Biochem. J. (1984) 222, 737-748 737 Printed in Great Britain Inhibition of uroporphyrinogen decarboxylase by halogenated biphenyls in chick hepatocyte cultures Essential role for inducdon of cytochrome P-448 Peter R. SINCLAIR,*t§ William J. BEMENT,* Herbert L. BONKOVSKY,*j and Jacqueline F. SINCLAIR*t * VA Medical Center, White River Junction, VT 05001, U.S.A., and the Departments of fBiochemistry and tMedicine, Dartmouth Medical School, Hanover, NH 03756, U.S.A. (Received 16 April 1984/Accepted 23 May 1984) Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was assayed in cultures of chick-embryo hepatocytes by the changes in composition of porphyrins accumulated after addition of excess 5-aminolaevulinate. Control cells accumulated mainly protoporphyrin, whereas cells treated with 3,4,3',4'-tetrachlorobiphenyl or 2,4,5,3',4'- pentabromobiphenyl accumulated mainly uroporphyrin, indicating decreased activ- ity of the decarboxylase. 3-Methylcholanthrene and other polycyclic-hydrocarbon inducers of the P-448 isoenzyme of cytochrome P-450, did not affect the decarboxylase in the absence ofthe biphenyls. Induction ofP-448 was detected as an increase in ethoxyresorufin de-ethylase activity. Pretreatment of cells with methylcholanthrene decreased the time required for the halogenated biphenyls to inhibit the decarboxylase. The dose response of methylcholanthrene showed that less than 40% of the maximal induction of cytochrome P-448 was needed to produce the maximum biphenyl-mediated inhibition of the decarboxylase. In contrast, induction ofthe cytochrome P-450 isoenzyme by propylisopropylacetamide had no effect on the biphenyl-mediated decrease in decarboxylase activity. Use of inhibitors of the P-450 and P-448 isoenzymes (SKF-525A, piperonyl butoxide and ellipticine) supported the concept that only the P-448 isoenzyme is involved in the inhibition of the decarboxylase by the halogenated biphenyls. The effect of preinduction with methylcholanthrene to enhance inhibition ofthe decarboxylase was also shown by the increased rate at which porphyrin accumulated from endogenously synthesized 5- aminolaevulinate after treatment ofcells with the combination ofpropylisopropylace- tamide and the biphenyls. Antioxidants, chelators of iron, and chromate affected the decrease in decarboxylase activity only if they prevented the induced increase in cytochrome P-448. We conclude that the P-448 and not the P-450 isoenzyme of cytochrome P-450 plays an obligatory role in the inhibition of uroporphyrinogen decarboxylase caused by halogenated biphenyls. Uroporphyrinogen decarboxylase (EC 4.1.1.37) that catalyses the decarboxylation of the four is an enzyme of the haem biosynthetic pathway acetate side chains of uroporphyrinogen to yield coproporphyrinogen (Mauzerall & Granick, 1958). Abbreviations used: ALA, 5-aminolaevulinic acid; A human disease, porphyria cutanea tarda, is MC, 3-methylcholanthrene; PBB, 2,4,5,3',4'-pentabro- associated with hepatic accumulation of the mobiphenyl; PIA, 2-propyl-2-isopropylacetamide; oxidized porphyrinogens, URO and heptacarboxyl PROTO, protoporphyrin; TCB, 3,4,3',4'-tetrachlorobi- . phenyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; porphyrins, and withw decreased activity of the URO, uroporphyrin; h.p.l.c., high-pressure liquid enzyme (for review, see Pimstone, 1982). Activity chromatography. of this enzyme is also decreased in liver homogen- § To whom reprint requests should be sent at the VA ates from animals treated with hexachlorobenzene Medical Center address. (Elder et al., 1976), TCDD (Jones & Sweeney, Vol. 222 738 P. R. Sinclair, W. J. Bement, H. L. Bonkovsky and J. F. Sinclair 1980) or with some congeners of polybrominated 1983). Thus, at present, the roles of particular P- biphenyls (Voorman et al., 1983). These halogenat- 450 isoenzymes in the loss of enzyme activity are ed compounds also cause hepatic accumulation of not clear. URO and heptacarboxyl-porphyrins. Evidence We have reinvestigated the role ofisoenzymes of from immunological studies indicates that, in cytochrome P-450 in the mechanism by which animals accumulating these porphyrins, the con- individual halogenated biphenyls (TCB and PBB) centration of immunoreactive enzyme is equiv- inhibit uroporphyrinogen decarboxylase. We alent to that in controls (Elder & Shepperd, 1982), found that induction of the MC-inducible isoen- suggesting that decreased enzyme activity is due to zyme was an obligatory requirement for inhibition inactivation rather than a decrease in amount of of the enzyme. Furthermore, we found that some the enzyme. Iron has been implicated in both these compounds previously used for investigating the animal and human porphyrias, since iron overload mechanism of the inhibition, such as antioxidants exacerbates