Arch Toxicol (1988) 62:479-481 Archives of

Toxicology �9 Springer-Verlag 1988

Short communication

Relative induction of molecular forms of cytochrome P-450 in y-Hexachlorocyclohexane exposed rat liver microsomes

Ashwani Kumar and Prem Prakash Dwivedi

Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow 226001, India

Abstract. The effect of y-hexachlorocyclohexane (HCH), (25 mg/kg body weight, i.p., administered for 4 consecu- tive days) on the induction of types of cytochrome P-450 in rat liver microsomes was studied. Induced proteins were characterized by "Western" blot analysis, using anticy- tochrome P-450 antibodies. It was observed that HCH is a "mixed-type" inducer and mediates induction of cyto- chrome P-450 b /e forms by several fold and of cyto- chrome P-450 c and d forms by nearly three fold.

Key words: Hexachlorocyclohexane - Liver - Cyto- chrome P-450 forms - Induction - Protein blots

Introduction

y-Hexachlorocyclohexane, an organochlorine com- pound, popularly known as lindane, is used extensively as a broad spectrum insecticide for controlling the vector borne diseases of humans and domestic animals, and also pests that cause great damage to the agriculture crops (Alexander 1981). It is a recalcitrant compound and re- peated usage lead to an accumulation of the residues of HCH to substantial levels in the soil and water. Residues of HCH have also been found in different varieties of food, and gain entry into human beings via the food chain (Hayes 1982). Due to its toxicity, a global attempt has been made to identify microorganisms capable of biodegrading lindane (MacRae et al. 1969; Jagnow et al. 1977; Macholz and Kujawa 1985).

In higher animals, lindane is a potent inducer of cyto- chrome P-450 and related mixed function monooxygenase activities (Schulte-Hermann 1974; Oesch et al. 1982; Srini- vasan and Radhakrishnamurty 1983), which, in turn, me- diate the biotransformation of lindane into nontoxic me- tabolites (Tanaka et al. 1979; Macholz and Kujawa 1985). It is now known that cytochrome P-450 represents a family of multiple molecular forms that have distinct enzymatic activities and are induced differentially, such as (i) cyto- chrome P-450 b and e, inducible by phenobarbital, (ii) cy- tochrome P-450 c and d, inducible by polycyclic aromatic hydrocarbons, and (iii) cytochrome P-450 pen, inducible by steroid hormone analogs. These P-450s differ immuno-

Offprint requests to: A. Kumar

logically, have different amino acid sequences (Lu and West 1979), and are independent gene products (Kumar et al. 1983; Adesnik and Atchison 1985). No information, however, is available on the forms of P-450 inducible by lindane. The objectives of this study were, therefore, to characterize the induction of different molecular forms of P-450 in rat liver microsomes on exposure to HCH, using immunochemical techniques.

Materials and methods

Animals and treatment. Male Wistar rats (100-125 g), ob- tained from the inhouse breeding facility of Industrial Toxicology Research Centre, Lucknow, India, were used for the studies. Five groups of animals were injected in- traperitoneally, equal volumes of either normal saline or peanut oil (controls), or phenobarbital (PB, 80 mg/kg body weight), HCH 25 mg/kg body weight) or 20-methyl- cholanthrene (MC, 35 mg/kg body weight), once daily for 4 consecutive days. Animals were starved overnight before sacrificing, by decapitation, on the 5th day. Livers were excised quickly and kept over ice till use. Hepatic micro- somes were prepared (Baker et al. 1973) and protein con- tent was estimated (Lowry et al. 1951) using bovine serum albumin as the standard. All experiments were done in triplicate and results of a typical experiment are presented.

Enzyme assay. 7-Ethoxyresorufin-0-deethylase assay was done as described by Burke et al. (1977), except that the post-mitochondrial supernatant fractions were used as the source of enzyme.

Primary and secondary antibodies. Antibodies, raised against purified cytochrome P-450 b /e (PB induced) and P-450 c/d (MC-induced) were a generous gift from G. Padmanaban, Indian Institute of Science, Bangalore, India. The preparation, purification and characterization of these antibodies have been reported earlier (Kumar and Padmanaban 1980).

Secondary, goat anti-rabbit IgG antibodies, coupled to enzyme horse radish peroxidase, were obtained from Cap- pei Laboratories Inc., Philadelphia, Pa, USA.

