Cytochrome P-450 levels and EROD activity in the Adriatic sturgeon (Acipenser naccarii, Chondrostei) and its induction by β-Naphthoflavone

The cytochrome P-450 enzyme system was studied in juvenile Acipenser naccarii (317.6±77 g initial live weight) reared at 23°C and at a standard diet over a period of two month before using for experiments (final weight 546.3±148.8 g). While determining normal levels of cytochrome P-450 in liver tissue its induction was also studied under exposure to P-naphthoflavone (BNF; single dose at 80 mg/Kg body weight 52 hrs prior to sampling). Control fish showed total cytochrome P-450 levels in liver microsomes around 0.40±0.12 nmoles/mg proteins (n=12) while in BNF exposed fish the values increased significantly (0.55±0.11nmoles/mg proteins; n=12). Ethoxyresorufin-O-deethylase (EROD) in control fish was 0.015±0.02 nmoles/mg proteins/min (n=12); it was found to be NADPH dependent and inducible by BNF. Western Immunoblotting and enzyme-linked-immunosorbent (ELISA) analyses of liver microsomes from untreated and BNF-exposed fish, using an antibody to rat cytochrome P-450 1A1 isoform (P-450 1A1) (Nelson et al.,1996) suggested the presence of a constitutive and BNF-inducible cytochrome P-450 homologous to rat P-450 protein.
Measurement of cytochrome P-450-dependent EROD activity and P-450 1A1 expression levels in A. naccarii may be a useful parameter in monitoring environmental pollution.     

Further consideration of phenobarbital effects on cytochrome P-450 activity in the killifish, Fundulus heteroclitus

1. Ethoxyresorufin O-deethylase (EROD) activity, aldrin epoxidase (AE) activity, cytochrome P-450 content, and levels of cytochrome P-450E (the major BNF-inducible P-450 form and primary EROD catalyst in scup) or its homologues were measured in hepatic microsomes isolated from Fundulus heteroclitus, scup (Stenotomus chrysops) and brook trout (Salvelinus fontinalis) treated with beta-naphthoflavone (BNF) or phenobarbital (PB). 2. In all three teleost species, BNF treatment caused expected increases in P-450 content, EROD activity and P-450E level; but either no change or a slight decrease in AE turnover rate (nmol/min/nmol P-450). 3. Polyclonal antibodies to P-450E did not inhibit AE activity in microsomes from BNF-treated scup, confirming that this major BNF-inducible P-450 form does not catalyze AE activity in fish. 4. In contrast, PB treatment did not affect hepatic AE activity, P-450 content or levels of "P-450E" in F. heteroclitus, but did variably affect EROD activity which was suppressed in one experiment and elevated in another. 5. The results indicate that (i) contrary to previous reports, neither PB nor MC-type inducers increase AE activity in F. heteroclitus, (ii) MC-type inducers do not affect AE activity in the other teleost species examined, and (iii) AE activity is not a reliable indicator of P-450 induction by environmental chemicals. 6. We emphasize the need to establish the mechanism of PB action, and the nature of any fish P-450 forms analogous to PB-inducible forms in mammals in order to conclusively evaluate PB-responses in fish.     

Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor

The synergistic effect of dexamethasone (DEX) and polycyclic aromatic hydrocarbons on the induction of cytochrome P450IA1 (P450IA1) was examined in H4IIEC3/T Reuber hepatoma cells. P450IA1 activity was determined by the hydroxylation of benzo[a]pyrene (AHH) and deethylation of 7-ethoxyresorufin (EROD). The amount of Ah receptor, i.e. the specific cytosolic binding protein of 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in H4IIEC3/T cells was characterized and quantitated by high performance gel filtration. Benz[a]anthracene and TCDD induced AHH and EROD activities, respectively, about 20-fold within 4 h. The increase was about 100-fold when cells were pretreated with DEX. The glucocorticoid alone induced P450IA1 activities 3-4 fold. DEX elicited half maximum AHH induction at a concentration of 20 nM in the presence or absence of benz[a]anthracene. Maximal potentiation of AHH induction required treatment with DEX for at least 32 h prior to the exposure to benz[a]anthracene. Treatment of H4IIEC3/T cells with DEX for 20 h caused a 2-3-fold increase in the amount of Ah receptor. The results suggest that the synergistic effect of DEX and polycyclic aromatic hydrocarbons on P450IA1 induction involves a time-consuming process which may consist of the synthesis or modification of a factor, possibly the Ah receptor.     

