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.     

Age-dependent expression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

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.     

Induction of cytochrome P-450IA1, IA2, IIB1, IIB2 and IIE1 by broccoli in rat liver and colon

Ingestion of broccoli or other cruciferous vegetables inhibits the induction of cancer by chemicals and modifies some cytochrome P-450 enzyme activities. The effect of dietary broccoli on the levels of P450IA and IIB mRNA and proteins in rat liver and colon has been studied. Rats were fed a ten percent broccoli diet for 7 days. The expression of the cytochrome P-450 forms was altered to a different extent in the liver and colon. The level of total P450IA mRNA in the liver was increased by the broccoli together with the P450IA1 and IA2 proteins. Colonic P450IA1 mRNA and protein were induced by the broccoli diet, whereas only P450IA2 protein and not mRNA was detectable in colon, but the protein level was unaffected by the broccoli diet. Liver P450IIB and IIE1 proteins were increased by the broccoli diet, whereas the level of P450IIB mRNAs was not affected. In contrast, the P450IIB mRNA levels were reduced but the protein levels were increased in colon and we suggest that a feedback mechanism caused the decrease of the P450IIB mRNAs levels. Because the ratio between activation and deactivation may be an important risk determinant, we conclude that the protective effect of the broccoli diet on chemically induced tumors in rodents may be caused by the broccoli-induced changes in P450IA and IIB associated enzyme activities.     

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.     

The effect of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes]

Effects of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes have been studied. Pyridine (200 mg/kg) increased total cytochrome P-450 content and activated metabolism of some specific substrates 24 hours after injection. There was an increase in the degree of p-nitrophenol and chlorzoxazone hydroxylation due to increasing ethanol-induced cytochrome P-450IIE1 content. Pyridine was also able to induce cytochrome P-450IIB1 in rat microsomes; this reaction was accompanied by acceleration of 7-pentoxyresorufin 0-dealkylation. Cytochrome P-450IA1 appearance in liver microsomes was associated with increasing content of cytochrome P-450IA2. Dealkylation rates for specific substrates (7-ethoxyresorufin and 7-methoxyresorufin) were also increased. Similar to pyridine, pyridine-7-oxide induced cytochromes P-450IIE1, P-450IIB1/B2, and P-450IA1/A2, resulting in activation of specific substrate metabolism. Hence, pyridine and its derivative pyridine-N-oxide can be regarded as effective inducers of cytochrome P-450.     

Induction of cytochrome P-450IA1 gene expression in human breast tumour cell lines

The induction of cytochrome P-450IA1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in eight human breast tumour cell lines. The cells were treated with various concentrations of TCDD for 24 h, and total RNA was isolated. The level of P-450IA1 RNA induced by 1 nM TCDD followed the order: MCF-7 greater than T47-D greater than ZR-75-1 greater than 3909 greater than 3522. AL-1, BT-20 and CAMA-1 did not respond to TCDD at the concentrations used. Northern blot analysis revealed 2 bands at 2.7 and 2.0 Kb, respectively, with the larger band being 6-fold more intense. The ratio was not changed by the TCDD treatment. TCDD induction did not change the benzo[a]pyrene-7,8-diol (BP-7,8-diol) metabolite profile compared with control cells, when cells were incubated with [3H]BP-7,8-diol for 24 h following the treatment with TCDD. These results demonstrate that different breast tumour cell lines vary greatly with respect to the basal expression levels of P-450IA1 RNA and its inducibility by TCDD. Furthermore, TCDD treatment does not change the relative distribution of BP-7,8-diol metabolites.     

Induction of hepatic cytochrome P-450IA1 in pigeons treated in vivo with Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs).

