Document evZ52jdBzMrLQjDGadGpq36nE

Voi. 14. No. 5 cliunis-s). 197?, ger, New irmoenJ Exp. 1. (I960. Clin, . Path, and 13-31*7 (1970). 13? (397*). 03. 110 2nd Edition idles, y, Life Sciences Vol. 14, pp. 853-860, 1974. Printed in Great Britain Perg&mon I'tiss INDUCTION OF DRUG-METABOLIZING ENZYMES AND ARYL HYDROCARBON HYDROXYLASE BY MICROSCOPE 1M4ERSION OIL Alvito P. Alvares1, David R. Bickers and Attallah Kappas The Rockefeller University, New York 10021 and Columbia University College of Physicians and Surgeons New York 10032 (Received is final Ions 14 January 1974) Summary Microscope immersion oil when adainistered intraperitoneally or applied to skin in experimental animals substantially Increased llvor woight, Microsomal protein, NADPH-cytochroms c reductase ac tivity, cytochrome P-450 content end the metabolism of the model substrates, cthylmorphlna end benzo(a)pyrene. Immersion oil caused the induction of the polycyclic hydrocarbon type of hemoprotoin, cytochroue P-448, When applied to skin, the oil also caused an 11-fold increase in bento(a)pyrene hydroxylase activity at the skin sites. The duration and intensity of action of many hormones, drugs, carcinogens and other environmental chemicals depend to e large extent on the rates at which they are metabolized by the cytochrome P-4S0 containing mixed-function oxidase system in liver cells. In this enzyme complex reducing equivalents from NADPH flow via a flavin enzyme, known as NADPH-cytechromc c reductase to the iron moiety of the hemoprotein, cytochrome P-4S0. Cytochrome P-450 and certain drug-metabolizing enzyme activities coupled to the mixed-function oxi dase system, are readily Inducible by drugs such as barbiturates, and aryl hy drocarbons, such as 3-othylcholanthrene and benzo(a)pyrene (1). The poly chlorinated biphenyls (PCBs), which have recently been recognized to bo wide spread and persistent environmental contaminants (2,3), have been shown in our previous studies to comprise a new and potent category of Inducers of Recipient of Irma T. Hirschl Career Scientist Award 853 HUNS 081804 J. 854 Induction of Drug Metabolism by Microscope Immersion Oil Vol. 14, No. S ruicrosomtl drug-metabolizing enzyme activities, having characteristics of botli the drug and tho carcinogen types of inducing agents (4). In a recont report nine samples of widely used microscope iwaorsinn oil marketed in Europe, North America and Japan were found to contain 30-45 per cent PCBs (5). Since these immersion oils frequently come in contact with skin, it was of interest to study their possible inducing effects on the he patic mixed-function oxidase system following parenteral and percutaneous administration of the oils in animals. Inducing affacts of immersion oil would be of particular importance since a study carried out in Japan showed that in subjects handling carbonless copy paper, which contained PCBs, only e third of tho PCBs which adhered to the fingers could be removed by ordinaiy handwashing with soap and water (6). METHODS Male Sprague-Dawley rats were used. Immersion oil, type A, containing 30 percent PCBs, purchased from R. P. Cargille Laboratories was diluted with mineral oil and administered i.p. at a dosaga of 10ul of immersion oil per 100 g body weight per day for 6 days. For the percutaneous experiments, !Chl of iwicrsion oil per 100 g body weight was applied daily for 6 days to a 3 x 3 cm area of the shaved nuchal region of the rets. Controls received mineral oil only. On day 7, the animals were killed and washed hepatic microsomc* wore prepared. Cytochrome