Destruction of hepatic mixed-function oxygenase parameters by CCl4 in rats following acute treatment with chlordecone, Mirex, and phenobarbital

Previous work has established the marked potentiation of CCl4 hepatoxicity by prior exposure to chlordecone (CD). This study was conducted to determine if prior exposure to CD results in enhancement of CCl4-induced destruction of the hepatic microsomal mixed-function oxygenase (MFO) system. Male Sprague-Dawley rats received a single oral dose of CD (10 mg/kg) or corn oil vehicle alone (1 ml/kg) 24 hr prior to a single ip injection of CCl4 (0-100 microliter/kg). Mirex (M; 10 mg/kg) and phenobarbital (PB; 80 mg/kg/day for two days) were used as negative and positive controls respectively for the potentiation of CCl4 hepatotoxicity. Hepatotoxicity was evaluated 24 hrs after CCl4 administration by elevations of three serum enzymes (GPT, GOT, and ICD). The key hepatic microsomal MFO parameters measured were microsomal protein, cytochrome P-450 content, glucose-6-phosphatase (G-6-Pase), and aminopyrine demethylase (APD). As previously demonstrated using a subchronic dietary pretreatment protocol, CD potentiated CCl4 hepatotoxicity over a range of CCl4 doses to a greater extent than PB or M, as judged by elevations in serum enzymes. PB caused the greatest increase in total P-450 content and the greatest increase in CCl4-mediated destruction of microsomal protein and APD activity. M caused the least destruction of total hepatic cytochrome P-450, despite the same level of cytochrome P-450 as in the PB group. CD treatment caused the greatest decrease in G-6-Pase activity in comparison to PB or M pretreatments and a similar degree of P-450 destruction as observed with the PB group. These findings suggest that in general, CCl4-induced destruction of hepatic MFO parameters measured in this study is disproportional to the known degree of potentiated hepatotoxicity by the pretreatments and does not accurately reflect the potentiation of CCl4 hepatotoxicity by CD.     

Inhibition of cholesterol 7 alpha-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC.

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification.     

Characterization of electron transport enzymes in the envelope of rat liver nuclei.

Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and beta-naphthoflavone (beta-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by beta-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or beta-NF as their microsomal counterparts are.     

Dynamics of androstenedione metabolite formation in rat liver microsomes during temporal phenobarbital induction.

1. Formation of androstenedione (AD) 7 alpha-, 16 alpha-, 16 beta- and 6 beta-hydroxymetabolites produced in rat liver microsomes and differing by the duration of phenobarbital (PB) induction (temporal induction) has been studied. 2. Formation of 7 alpha-, 16 alpha- and 6 beta-metabolite is sexually differentiated during PB-induction. 3. The most dramatical changes were observed in the 16 beta-hydroxylase activity specific for cytochrome P-450b which increased in all rat groups investigated. 4. The immunochemical method using antibodies against P-450b/e was applied to measure its content in microsomes. 5. It was shown that the microsomal level of P-450b/e correlated (r = 0.63) with a 16 beta-hydroxylase activity in a narrow range of enzyme concentrations (from 0.16 to 0.32 nmol/mg). 6. In microsomal preparations with a higher level of P-450b/e the correlation is lower (r = 0.4). 7. The dependence of the P-450b catalytic activity on the P-450b to NADPH-cytochrome P-450 reductase relation is discussed.     

Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes

Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.     

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.     

Attenuation of low dose phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in rats by cimetidine

Cimetidine, a substituted imidazole, is an inhibitor of hepatic cytochrome P-450-mediated drug metabolism in rats and humans. We investigated the effect of cimetidine on phenobarbital induction of hepatic microsomal aminopyrine N-demethylase activity in the rat. Phenobarbital induction of aminopyrine N-demethylase was log-linear in the range of 1-6 mg/kg/day and the ED50 was approximately 3 mg/kg/day. Cimetidine 75 mg/kg (four times a day) attenuated the induction of aminopyrine N-demethylase activity by 58% in low dose (3 mg/kg/day) but not in high dose (40 mg/kg/day) phenobarbital treated rats. This result could not be explained by residual inhibition of enzyme activity by cimetidine and suggests that cimetidine affects the induction of hepatic cytochrome P-450 by low dose phenobarbital.     