1. Treatment with a commercial mixture of polychlorinated biphenyls (PCBs) resulted in highly significant increases in pigeon hepatic microsomal proteins (100-fold), cytochrome P-450 (11-fold), cytochrome b5 (7-fold), NADPH-cytochrome c-(P450) reductase (7-fold), ethoxycoumarin-O-deethylation (9-fold), aldrin epoxidase (22-fold), ethoxyresorufin-O-deethylation (48-fold), N-demethylation of dimethylnitrosamine (28-fold) but not of lauric acid 12-hydroxylation. 2. SDS-PAGE analysis of pigeon hepatic microsomal proteins induced by Aroclor 1254 suggested highly significant increases in the density of staining in bands of estimated Mr 51-52 kD, 54-54.5 kD, 57-58 kD, 59-60 kD and of 77.5-78.5 kD. 3. The induction of cytochrome P-450IA1 was confirmed by Western immunoblotting using the monoclonal antibodies MAB 1-12-3 and MAB 1-8-4. 4. There was agreement between the 8-fold increase in cytochrome P-450IA1 increased staining of microsomal proteins, as judged by SDS-PAGE, and the 24-fold increase in the amount of protein that reacted with the monoclonal antibodies MAB 1-12-3 and MAB 1-8-4, as judged by Western immunoblotting. 5. It is concluded that treatment with a commercial PCB mixture resulted in the induction of several isoforms of pigeon hepatic cytochrome P-450 in a fashion that is likely to be similar to that reported for mammals.     

Expression of cytochrome P-450d by Saccharomyces cerevisiae

Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d. The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d. The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.     

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.     

Age-dependent exression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2α-, 2β-, 15α-, 16α-, and 16β-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7α-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible form) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6β-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

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.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.     

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.     

Identification of cytochrome P450IA2 as a human autoantigen

Autoantibodies occurring in a patient with idiopathic autoimmune type chronic active hepatitis (CAH) were found to react with purified rabbit cytochrome P450IA2 and to a much lesser extent with P450IA1. Both cytochrome P450s are known to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rabbit, and the expression of the microsomal protein recognized by the patient serum was induced in adult rabbit livers after treatment with TCDD. This protein is only weakly detected in liver microsomes from neonatal rabbits exposed to TCDD in utero, which is consistent with the age-dependent induction of P450IA2 by TCDD. The serum specifically reacted with a protein of similar size in microsomes prepared from COS-1 cells transfected with an expression vector containing the full length human P450IA2 cDNA. This reactivity was not detected in the cells transfected with the vector alone, indicating that the antibody recognizes human P450IA2. In addition, the serum extensively inhibited 7-ethoxyresorufin O-deethylation catalyzed by isolated human liver microsomes. This catalytic activity is associated with class IA P450s in other species. A screen of sera from patients with various hepatic and nonhepatic diseases indicates that the autoantibody to P450IA2 occurs rarely in CAH. Cytochrome P450IA2 becomes the third P450 identified as an autoantigen in inflammatory liver diseases.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic and renal prostaglandin synthetase

This study examines the possibility that the very toxic compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces its toxic effects through induction or repression of microsomal prostaglandin synthetase (cyclooxygenase). The effects of TCDD on microsomal synthesis of prostaglandin from [¹⁴C]arachidonic acid in rabbit liver and kidney medulla were examined 24 and 72 hr after TCDD administration. A hepatotoxic dose of TCDD (30 μg/kg) did not affect prostaglandin synthetase activity of rabbit liver or kidney medulla microsomes at either time point, although other microsomal enzymes (cytochrome P-488) were altered in both tissues.     

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.     

Purification and characterization of cytochromes P-450 from liver microsomes of 3-methylcholanthrene-treated crab-eating monkeys

Two forms of cytochrome P-450 (P-450MC1 and P-450MC2) were purified from liver microsomes of crab-eating monkeys (Macaca irus) treated with 3-methylcholanthrene (MC). Monkey P-450MC1 preparation had a specific content of 14.0 nmol/mg protein and showed a main protein band with a minimum molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monkey P-450MC2 preparation had a specific content of 12.1 nmol/mg protein and a minimum molecular weight of 54,000. The carbon monoxide-reduced difference spectral peaks of monkey P-450MC1 and P-450MC2 were at 448 and 447 nm, respectively. In the reconstituted system, monkey P-450MC2 had high activities for benzo[a]pyrene 3-hydroxylation and 7-ethoxycoumarin O-deethylation. Monkey P-450MC1 had low activities toward these two substrates and a high activity for benzphetamine N-demethylation. Monkey P-450MC1 and P-450MC2 were detected by immunoblotting using an antibody prepared against rat cytochrome P-450c, which is a major form of cytochrome P-450 in liver microsomes of MC-treated rats. These results suggested that the molecular properties of cytochrome P-450 in liver microsomes of crab-eating monkeys treated with MC are similar to those in rats.