Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.     

Mechanism of maintenance of liver-specific functions by DMSO in cultured rat hepatocytes

The present study was undertaken to investigate the mechanism by which dimethylsulfoxide (DMSO) exerts its protective action on cytochrome P450-dependent activities and differentiation in cultured rat hepatocytes. Loss of cytochrome P450 is associated with a shortage of heme and reduced activity of delta-aminolaevulinic acid dehydratase: the addition of DMSO, which induces this enzyme in human hepatoma cells, is not able to affect it in hepatocytes in primary culture. DMSO is a strong scavenger of hydroxyl radicals and may destroy the reactive oxygen species formed under conventional culture conditions (i.e., 95% air and 5% CO2). In fact other powerful scavengers of oxygen radicals like dimethylthiourea, desferal, and catalase itself maintain higher levels of cytochrome P450 and higher activities of 7-ethoxycoumarin O-deethylase during 3 days of culture. DMSO and the other scavengers are also able to retain features of the morphological and biochemical differentiation of hepatocytes such as the ability to induce tyrosine aminotransferase activity in response to glucocorticoids.     

Xenobiotic biotransformation in the rainbow trout liver and kidney during starvation

Microsomal cytochrome P-450-dependent activities in the kidney of fish starved for 6 weeks were significantly lower than in fed fish whereas these activities in the liver were only depressed after 12 weeks of starvation. Hepatic cytochrome P-450-dependent activities were depressed to varying extents after 12 weeks of starvation when different substrates were used. The content of hepatic cytochrome P-450 was not affected by starvation. Hepatic UDP-glucuronosyl transferase activities were not affected by starvation. Induction of several hepatic cytochrome P-450-dependent activities by treatment of fish with beta-naphthoflavone was not influenced by starvation. In the kidneys of fish starved for 12 weeks induced levels of cytochrome P-450-dependent benzo(a)pyrene hydroxylase activities were significantly lower than in the kidneys of fed induced fish.     

Microsomal monooxygenase system in Morris hepatoma: purification and characterization of cytochromes P-450 from Morris hepatoma 5123D of 3-methylcholanthrene-treated rats

Two forms of cytochrome P-450 (hepatoma P-450MCI and P-450MCII) were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P-450MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P-450MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxide-reduced difference spectral peak of hepatoma P-450MCI was at 446.5 nm, whereas the peak of hepatoma P-450MCII was at 451 nm. In the reconstituted system, hepatoma P-450MCI catalyzed 3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin, but showed low activities for N-demethylation of benzphetamine and aminopyrine, O-demethylation of p-nitroanisole, and p-hydroxylation of aniline. On the other hand, hepatoma P-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P-450MCI was immunologically indistinguishable from rat liver cytochrome P-450c, but hepatoma P-450MCII was distinct from hepatoma P-450MCI and rat liver cytochrome P-450c. Peptide maps of hepatoma P-450MCI and rat liver cytochrome P-450c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P-450.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Rat pulmonary microsomal cytochrome P-450 enzymes involved in the activation of procarcinogens.

Rat lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic specificities towards activation of several procarcinogens to genotoxic metabolites in Salmonella typhimurium TA1535/pSK1002. We first examined the roles of rat liver microsomal P-450 enzymes in the activation of benzo[a]pyrene and its 7,8-diol enantiomers to genotoxic products, and found that P-450 1A1 is a major catalyst for the activation of these potential procarcinogens in rat livers. Using lung microsomes isolated from rats treated with various P-450 inducers we obtained evidence that at least three P-450 enzymes are involved in the activation of several procarcinogens. Immunoinhibition studies support the view that benzo[a]pyrene and its 7,8-diol derivatives, other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole are activated to genotoxins mainly by rat P-450 1A1, which is inducible in rat lungs by 5,6-benzoflavone and the polychlorinated biphenyl mixture Aroclor 1254. Activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline and 2-amino-3-methylimidazo[4,5-f]quinoline may be catalyzed by another P-450 enzyme because the activities were not induced by treatment with 5,6-benzoflavone or Aroclor 1254. The observation that both activities were inhibited by antibodies raised against P-450 1A2 and by 7,8-benzoflavone suggests a role for an enzyme of P-450 1A family, probably P-450 1A2, in rat lung microsomes. The activation of aflatoxin B1 and sterigmatocystin appears to be catalyzed by other P-450 enzyme(s) rather than the P-450 1A family as judged by the different responses of activities to the P-450 inducers and the specific antibodies in rat lung microsomes. Interestingly, lung microsomal activation of several procarcinogens was found to be suppressed in rats treated with isosafrole and pregnenolone 16 alpha-carbonitrile. Thus, the results support the roles of different P-450 enzymes in the activation of procarcinogens in rat lung microsomes.     

The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line

When grown in the standard Dulbecco's medium the human liver derived Hep G2 hepatoma cell line shows only 10-20% of the cytochrome P-450-dependent mixed function oxidase (MFO) activity of freshly isolated human adult hepatocytes. However, the MFO activities and, to a lesser extent, the activities of UDP-glucuronyltransferase and glutathione-S-transferase can be increased by altering the composition of the growth medium. Modified Earle's medium was more effective in this respect than Williams' E medium and increased the O-dealkylations of ethoxyresorufin, benzyloxyresorufin and pentoxyresorufin 50-, 30- and 10-fold, respectively.     