Protein blotting. For protein "Western" blotting, solubi- lized microsomal proteins were separated by SDS-poly- acrylamide gel electrophoresis, transferred to nitrocellulose paper under electric current and the cross-reactive protein

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HCH

12

Fig. 3. Densitometric analysis of the "Western" blot in Fig. 2. I and H represent the position of P-450 c and d, respectively, in control (C) and HCH-treated (HCH) animals

Fig. 1. Protein "Western" blot of microsomes probed with anti- P-450 b and e antibodies. Samples of solubilized total micro- somal protein (2 p~g each), obtained from hexachlorocyclohexane (lane a), phenobarbital (lane b) and control (lane c) groups were separated on SDS-polyacrylamide gel electrophoresis, blotted on to nitrocellulose and probed with anti P-450 b and e antibodies. Arrow and arrowhead indicate the positions of purified cyto- chrome P-450 b/e and dye front, respectively

Fig. 2. Protein "Western" blot of microsomes probed with anti P-450 c and d antibodies. Samples of microsomal protein from HCH (10 Ixg, lane a), MC (10 Ixg, lane b; 1 Ixg, lane c) and control (10 Ixg, lane d) were used and probed with anti P-450 c and d an- tibodies. I and II indicate the positions of purified cytochromes P-450 c and d, respectively, and arrowhead represents the dye front

bands were detected immunochemically, as described by Nielson et al. (1982) with some modifications. Briefly, the nitrocellulose sheets, containing microsomal proteins, were incubated with either anticytochrome P-450 b /e an- tiserum (1/200 dilution) or anticytochrome P-450 c /d antiserum (1/100 dilution), followed with enzyme-coupled goat anti-rabbit IgG antibodies (1/1000 dilution). Visuali- zation of bound antibodies was achieved by treatment with diaminobenzidine and hydrogen peroxide and was quanti- rated with the aid of a EC densitometer attached with a 3390 A Hewlett Packard integrator.

Results and discussion

Xenobiotic compounds that induce hepatic microsomal cytochrome P-450 and associated mixed function mono- oxygenase activity, have broadly been classified as PB-type

and MC-type inducers (Conney and Burns 1972; Schulte- Hermann 1974; Linko et al. 1986). Organochlorine com- pounds, including lindane, accordingly, have been grouped as PB-type inducers. Studies in the recent past have, however, revealed that the multiplicity of P-450 types is more complex and using immunochemical tech- niques, multiple molecular forms of P-450, inducible by various chemicals and drugs have been described (Thomas et al. 1983; Linko et al. 1986). In the present study, at- tempts have, therefore, been made towards the character- ization of distinct forms of P-450 induced by HCH.

Protein blots of microsomes, obtained from differ- ent group of animals, were probed with anticytochrome P-450 b /e antibodies. Although no cross-reactive protein was present in control microsomes, a substantial amount, co-migrating with purified P-450 b/e, was present in microsomes obtained either from HCH or PB-treated animals, indicating an induction of this form by at least several fold by both the inducers (Fig. 1). Induction of cyto- chrome P-450 b / e by HCH is in agreement with earlier observations, where pretreatment of animals with PB has been shown to induce the metabolism of HCH (Koransky et al. 1964).

Similarly, to study the induction of cytochrome P-450 c and d forms, blots of microsomal proteins were probed with anticytochrome P-450 c /d antibodies. Distinct levels of P-450 c and d were present in control microsomes and a

Table 1. Induction of 7-ethoxyresorufin-0-deethylase activity by y-hexachlorocyclohexane treatment in rat liver

Treatment pmole resorufin formed/ min/mg protein*

Control 83.7 + 3.9 Phenobarbital 138.6+ 6.2 3-Methylcholanthrene 739.3 _+_ 28.1 y-Hexachlorocyclohexane 356.5 + 15.6

7-ethoxyresorufin-0-deethylase activity measurements were done in hepatic post-mitochondrial supernatant fractions, obtained from different groups of animals. Incubation time 10 min * Mean + SE, n = 6 (duplicate assays)

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relatively small but significant induct ion (2 .5-3 fold) in both the forms was observed in H C H treated group (Figs. 2 and 3). As expected, substantial levels of P-450 c and d were present in microsomes from MC-trea ted ani- mals. Some low molecular weight peptides, cross-reactive with ant i -cytochrome P-450 ant ibodies, were also observed along with the major P-450 prote in bands (Figs. 1 and 2) and might represent protease digestion products formed during the experiment. Treatment of HCH also caused a round a 5-fold increase in ethoxyresorufin-0-deethylase activity (Table 1) which is pr imar i ly media ted by P-450 c and d forms (Burke et al. 1977). Induct ion of P-450 c and d forms by H C H can possibly explain its carcinogenic potent ia l in some strains of rodents (Oesch et al. 1982).