Mixed-function oxygenase in juvenile rainbow trout exposed to hexachlorobenzene or 3,3',4,4'-tetrachlorobiphenyl.

1. 3,3',4,4'-tetrachlorobiphenyl (PCB 77), but not hexachlorobenzene, induced liver microsomal cytochrome P-450 (Cyt P-450), ethoxycoumarin-O-deethylase (ECOD) and ethoxyresorufin-O-deethylase (EROD) in rainbow trout. Maximum induction was observed in a PCB 77 injected group of fish (1.0 mg/kg, i.p. injection) 13 days after the injections being 2, 10 and 50 times the value of non-induced fish, respectively. 2. The apparent Km value of ethoxyresorufin of this induced group of fish differed only slightly from that of non-induced fish. The apparent Vmax value (EROD) was 50 times higher. 3. Freezing small pieces of liver in liquid nitrogen did not produce cytochrome P-420. 4. Fluorimetric and spectrophotometric measurements of EROD correlated.     

Cytochrome P450 1A induction in gudgeon Gobio gobio : Laboratory and Field Studies

The induction of cytochrome P450 1A was studied in gudgeon (Gobio gobio), a common European cyprinid, using both farm-raised and field-caught fish. The effects of sex, reproductive status and past exposure to xenobiotics were assessed. When exposed to beta-naphthoflavone (bNF), reared gudgeon showed a dose-dependent increase of EROD activity with a plateau observed at doses from 20 mg kg-1 (females) and 5 mg kg-1 (males). The sexual difference in EROD activity was related to the gonadosomatic index (GSI) of the female whatever the level of induction. Dose and sex effects were confirmed by the immunodetection of CYP1A protein. More than 1 month was necessary for EROD activity to decrease to baseline levels. A second bNF injection after 32 days gave similar levels of induction, suggesting that EROD induction by bNF was not impaired by a pretreatment. Wild fish were brought from two sites in the Rhone river basin: a low contaminated site (Ain) and a highly contaminated site (Rhone). Wild gudgeon were highly induced by bNF in laboratory conditions, except males from the Rhone site which exhibited EROD levels as high as the EROD plateau found in laboratory conditions. A 2- month depuration period in clean water was necessary for EROD activity in wild gudgeon to decrease to baseline levels. These results provide better knowledge of the main factors of modulation of the induction in gudgeon as well as on the influence of the history of exposure to inducers.     

Mixed-function oxidase induction as a test for the biological monitoring of water: limitations and prospects]

The induction in fish liver of some enzyme activities, and typically of microsomal mixed-function oxidases (MFO), provides the earliest biological warning signal of exposure to pollutants. Our studies provided evidence that the basal levels of cytochrome P-450 and specific MFO activities, such as arylhydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EROD), were strongly influenced by the diet in freshwater fish (Oncorhynchus mykiss). The response of fish liver to a known enzyme inducer, i.e., beta-naphthoflavone, was also affected by the diet, which therefore should be carefully controlled in laboratory studies. Under field conditions MFO activities were significantly enhanced in the liver of O. mykiss kept in polluted river water as well as in the liver of the seawater fish Diplodus annularis collected from a polluted harbour area, as compared to specimens of the same species collected from an unpolluted reference area.     

The effect of sex steroids and pregnenolone-16 alpha-carbonitrile on the hepatic microsomal monooxygenase system of rainbow trout (Salmo gairdneri)

Estradiol benzoate (EB-5 mg/kg) or testosterone propionate (TP-50 mg/kg) administration to sexually immature rainbow trout resulted in an increase in liver weight to body weight ratios, and a diminution in hepatic microsomal benzphetamine-N-demethylation (BeND), ethoxycoumarin- and ethoxyresorufin-O-deethylations (ECOD, EROD) and cytochrome(s) P-450 content when compared to corn oil-pretreated controls. A low dose of TP (2 mg/kg) caused an increase in cytochrome(s) P-450 content but had no effect on the selected monooxygenase activities. EB administration prior to treatment with 150 mg/kg beta-naphthoflavone caused a dose-dependent decrease in the magnitude of induction of cytochrome(s) P-450 and associated catalytic activities. These data suggest that the sex differences in monooxygenation observed in prespawning trout are mediated via the sex steroids and that fish may respond differently to inducers depending on their reproductive state at the time of exposure. Administration of the synthetic steroid, pregnenolone-16 alpha-carbonitrile (PCN), resulted in an increase in BeND and ECOD but had no effect on EROD or cytochrome(s) P-450 content.     