the decrease in decarboxylase (Tal- and chelators of iron, act by preventing induction jaard et al., 1971), iron deficiency prevents it of the cytochrome P-448. (Jones et al., 1981), and the symptoms of porphyria Part of this work has already appeared in cutanea tarda are relieved when patients are bled abstract form (Sinclair et al., 1984a). to remove iron (Pimstone, 1982). Sinclair & Granick (1974) showed that cultured Materials and methods chick-embryo hepatocytes accumulate URO when exposed to a number of halogenated cyclic com- Materials pounds. Chelators of iron prevented the URO Benzanthracene, benz[a]pyrene, ,B-naphthofla- accumulation, suggesting that, in the culture as in vone, MC, metyrapone and 2-acetylaminofluorene the human disease and experimental porphyrias, were purchased from Aldrich (Milwaukee, WI,, iron is somehow involved. Effects of cyclohexi- U.S.A.). ALA, butylated hydroxyanisole, cyclo- mide and inhibitors ofcytochrome P-450-mediated heximide, and a-tocopherol were purchased from activities implied that the mechanism ofthis URO Sigma (St. Louis, MO, U.S.A.). Ellipticine was accumulation also involves an induction and from Sigma or was a gift from Dr. P. Lesca, action of cytochrome P-450, possibly via metabo- CNRS, Pharmacology Laboratory, Toulouse, lism ofthe halogenated compounds to inhibitors of France. TCB and piperonyl butoxide were pur- uroporphyrinogen decarboxylase. chased from Ultra-science (Hope, RI, U.S.A.). Initially, no particular isoenzyme of cytochrome Spectral-grade dimethyl sulphoxide was from P-450 was implicated in the mechanism of the Fisher (Boston, MA, U.S.A.). Williams E medium decrease in uroporphyrinogen decarboxylase. Sub- was purchased as powder from Flow Laboratories, sequently, phenobarbital and MC were shown to (McLean, VA, U.S.A.). URO and PROTO induce different isoenzymes of cytochrome P-450 standards were purchased from Porphyrin Pro- in chick liver culture (Althaus et al., 1979), similar ducts (Logan, UT, U.S.A.). Desferrioxamine me- to the well-known inductions by these chemicals in sylate was purchased from Ciba-Geigy (Summit, animals (for review, see Lu & West, 1980). NJ, U.S.A.). 7-Ethoxyresorufin (ethoxyphenoxa- Phenobarbital has been reported to cause synergis- zone) was purchased from Pierce (Rockford, IL, tic decrease in uroporphyrinogen decarboxylase in U.S.A.). Aminopyrine was purchased from Merck chick liver culture when combined with TCDD (de (Darmstadt, Germany). PBB, purified from the Verneuil et al., 1983a). This synergism was crude polybrominated biphenyls (Firemaster) mix- postulated to be due to involvement of a phenobar- ture, was a gift from Dr. S. Aust, Michigan State bital-inducible isoenzyme ofcytochrome P-450. In University (East Lansing, MI, U.S.A.). SKF-525A contrast, others had reported that the rate of URO (2-dimethylaminoethyl-2,2-diphenylvalerate hy- production after treatment of the culture with drochloride) and cimetidine hydrochloride were hexachlorobenzene or mixtures of polybrominated gifts from Smith, Kline and French (Philadelphia, biphenyls was increased by pretreatment of cells PA, U.S.A.). Rhodotorulic acid was a gift from Dr. with MC orP-naphthoflavone (Debets et al., 1980). H. Akers of Rockefeller University, New York, These latter two compounds induce an isoenzyme U.S.A. PIA was a gift from Hoffmann-La Roche that will be referred to in the present paper as (Nutley, NJ, U.S.A.). N-[9-14C]-2-Acetylamino- 'cytochrome P-448'. In contrast with these mech- fluorene (47.6mCi/mmol) was purchased from anisms involving isoenzymes ofcytochrome P-450, New England Nuclear (Boston, MA, U.S.A.). Kawanishi et al. (1981, 1983) have reported that uroporphyrinogen decarboxylase is directly inhib- Hepatocyte cultures ited by chlorinated biphenyls. This direct inhi- Primary cultures from livers of 16-day White bition was said to account for URO accumulation Leghorn chicken embryos were prepared and in chick liver cultures (Kawanishi et al., 1981, maintained in Williams E medium as described 1984 Halogenated biphenyls and chick hepatocyte uroporphyrin 739 previously (Sinclair et al., 1982). Cells were radioactivity determined. The 1 h incubation was cultured in 3.5cm-diameter plastic dishes unless chosen because ofthe relatively rapid rate at which otherwise indicated. Dishes contained 0.5-0.6mg cells metabolized acetylaminofluorene. The single of protein per 3.5cm dish or 1.5-1.7mg of protein/ extraction removed more than 90% of the extract- 6.0cm dish. Chemicals were added during day 2 of able radioactivity. The organic-soluble metabolites culture after rinsing cells twice with Williams E were separated by t.l.c. (hexane/tetrahydrofuran, medium containing dexamethasone and tri-iodo- 1: 1, v/v) on silica gel (EM Laboratories, thyronine