Content of cytochrome P-450 and its induction capacity in primary liver tumors in rats

The content of cytochrome P-450 has been measured in primary hepatomas induced by diethylnitrosamine. As a rule, the enzyme content in hepatomas was decreased, as compared to normal liver and tumor-affected liver, but some hepatomas contained cytochrome P-450 in greater amount than normal tissue. Aroclor 1254 induced an increase in cytochrome P-450 content, which was identical in hepatomas, normal liver and tumor-affected liver. The dependence of hepatoma morphology on cytochrome P-450 content was not detected.     

Biochemical bases of ftorafur-potentiating effects of perfluorodecalin, inducer of cytochrome P-450 dependent microsomal monooxygenases]

Activity of cytochrome P-450-dependent monooxygenase system is significantly lower in hepatoma H-2-73 and Lewis lung carcinoma-bearing mice as compared to that in control animals. An inhibition of the cytochrome P-450 system was observed after the injection of ftorafur. Treatment of the mice with perfluorodecalin markedly increased the antineoplastic activity of ftorafur determined by a loss of the leucocytes and of the weight hepatoma H-2-73- and Lewis lung carcinoma resistant to ftorafur alone.     

Genotoxicity of ethyl carbamate in the Drosophila wing spot test: dependence on genotype-controlled metabolic capacity

Ethylcarbamate has a clear genotoxic potential in Drosophila melanogaster. In the somatic mutation and recombination test (SMART) it induces, in a dose-dependent manner, single spots as well as twin spots. The twin spots indicate a recombinogenic activity of ethyl carbamate. A strong strain difference demonstrates that the metabolic activation of ethyl carbamate to DNA binding metabolites most probably also in Drosophila involves cytochrome P450-dependent enzyme activities.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

Effect of magnetic fields on rodent monooxygenase enzymes

The effects of 50 Hz, 1.2 mT magnetic fields (MFs) were tested on hepatic monooxygenase enzymes of basal and β-naphthoflavone-phenobarbital-preinduced rats and mice. An inductive effect on cytochrome P-450 level and on some enzymatic cytochrome P-450-dependent activities was observed in basal mice after MF exposure. Enzymatic activities in preinduced mice and rats were reduced by MFs, the degree of reduction depending on the enzyme. A specific inhibitory effect was determined in some of the assayed activities and in the relative peculiar P-450 isoforms detected by Western blot analysis. © 1995 Wiley-Liss, Inc.     

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.     

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. I. Chromosomal determination of some cytochrome P-450-dependent reactions

The genetic regulation of some cytochrome P-450-dependent enzyme activities has been studied in adult Drosophila. Strains having genetically determined high or low enzyme activities were crossed with a marker strain and the metabolism was analyzed in microsomes from hybrids carrying different combinations of chromosomes from the strain under test. High p-nitroanisole (PNA) N-demethylation, biphenyl 3-hydroxylation and an increased amount of a protein with an apparent mol. wt. of 54 000, after SDS-gel electrophoresis of the microsomes in insecticide-resistant Drosophila strains, are inherited as dominant second chromosome traits. A low capacity for benzo[a]pyrene (BP) hydroxylation and 7-ethoxycoumarin O-deethylation in the Hikone R strain is semidominantly inherited in both cases and determined by gene(s) on the third chromosome. A semidominantly inherited high 4-hydroxylation of biphenyl and a high amount of a protein with an apparent mol. wt. of 56 000 in the Oregon R strain are also localized to the second chromosome. The results indicate that several other cytochrome P-450-dependent activities are not regulated by the genes mentioned above. In conclusion, at least three genes regulating the cytochrome P-450 system in Drosophila have been identified.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Xenobiotic-metabolizing enzymes and benzo[a]pyrene metabolism in the benzo[a]pyrene-sensitive mutant strain of Drosophila simulans.

Basal levels of aryl hydrocarbon hydroxylase, epoxide hydrolase and glutathione S-transferase enzyme activities, cytochrome P-450 content and inducibility of enzymes with phenobarbital were found to be similar in the microsomes of D. simulans mutant strain 364yv, which is sensitive to the toxic and mutagenic effects of benzo[a]pyrene (BP), and of the wild resistant Turku strain. In contrast, increases in the rate of BP turnover per molecule of cytochrome P-450, intensity of the hemoprotein band with apparent molecular weight 56,000 and the yield of BP 7,8-dihydrodiol and 9,10-dihydrodiol occurred only in microsomes of BP-pretreated 364yv flies but not of Turku ones. It is likely that BP induces an aberrant form of cytochrome P-450 in 364yv flies with a rare mutation in one of the P-450 regulating genes.     

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.     

The alkane-hydroxylating enzyme system of the yeast Candida guilliermondii.

The growth of the investigated Candida guilliermondii strain on n-alkanes induces an alkane-hydroxylating enzyme system, which consists of a cytochrome P-450 and a NADPH-dependent reductase. The cytochrome P-450 was purified to 4 nmoles per mg protein. Long-chain alkanes, preferably hexadecane to octadecane, are hydroxylated to the corresponding primary alcohol by this enzyme system. The substrate induces a type I spectrum, other compounds checked type II spectra.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the $\text{in}$$\text{vitro}$ modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).