EROD activity in gill filaments of anadromous and marine fish as a biomarker of dioxin-like pollutants

The applicability of a gill filament-based ethoxyresorufin O-deethylase (EROD) assay, originally developed in rainbow trout, was examined in Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens) and spotted wolffish (Anarhichas minor). All species but spotted wolffish showed strong EROD induction in tip pieces of gill filaments following 48 h of exposure to waterborne beta-naphthoflavone. Atlantic salmon parr, smolts held in freshwater and smolts transferred to seawater showed EROD induction of similar magnitude. Arctic charr, differing 11-fold in body weight, showed similar EROD activities as expressed per gill filament tip. Laboratory exposure of saithe to water and sediments collected at polluted sites, resulted in strong EROD induction. In conclusion, the gill filament assay seems useful for monitoring exposure to aryl hydrocarbon receptor agonists in various species. Furthermore, smoltification status, water salinity and body size proved to have minor influence on gill filament EROD activity. However, the results in spotted wolffish show that some species may be less suitable for monitoring using the gill assay. Assessment of gill filament EROD activity in fish exposed to polluted water and sediments in the laboratory proved to be an easy and cost-effective way to survey pollution with dioxin-like chemicals.     

Food restriction prevents the loss of isosafrole inducible cytochrome P-450 mRNA and enzyme levels in aging rats

The influence of food restriction (FR) on the drug-inducible capacity of liver microsomal cytochrome P-450s IA1, IA2 and IIB1 and IIB2 was studied in 20-month-old male Fischer-344 rats. ELISA and Western Blotting revealed that the induction of the cytochrome P-450-IA1/IA2 and P-450-IIB1/IIB2 enzymes was considerably higher in the liver microsomes of FR rats than in their ad libitum (AL) fed counterparts. In order to determine whether the higher P-450 enzyme levels in FR rats were a reflection of an increased synthesis rate or a stabilization of these enzymes, hybridization studies were performed with a cDNA probe for P-450-IIB1/IIB2. These studies show markedly higher levels of P-450-IIB1/IIB2 mRNAs in the livers of FR rats as compared to AL animals. These results suggest that it is possible to prevent the age-dependent loss of drug-induced cytochrome P-450s by 40% dietary restriction which suggest FR may improve the drug-metabolizing capacity during aging.     

An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity.

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.     

Serum and extracellular calcium modulate induction of cytochrome P-450IA1 in human keratinocytes

Culture conditions allowing for cytochrome P-450IA1 induction by 2,3,7,8-tetrachlorodibenzofuran (TCDF) in normal human keratinocytes (HK) were investigated. HK grown in serum-free low extracellular Ca2+ (0.1 mM) medium did not accumulate P-450IA1 mRNA in response to TCDF. If, however, the cultures were pretreated for more than 24 h with either serum or elevated extracellular Ca2+ (2.0 mM), induction of P-450IA1 was obtained by TCDF. Serum and elevated Ca2+ concentrations were found to be additive in this respect. When analyzing HK derived from five individuals, no apparent difference was found in the relative induction of P-450IA1 by increasing concentrations of TCDF, giving an EC50 of approximately 2 nM. The permissive effect of serum and elevated Ca2+ could be conferred to a reporter gene by the -1140 to +2435 part of the human CYPIA1 gene. Culture conditions allowing for P-450IA1 induction correlated with conditions that induced mRNA corresponding to the differentiation specific enzyme epidermal transglutaminase. This finding, together with the known differentiation promoting effects of serum and elevated Ca2+, suggest that terminal differentiation is necessary for P-450IA1 induction in HK by Ah receptor ligands.     

