Vorkommen von chiorierten Uiphcnylrn tmd HeKarhlnrbenr.nl neben chiorierten Insekliziden in Humanmilrh und menschlichetn Fetlgewcbe, NaturwiKseuRchaflen, 07 (1970)497. 7 F.,1. lliros. A.C. Walker and A. 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Srnml., Suppl., 440 (1976) 133. 28 D.R. Bickers, J. Eiscman, A. Kappa* and A.P. Alvarcs, Microscope immersion oils; Effects of akin application on cutaneous and hepatic drug-metabolizing enzymes, Biochcm. Pharmacol., 24 (1975) 779. 29 K.J. Felrimnnn and If.l. Malbach, Percutaneous penetration of some pesticides and herbicides in man,Toxicol. Appl. Pharmacol., 28 (1974) 126. MONS 00U60