Analysis of debromination of 1,2-dibromoethane by cytochrome P-450-linked hydroxylation systems as observed by bromide electrode

Debromination of 1,2-dibromoethane (DBE) by a rabbit liver microsomal preparation and a reconstituted cytochrome P-450 enzyme system was investigated. The reaction was performed in our newly constructed reaction vessel, in which a bromide electrode was installed. During the reaction, the liberated bromide ion was continuously measured by the bromide electrode, and the amount was recorded. In the microsomal preparation, the DBE-debromination rate per nmol cytochrome P-450 was enhanced by phenobarbital-pretreatment of rabbits compared with the untreated microsomes, whereas it was diminished by 3-methylcholanthrene-pretreatment. The debromination reaction was reconstituted in a purified enzyme system containing phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450PB), NADPH-cytochrome P-450 reductase, and NADPH. The optimum conditions required the presence of dilauroylphosphatidylcholine and cytochrome b5. Cytochrome b5 was found not to be an obligatory component for the DBE-debromination in the reconstituted system, but it stimulated the activity about 3.4-fold. Preincubation of the reconstituted mixture with guinea pig anti-cytochrome P-450PB antiserum markedly inhibited the debromination reaction.     

Cytochrome P-450—Mediated denitrification of 2-nitropropane in mouse liver microsomes

Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD₁ mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Effect of excessive intake of ascorbic acid on hepatic and extra-hepatic phase I and phase II drug metabolism in rat

Guinea pig is the animal model of choice for studies on effects of ascorbic acid (AA). However, rat is one of the largely used animals for investigations related to chemical carcinogenesis. Therefore, the present study was designed to evaluate the changes induced by high intake of the vitamin in xenobiotic and carcinogen metabolizing status of the organs. Male Wistar rats, dosed daily with 50 mg AA/100 g body weight for 10 weeks, demonstrated a small non-significant increase in hepatic, pulmonary and colon cytochrome P-450 (Cyt. P-450) contents, which was accompanied with a significant increase in hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities. Phase II enzymes of drug metabolism responded in different ways to increased intake of AA. UDP-glucuronyltransferase (UDPGT) activity was unaffected in liver and colon, but it was increased (p less than 0.005) in lung. Activities of glutathione S-transferase (GST) were decreased in the three organs. Inducibility of AHH by 3-methylcholanthrene (MCA) or phenobarbital (PB) was largely reduced due to AA feeding. Besides this, MCA and PB had differential effects on enzymatic levels in AA fed rats. When compared with our earlier observations in guinea pig, it was found that rat responded similarly to guinea pig to increased intake of AA with regard to hepatic AHH, Cyt. P-450, UDPGT and GST, pulmonary AHH, Cyt. P-450 and Cyt. b5, and all studied colon enzymes, except GST.     

Coordinate elevations of liver delta-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: the effects of heme

1. The role of heme in the coordinate elevations of liver delta-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P-450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Luteinizing hormone and cyclic AMP-mediated induction of microsomal cytochrome P-450 enzymes in cultured mouse Leydig cells