Specificity and cross-reactivity of monoclonal and polyclonal antibodies against cytochrome P-450E of the marine fish scup

Monoclonal antibody (MAb) 1-12-3 generated against liver cytochrome P-450E (P-450E), an aryl hydrocarbon hydroxylase of the marine fish Stenotomus chrysops (scup), reacted only with P-450E when tested in immunoblot analysis with five P-450 fractions from scup liver. This and six other MAbs against P-450E recognized purified P-450E, as well as a single band in beta-naphthoflavone (BNF)-induced scup microsomes that comigrated with authentic P-450E. Like MAb 1-12-3, polyclonal anti-P-450E reacted with P-450E but not with other scup P-450 fractions and reacted strongly with a band coincident to P-450E in BNF-treated scup microsomes. However, the polyclonal antibody (PAb) also faintly recognized additional microsomal proteins. MAb 1-12-3 recognized P-450E induced by 3,3',4,4',5,5'-hexachlorobiphenyl and by polychlorinated biphenyl mixtures in scup, and a single band induced by BNF or 3-methylcholanthrene (MC) in microsomes of other teleosts, including two trout species, killifish and winter flounder. The content of the P-450E counterpart in these fish and also in untreated scup coincided with induced ethoxyresorufin O-deethylase (EROD) activity. Induced EROD activity in scup and trout was strongly inhibited by MAb 1-12-3, further demonstrating the relationship between P-450E and induced P-450E in trout. MAb 1-12-3, two other MAbs, and anti-P-450E PAb recognized a band comigrating with P-450c in BNF-induced rat microsomes. MAb 1-12-3 also recognized purified rat P-450c. MAb 1-12-3 and anti-P-450E PAb recognized a second band of lower molecular weight than P-450c in BNF rat microsomes which may correspond to P-450d, the MC- and isosafrole-inducible rat isozyme. The results firmly establish the identity of scup P-450E, the relationship of BNF-induced P-450 in other teleosts with P-450E, and the immunochemical relationship of P-450E with rat P-450c. Furthermore, results with untreated fish suggest that effects of environmental chemicals may be detected by immunoblotting with monoclonal anti-P-450E.     

Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants.

The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide.     

Differential enzyme induction of mouse liver and lung following a single low or high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The induction response of cytochrome P-450-dependent enzyme activities to a single low (5 nmol/kg) or high (50 nmol/kg, intraperitoneal [ip] dose of TCDD was examined in liver and lung homogenates over a 12-week time course in an outbred, Ah-responsive strain of mice (National Institutes of Health [NIH] Swiss). Total hepatic cytochrome P-450 was quantified, and the dealkylation of ethoxy- and benzyloxyresorufin (activities of P-450 IA1 and IIB1, respectively) were measured in both tissues at 48 and 96 hr and at 1, 4, and 12 weeks post-TCDD administration. Western immunoblotting with monoclonal antibody 1-7-1 was conducted to confirm the specific IA1-inductive effects of each dose of TCDD over the same time course. Following the low dose, specific IA1 induction was apparent in liver at the earliest time point, was maximal at 1 week, and declined to control values at 12 weeks. Pulmonary IA1 was near-maximally induced at 48 hr, and remained at that level for 4 weeks. In contrast, a tenfold higher dose of TCDD elicited similar IA1 induction profiles for both tissues, with a maximum at 1 week and a progressive loss at 4 and 12 weeks postexposure. P-450 IIB1 activity was elevated in TCDD-treated animals by enzymatic assay; however, Western immunoblotting did not confirm this finding. These data demonstrate persistent dose-dependent P450 induction over many weeks by a single TCDD dose, with significant organ-specific differences: (a) lung is more sensitive than liver to a nonmaximal inducing dose of TCDD, and (b) at a maximally inducing dose of TCDD, lung is very similar to liver in both the level and time course of IA1 induction.     

Induction of cytochrome P-450 isozymes by mirex and chlordecone

The effect of the insecticides, mirex and chordecone (Kepone), on the cytochrome P-450 monooxygenase system in C57BL/6N mouse liver microsomes was studied. Mice were treated intraperitoneally with low (6 mg/kg) and high (30 mg/kg) doses of mirex and chlordecone in corn oil for 2 days. For comparison, mice were also treated with either phenobarbital (PB) or 3-methylcholanthrene (3-MC). All treatments significantly increased the hepatic microsomal P-450 content over that of controls. Benzphetamine N-demethylase, ethoxyresorufin O-deethylase, benzo[a]pyrene hydroxylase, and acetanilide hydroxylase activities were also determined. Mirex and chlordecone resembled phenobarbital with respect to the induction of monooxygenase activities. Immunoquantitation with antibodies to purified P-450 IIB1 (Pb-induced P-450) and P-450 IA1 (3-MC-induced P-450) indicated that mirex and chlordecone induced P-450 IIB1 in a dose-dependent manner. The high dose of mirex also induced a small amount of a protein cross reacting with the antibody to IA1. The induction of this isozyme did not, however, contribute significantly to the monooxygenase activities measured.     