P-4S0 content (7), ethyl isocyanide dif ference spectre (B), HADPH-cytoehrome c Teductase (9) and ethylmorphine Ndemothylaso (10) activities were determined as described previously. Hepatic aryl hydrocarbon (benio(a)pyrene) hydroxylase activity was determined on the post-mitochondrial fraction using a fluoroaetrlc assay (11). To dotermine the benzo(a)pyrene hydroxylase activity in skin, the skin sites to which im mersion oil or mineral oil had been applied were excised and homogenised as reported earlier (12). The whole skin homogenate thus prepared was diluted to 1Q\ with 0.01 M phosphate buffer, pH 7.4 and D.S ml, containing 7-3 mg Vol. H. No. 5 protein. a<i ' Inducer' ' have been cetei which phenobart of substrates methylcholanth notarbltal adr chroma P-450 othylmorphina nethylcholant' tent but eaus induce a spet p-441 (13-1?) tion of VOvl hydroxylase cent, micros activity by fold inctca? Tate of met; These data of both the Whan e hemoprotcit carbons, s< the 4SV41 untreated Table 1. > in increa' VoJ. 14, fio, 5 tic* of 'ersion oil 50-45 porCt with on the hotaneous ion oi1 v> showed #*, only >' ordin- iiainlng tod with i 1 per nit, days to calved :lc ai de difna NHrpatlc on tha mi ne ieh in. d at luted Vol. 14, No. 5 Induction of Drug t'eiabolisrc by K'icrosc:?' Innctsion Oil 855 protein, was assayed for benzo(s}pyrer.e hydroxylase activi:;* (11). RESULTS ASb DISCUSSION Inducers of cytochrome P-4S0 and drug-retaboliiiri enrynes in the livor have been categorited into two rain groups of compounds (1)- One group, of which phonobarbital is a prototype, enhances the metabslisn of a large veriaty of substrates by liver cells; a second group typified by the carcinogen, 3cthylcholanthrene, stimulates the metabolism of only a few substances. Phenobarbital administration increases liver weight, microsomal protein, cyto chrome P-450 concentration and enhances NADPH-cytoehrcse c reductase and ethyisaorphine K-deaethylase activities. Polycyclic hydrocarbons, such as 3methylcholanthrene, do not induce the above parar.eters to try significant ex tent but cause e marked enhancement of ben:o(a)pyrene hydroxylase; they also Induce a spectrally and catalytically distinct liver hemoprrtcin, cytochrome P-448 (13-15). As shown in Table 1, skin application or parenteral administra tion of lOul of iaiaersion oil, type A, to rats not onl.' induced benzo(a)pyrono hydroxylaso activity ten-fold but also increased liver weight about 40 per cent, microsomal protain about 20-40 percent and VADI'I.'-eytcchrome c reductase activity by 40 percent. In addition, the immersion oil elso caused a 3-4 fold increase in cytochrome P-450 concentration and a 2-3 fold increase in the rate of metabolism of the model drug substrate, ethylmorphine (Table 1). These data show that iimaersion oil, like the PCBs (4), shares tho properties of both tho cercinogen end the drug types of Inducer compounds. When ethyl isocyanide is used is the ligand for the reduced microsomal heaoprotcJn, spectral peaks at 430 and 455 nm are observed. Polycyclic hydro carbons, such as S-rrethyleholanthrcne, causa a marked increase In tho ratio of the 455:430 na peaks when compared to tho ratio obtained with microsomes fro* untreated rats or rats pretreated with phenobnrbital (13,14). As shown in Table 1, imr.orsion oil resombles the polycyelic hydrocarbon class of inducors in increasing tho 455:430 ratio from 0.64 to about 1.25. huns oaiaoa 856 Induction of Drag Metabolism by Microscope Immersion Oil Vol. 14, No. 5 TABLE 1 Effect of skin application or i.p. sdninistretion