Treatment of mouse Leydig cell cultures with luteinizing hormone (LH) or with 8-bromo-cAMP (8-Br-cAMP) for 5 days elicited a dose- and time-dependent increase in the microsomal cytochrome P-450 enzyme activities. 17 alpha-Hydroxylase and C17-20 lyase as well as a parallel increase in testosterone production. Reduction of the oxygen tension from 19 to 1% resulted in a greater increase in enzyme activity. Induction of microsomal cytochrome P-450 activities was 35 to 50% greater with 8-Br-cAMP than with LH and the increase in C17-20 lyase activity was 4-fold greater than that of 17 alpha-hydroxylase. Maximal induction of P-450 enzyme activities was observed between 3 and 5 days of continual treatment with 8-Br-cAMP or LH. Removal of 8-Br-cAMP from the culture medium inhibited any further increase in C17-20 lyase activity and testosterone production. The role of protein synthesis in the induction process was investigated by incubating Leydig cell cultures with and without cycloheximide between 24 and 48 h of treatment with 8-Br-cAMP. Cycloheximide completely inhibited the induction of C17-20 lyase activity and the increase in testosterone production. After removal of the inhibitor, cultures responded in a manner that paralleled induction in cultures that had not been treated with cycloheximide. In both cases, a 24-h lag period occurred prior to an increase in cytochrome P-450 activity. These data suggest that the increase in microsomal cytochrome P-450 activities represents an increase in enzyme synthesis and, furthermore, that reduction of oxygen tension decreases degradation of newly synthesized Leydig cell microsomal cytochrome P-450 activities as recently reported (Quinn, P.G., and Payne, A.H. (1984) J. Biol. Chem. 259, 4130-4135).     

Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P-450b/e IgG established that low levels of "b-like" P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450's b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti-P-450b/e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (greater than 2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (less than 1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (approximately 40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti-P-450b/e. A protein comigrating with P-450e was well above detection (6-7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

A requirement for dietary lipids for induction of cytochrome P-450 by phenobarbitone in rat liver microsomal fraction

1. Rats fed on purified synthetic diets have a markedly lower cytochrome P-450 concentration and hydroxylating enzyme activity in liver microsomal fraction than rats fed on stock pellets. 2. When both groups are treated with phenobarbitone the difference is even greater, the purified diet allowing only 50% of the cytochrome P-450 concentrations of controls. 3. Addition of herring oil, linoleic acid or 0.1% oxidized sitosterol to the diets allows induction of cytochrome P-450 to take place. 4. Addition of coconut oil to the diet does not permit induction of cytochrome P-450. 5. The interactions between dietary protein and the lipid substances are explored. 6. The mechanism of induction of microsomal hydroxylation enzymes by drugs is discussed in the light of these requirements.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach

The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gamma-glutamyl transpeptidase (GGT), beta-glucuronidase (beta-G1), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT50, PB3a, MC1, MC2) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and beta-G1 activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers.     

3-Methoxy-4-aminoazobenzene, a unique carcinogenic aromatic amine as a substrate for cytochrome-P-450-mediated mutagenesis

Rat liver microsomal enzyme(s) that catalyze mutagenic activation of a carcinogenic aminoazo dye, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), was studied by virtue of the Salmonella typhimurium TA98 assay using o-aminoazotoluene (OAT) as the control. Male Wistar rats were pretreated with phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyl (PCB), and the liver microsomal activities for mutagenic activation of 3-MeO-AAB and OAT were examined. In agreement with the reported results on several carcinogenic aromatic amines, MC pretreatment resulted in greater activation of microsomal activity in the OAT mutagenesis (about a 4-fold increase as compared to the untreated control) than did PB (1.5-fold increase). By contrast, the mutagenic activation of 3-MeO-AAB is found to be more efficiently catalyzed by those enzyme(s) that are induced by PB pretreatment (4-fold increase) than by those that are induced by MC (1.8-fold increase). The induced enzymes that principally mediate the mutagenic activation of these azo dyes are indicated to be cytochrome P-450s, because the mutagenic activation was strongly inhibited by addition of cytochrome P-450 inhibitors such as 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525A) and 7,8-benzoflavone. These data suggest that 3-MeO-AAB is a unique carcinogenic aromatic amine as a substrate for mutagenic activation via catalysis of those cytochrome P-450s that are induced by PB pretreatment.