Musk xylene is a novel specific inducer of cytochrome P-450IA2.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.     

Immunological characterization of constitutive isozymes of cytochrome P-450 from rainbow trout. Evidence for homology with phenobarbital-induced rat P-450s

Immunoglobulin G fractions (IgGs), isolated from rabbits immunized against hepatic cytochrome P-450 isozymes were used to investigate the immunochemical homology among trout P-450s and between trout and rat P-450s. The antigens used for immunization were five constitutive trout P-450s (LMC1 to LMC5), one beta-naphthoflavone (BNF)-inducible trout P-450 (LM4b), and one phenobarbital-induced rat P4500IIB1 (PB-B). In the enzyme-linked immunosorbent assay (ELISA), strong cross-reactivity was observed between anti-LMC2 IgG and P-450 LMC1, and between anti-LMC3 IgG and P-450 LMC4. There was little or no cross-reactivity of anti-LMC5 IgG with other trout P-450s. Trout P-450 LM4b was not recognized by any of the antibodies against constitutive trout P-450s. Antibodies to P-450 LMC1 and P450 LMC2 cross-reacted strongly with rat P450IIB1 and with proteins of PB-induced rat liver microsomes. Rat P450IA1 (BNF-B) did not cross-react with anti-LMC1 or anti-LMC2 IgG. These cross-reactions were essentially confirmed by immunoblot (Western blot) analysis. Western blots of PB-induced rat liver microsomes probed with anti LMC1 revealed two major immunoreactive proteins in the P-450 region, one of which co-migrated with rat P450IIB1. P450IIB1 itself cross-reacted strongly with anti-LMC1 IgG. In control rats, a single protein band cross-reacted poorly with anti-LMC1 IgG. Antibodies to LMC1 and LMC2 did not cross-react with rat P450IA1 in Western blots. The antigenic epitopes in rat P450IIB1 recognized by anti-LMC1 IgG and anti-LMC2 IgG are probably not located at or near the active site of the enzyme since these antibodies did not inhibit benzphetamine N-demethylase activity of P450IIB1 or of PB-induced rat liver microsomes. In general, our results demonstrate: (1) the presence of a significant homology between LMC1 and LMC2, and between constitutive trout P-450 (LMC1) and PB-induced rat P-450 (P450IIB1); and (2) distant homology between constitutive trout P-450s and constitutive rat P-450s or BNF-induced rat P-450s.     

Intralobular localization of different cytochrome P-450 form dependent monooxygenase activities in the liver of normal and inducer-treated rats.

1. Isolated periportal (PP) and perivenous (PV) hepatocytes from normal and inducer-treated rat livers were used to examine the following: intralobular localization of cytochrome P-450IA, P-450IIB, P-450IIE and P-450IIIA dependent monooxygenase activities and effects of phenobarbital (PB), beta-naphthoflavone (BNF) and pregnenolone-16 alpha-carbonitrile (PCN) on the zonal induction of these monooxygenases. 2. 7-Ethoxyresorufin O-deethylase (7EROD), 7-pentoxyresorufin O-dealkylase (7PROD) and N-nitrosodimethylamine N-demethylase (NAND) activities of PP hepatocytes were not significantly different from those of PV hepatocytes. 3. Ethylmorphine N-demethylase (EMND) activity was significantly higher in PV hepatocytes than in PP hepatocytes of normal rats. 4. EMND activity was induced by PCN and PB treatments. The response of EMND activity to PCN treatment was higher in PP hepatocytes than that in PV hepatocytes, and as a result the PV dominance disappeared following PCN treatment. 5. Extents of the response of this activity to PB treatment were similar in PP and PV hepatocytes, and PV dominance remained unchanged even after induction.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.