The present studies suggest that H C H is "mixed type" inducer. Besides the induct ion of four dist inct forms of P-450 studied here, whether it induces any other forms re- mains to be seen. Since cytochrome P-450 represents the major route of b io t ransformat ion of foreign chemicals (Conney and Burns 1972), character izat ion of the induced P-450 forms should be of help towards better unders tand- .ing of the toxicokinet ics of HCH and its influence on the toxici ty of other foreign chemicals. This should also lead towards the cloning of dist inct P-450 genes in the micro- organisms that will be of use towards the b iodegrada t ion of the residues of HCH and possibly other organochlor ine compounds in different condit ions.

Acknowledgements. We wish to thank Prof. P. K. Ray, Director, Industrial Toxicology Research Centre, Lucknow, India, for his keen interest and support to this study and to Dr N. S. Srikanth for his help for the enzyme assays. Financial assistance to one of us (PPD) by Council of Scientific and Industrial Research is gratefully acknowledged.

References

Adesnik M, Atchison A (1985) Genes for cytochrome P-450 and their regulation. CRC Crit Rev Biochem 19:247-305

Alexander M (1981) Biodegradation of chemicals of environ- mental concern. Science 211 : 132-138

Baker RC, Coons LB, Hodgson E (1973) Low speed preparation of microsomes: a comparative study. Chem Biol Interact 6: 307-316

Burke MD, Prough RA, Mayer RT (1977) Characteristics of a microsomal cytochrome P-448 mediated reaction, ethoxy- resorufin-0-deethylation. Drug Metab Disp 5 : 1 - 8

Conney AH, Burns JJ (1972) Metabolic interactions among envi- ronmental chemicals and drugs. Science 178:576-586

Hayes WJ Jr (1982) Pesticide studies in man. Williams and Wil- kins, Baltimore, USA

Jagnow G, Haider K, Ellwardt PC (1977) Anaerobic dechlorina- tion and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria. Arch Microbiol 115:285-292

Koransky W, Portig J, Vohland HW, Klempau I (1964) Elimina- tion of 13 and T-hexachlorocyclohexane and the effects of liver microsomai enzymes. Arch Exp Pathol Pharmacol 247:49-60

Kumar A, Padmanaban G (1980) Studies on the synthesis of cyto- chrome P-450 and cytochrome P-448 in rat liver. J Biol Chem 255:522-525

Kumar A, Raphael C, Adesnik M (1983) Cloned cytochrome P-450 DNA: Nucleotide sequence and homology to multiple phenobarbital induced mRNA species. J Biol Chem 258: 11280-11284

Linko P, Yeowell HN, Gasiewicz TA, Goldstein JA (1986) Induc- tion of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (AH)-responsive mice. J Bio- chem Toxicol 1: 95-107

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275

Lu AYH, West SB (1979) Multiplicity of mammalian microsomal cytochromes P-450. Pharmacol Rev 31 : 277-295

Macholz RM, Kujawa M (1985) Recent state of lindane metabo- lism. Part III. Residue Rev 94:119-149

MacRae IC, Raghu K, Bautisa EM (1969) Anaerobic degradation of the insecticide lindane by Clostridium sp. Nature 221: 859-860

Nielson PJ, Manchester KL, Towbin H, Gordon J, Thomas G (1982) The phosphorylation of ribosomal protein $6 in rat tis- sues following cycloheximide injection, in diabetes, and after denervation of diaphragm. J Biol Chem 257: 12316-12321

Oesch F, Friedberg T, Herbst M, Paul W, Wilhelm N, Bentley P (1982) Effects of lindane treatment on drug metabolizing en- zymes and liver weight of CF-1 mice in which it evoked hepa- tomas and in nonsusceptible rodents. Chem Biol Interact 40: 1-14

Schulte-Hermann R (1974) Induction of liver growth by xeno- biotic compounds and other stimuli. CRC Crit Rev Toxi- col 3:97-158

Srinivasan K, Radhakrishnamurty R (1983) Induction of liver mixed function oxygenase system by 13 and T-hexachloro- cyclohexane. Ind J Biochem Biophys 20:84-91

Tanaka K, Kurihara N, Nakajima M (1979) Oxidative metabo- lism of tetrachlorocyclohexane, pentachlorocyclohexane and hexachlorocyclohexane with microsomes from rat liver and house fly abdomen. Pest Biochem Physiol 10:79-95

Thomas PE, Reik LM, Ryan DE, Levin W (1983) Induction of two immunochemically related rat liver cytochrome P-450 iso- zymes, cytochromes P-450c and P-450d, by structurally di- verse xenobiotics. J Biol Chem 258:4590-4598

Received August 5, 1988/Accepted September 26, 1988