of Microscopo Inversion Oil on Hepatic Cytochrome P-4S0 and Drug-metabolizing Enzyme* Skin Application Intreperitoneal injection Measurement liver wt., g/g body wt. Mineral Immersion oil oil 0.04S1 to.001 0.06S2 10.001 Mineral oil Immersion oil 0.042 10.007 0.0612 10.007 Microsomal protein. mg/g liver wet wt. Cytochrome P-450, nmol/mg protein Z1.60 to. 60 0.62S 10.033 50. M2 12.06 2.5172 10.140 23.92 11.20 0.692 10.014 29.12* 11.70 2.27J2 10.089 Ethyl isocyanide differeuce spectra, ratio of 455:450 peaks 0.64 10.01 1.30* 10.06 0.64 10.02 1.242 10.02 NAOPH-cyt. c reductase, nmol cyt. c reduced/mg protein/win. 72.S6 12.78 102.02 13.02 73.36 12.47 100.602 15.75 Ethylmorphina N-damethylase wmol HCHO/mg protein/hr 0.350 10. 029 0.940* 10.046 0.398 10.007 1.1222 10.04$ B*nio(a)pyrene hydroxylase nmol OIIBP/mg protein/hr. 2.284 10.107 25. MO2 11.019 2.462 10.141 12.2m7 10.514 1 Bach value represents mean t SB for 5 rats. 2 Values significantly different from the respective control values (P< 0.0S) Vol. H. f The rots tre untreat ever, f in the hemoprt A simi treate' these cyclic MQNS 081807 !. 14, No. 5 morsion njection rsion II 361 2 '07 22 0 71 2 Vol. 14. No. 5 Induction of Drug Metabolism by Microscope Immersion Oil 857 The CO-diffarence spectrs of liver aicrosomes froa untreated rats and rats treated with immersion oil are shown In fig. 1. Liver aicrosoncs froa l i; FIG. 1. CO-dIfference spectra of liver microsomes froa untreated rats and rats treated with immersion oil. untreated rats showed an absorption maximum at 450 no, as expected (7), How ever, following percutaneous or i.p. adainistration of isimersion oil, a shift in the absorption aaxinun occurred; the highly induced microsomal CO-binding heaoprotcin now exhibiting an absorption maximum at 448.5 nm instead of 450 na. A similar change in spectral properties of the hemoprotein is observed in rats treated with J-mcthylcholanthrene (14) as well as with the PCBs (4). Thus these studies show that immersion oil also shares the properties of the poly cyclic hydrocarbons in causing a change in the spectrel properties of the in- !t`'X lU-Y MOMS 08180a H."W V.*g ' fy'*' if t I :*!# -'-** 5T 'V'V 1 >,A> Ahiy<r *-*-* * ft58 Induction o( Utug Metabolism by Microscope Immersion OH doced liver hemoprotcin. Since skin is a point of contact with pany workers handling iaaersion oil In microscopy, the effect of the oil on bento(a)pyrene hydroxylase acti vity in skin was detenained following cutaneous application of 10 wl of the oil per 100 g body weight. As shown in fig. 2, insertion oil caused an Conlrot G3 Immersion o *.200 * 'so aT FIG. 2 Induction of skin benzo(a)pyrene hydroxylase activity following skin application of iseperslon oil. Each valua represents naan and S.E. of 5 rats. 11-fold induction of the hydroxylase in skin homogenate. The eryl hydrocar bon hydroxylase system transforms carcinogenic polycyclic hydrocarbons, such as bento(a)pyrene or J-nethylcholanthrene into Petabolites with altered car- als ia* bar as be ato the of t woul of f den< aHar pice tech tori' of tt MQNS 081809 m'9" Vol. H, NO. 5 immersion . leie ecti* ul of the Red on hydrocar* on*, tuch red car* VoJ. 14, No. 5 Induction of Dreg Metabolism by Microscope immersion Oil 859 cinogenlc properties. Recent evidence suggests that the induction of the hy droxylase system may be disadvantageous, in that intermediates, such as epox ides, derived from polycyclic hydrocarbons may be more active in the produc tion of malignant transformation in rodant cell culture than the hydrocarbons or their corresponding phenolic metabolites (16). In addition to other constituents and mineral oil, immersion ell as no ted, contains high concentrations of PCBs (S). Tha inducing properties of tho immersion oil are east probably due to the PCBs which they contain. The possibility does exist, however, that other components of iamersion oil may also have inductive properties for tha mixed-function oxideee system. Since immersion oil possesses the inducing properties of barbiturates and since barbiturates have bean found to atimulatt tha metabolism in man of drugs such as dlphcnylhydantoin, biahydroxycouaarin and phonylbutasono (17,18), it would be of interest to determine if individuals handling immersion oil in the labor atory or in tha factory have the capacity to motdbolise drugs more rapidly than the generel population. Moreover because Immersion oil also possesses certain of tha Inducing characteristics of the polycyclic hydrocarbon carcinogens, it would also be of great importance to examine tha carcinogenic potentialities of such oils. This question is of special significance in view of recent evi dence that the PCBs cause fatty degeneration and tha appearance of multiple adenomatous nodules in tho livers of rats (19), hepstomas in tho livers of mice (20) and neoplastic-Ube changes In the gastric mucosa of monkeys (21). ACKNOWLEDGEMENTS Supported In part by USPHS grant BS-00621. We thank Julie Eiseman for technical assistance. We thank John J. Cargill# of R. P. CargiLle Labora tories for his cooperation in supplying the information on the composition f the lmeersion oils. REFERENCES I. A. H. CONWY, Pharmacol. Rev. 19, 317-366 (1967). 7. A. L. HMMOND, Science 17S. 1SS-156 (1072). MONS 081810 -^yv I WO Induction of Drug Metabolism by Microscope Immersion Oil Vol. 14, No. 5 3. I). S. PEAKALL, Residue Kev. 44, 1-21 (1972). 4. A. p. ALVAKES, D. R. BICKERS and A. KAPPAS, Proc. Netl. Acad. Sci. 70, 1321-132S IWJ). ~~ 5. H. S. BENNETT and P. N. ALBRO, Science 1B1, 990 (1973). 6. M. KURATSUNfi and Y. HASt/DA, Environ. Health Perspact. l_, 61-62 (1972). 7. T.OMURA and R. SATO, J. Bio). Cbaa. 239, 2370-2385 (1964). 6. A. P. ALVARES. C. SCHILLING, W. LEVIN and R. KUNTZMAN, J. Pharmacol. Bap. Thor. 163, 417-424 (1968). 9. P. HAZEL In Fund--antal* of Drug Metabolism and Drug Disposition B. N. La Du, H. C. Handel and E. L. Nay, ods. p. 546-582. Willis-- and Wilkins, Baltimore, Md. (1971). 10. A. P. AtVARBS and C. J. MANNERING, Mol. Pharmacol. 206-212 (1970). 11. D. W. NEBERT and H. V. GELB01N, J. Biol. Cham. ill. 6242-6249 (1966). 12. D. ft. BICKERS, A. KAPPAS and A. P. ALVARBS, J. Pharmacol. Exp. Thar. In prass (1974). 13. N. 6. SIADEK and G. J. MAHKBRING, Biochan. Biophys. Rea. Co--un. 24, 661-674 (1966). 14. A. P. ALVARBS, G. R. SCHILLING, W. LEVIN and R. KUNTZMAN, Blochea. Blophyf. Ros. CO--un. 29, S21-S26 (1967). 15. A. P. ALVARBS, C. R. SCHILLING and R. KUNTZMAN, Biochem. Biophys. Rea. Co--un. SO, SB8-593 (1968). 16. ? t. WOVE*, P. SIMS, E. HUBERMAN, H. HAROUARDT, T. XUROX1 and C- HB1DELBERGER, Proc. Natl. Acad. Sci. 8, 1098-1101 (1971). 17. S. A. CUCINBLL, A. H. C0NNBY, M. SANSUR and J. J. BURNS, Clin. Pharmacol. Ther. 6, 420-429 (1965). 18. A. J. LEVI. S. SHERLOCK and D. WALKER, Lancet 1, 127S-1279 (1968) 29. N. T. XINURA and T. BABA, Gann 64. 105-10$ (1973). 20. H. NAGASAKI, S. T0NI1, T. MEGA, M. MARUGANI and N. ITO, Gann, 63, SOS (1972). 21. J. ft- ALLEN and 0. H. NORBACK, Science 179, 49B-499 (1973). Life S I'untt Ins It of giv r--TT'-TTii if" "II |71i| l"`"IT""i'.r" I .......... . i, M HONS Q818U __________ >.