The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Cytochrome P-450-dependent catabolism of triethanolamine in Rhodotorula mucilaginosa

The yeast Rhodotorula mucilaginosa was able to grow in media containing triethanolamine or diethanolamine as the sole nitrogen source. During growth in the presence of triethanolamine, extracts of yeast cells contained increased levels of cytochrome P-450 dependent monooxygenase which catalyzed the oxidative N-dealkylation of aminoalcohols. Formation of diethanolamine, ethanolamine and glyoxylate from triethanolamine was demonstrated, and the identity of the products was verified by thin layer chromatography. These observations suggested the following scheme of triethanolamine catabolism: triethanolamine diethanolamine + glycolaldehyde, diethanolamine ethanolamine + glycolaldehyde, ethanolamine NH₃ + glycolaldehyde glycolate glyoxylate glycerate pathway.     

Comparative studies on pharmacokinetics and metabolism of the anti-juvenile hormone precocene II

The cuticular penetration and pharmacokinetics of the anti-juvenile hormone precocene II were determined in a sensitive species (Oncopeltus fasciatus) and an insensitive species (Heliothis zea). Precocene was sequestered by the fat body and slowly metabolized in Oncopeltus, but rapidly metabolized and excreted in Heliothis. Studies in vitro using inhibitors for cytochrome P-450 and for cyt P-450-NADPH-reductase, confirmed the anticipated detoxification of precocene by a mixed-function oxidase via the 3,4-epoxide. Use of the inhibitors in vivo had no influence on the metabolism of precocene.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

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.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Induction of cytochrome P-450-dependent drug metabolism by juvenile hormone mimics in mammalian cell culture

The monooxygenase activity of fetal hepatocytes in culture shows a differential response toward juvenile hormone I and analogs. Juvenile hormone I, R-20458, and Methoprene increase the deethyiation of 7-ethoxyresorufin while not affecting or even inhibiting the N-demethylation of p-chloro-N-methylaniline. RO-203600, a 1,3-benzodioxole-containing analog, increases both the deethylase and the N-demethylase, whereas Hydroprene does not affect either activity. The inductive effect with juvenile hormone I is obtained with exposure periods of at least 30 min and is maximum when the concentration of the hormone is 14 μM in the medium. This amount results in the covalent binding to cellular macromolecules of 1.3 × 19^?18 moles/cell. The induction requires continuous protein synthesis but RNA synthesis only for a short initial period. It is concluded that juvenile hormone and mimics induce specific cytochrome P-450 species in fetal liver cells even if the culture conditions are not optimal. The toxicological implications of these results are briefly discussed.     

微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。     

Cytochrome P-450 monooxygenase,epoxide hydrolase and flavin monooxygenase activities in clara cells and alveolar type II cells isolated from rabbit

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC 7-ethoxycoumarin - DMA N,N-dimethylaniline     

The expression of cytochrome P-450 and cytochrome P-450 reductase genes in the simultaneous transformation of corticosteroids and phenanthrene by Cunninghamella elegans

The expression of cytochrome P-450 and cytochrome P-450 reductase (CPR) genes in the conterminous biotransformation of corticosteroids and PAHs was studied in Cunninghamella elegans 1785/21Gp. We had previously used this strain as a microbial eucaryotic model for studying the relationship between mammalian steroid hydroxylation and the metabolization of PAHs. We reported that cytochrome P-450 reductase is involved in the biotransformaton of cortexolone and phenanthrene. RT-PCR and Northern blotting analyses indicated that the cytochrome P-450 and CPR genes appear to be inducible by both steroids and PAHs. The expression of the cytochrome P-450 gene was increased ninefold and the expression of the CPR gene increased 6.4-fold in cultures with cortexolone and/or phenanthrene in comparison with controls. We conclude that the increase in cytochrome P-450 gene expression was accompanied by an increase in cytochrome P-450 enzymatic activity levels.     

Purification and some properties of cytochrome P-450 specific for steroid 17 alpha-hydroxylation and C17-C20 bond cleavage from guinea pig adrenal microsomes

The energy requirements for mitochondrial protein synthesis were investigated in isolated rat liver mitochondria. Controlled changes in coupling efficiency were obtained by titration with FCCP in the presence of various substrates. No relationship was observed between the efficiency of oxidative phosphorylation and the inhibition of protein synthesis. With succinate-ADP as the substrate the ADP:O ratio was decreased by 70–80% with no effect on protein synthesis. In contrast, with acetate-ADP as substrate, a 10–20% reduction in the ADP:O ratio gave complete inhibition of protein synthesis. The data suggest that the rate of ATP production is more important for maintenance of protein synthesis than the efficiency of coupling $\text{per se}$. Thus, certain substrates can support maximal rates of protein synthesis even in relatively poorly coupled mitochondria. Analysis of mitochondrial translation products formed in the presence of increasing FCCP concentrations also showed that decreased efficiency of oxidative phosphorylation had no influence on the nature of the products.     

Aniline is hydroxylated by the cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism

Hydroxylation of aniline, catalyzed by rabbit liver microsomal cytochromes P-450 in reconstituted systems, was inhibited by catalase, superoxide dismutase, catechol, mannitol, hydroquinone, dimethylsulfoxide and benzoate, whereas the cytochrome P-450-catalyzed O-demethylation of paranitroanisole, measured under the same conditions, was unaffected by these agents. A similar inhibition profile of the hydroxylation reaction was observed in reconstituted systems where cytochrome P-450 had been replaced by hemoglobin. The results indicate that aniline hydroxylation is mediated by hydroxyl radicals generated in an iron-catalyzed Haber-Weiss reaction between O₂^? and H₂O₂ and may explain some of the special properties of this reaction previously described.     

Cytochrome P-450- and Peroxidase-Dependent Activation of Procarbazine and Iproniazid in Mammalian Cells

Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metab olized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.     

Induction of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah)—Responsive mice

Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At > 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70X), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58X) and P-450d (8X), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ahresponsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P₃-450 and a very small increase in P₁-450 in the responsive strain. The increase in P₁-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P₃-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [³H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [³H]-TCDD only at very high concentrations (10^?6 to 10^?5 M).     

Cytochrome P-450- and Peroxidase-Dependent Activation of Procarbazine and Iproniazid in Mammalian Cells

Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metab olized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Cytochrome P-450 Family 1 in Rat Embryo Cell Culture Immortalized by Rausher Leukemia Virus

We studied comparative expression and activity of cytochrome P450 family 1 (CYP1) isoforms in rat embryo cells, both primary and immortalized by Rausher leukemia virus (RLV). In RLV-infected embryonal cells compared with the initial ones the expression levels of CYP1A1 and 1B1 mRNAs and benzo[a]pyrene (BP) hydroxylase activity were higher, regardless of their treatment with the CYP1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. The sensitivity to BP and 7,12-dimethylbenzo[a]anthracene was higher in the cells immortalized with RLV. The expression level of mRNAs of induction-mediating proteins aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator was the same in both cell cultures tested. Higher sensitivity of cells immortalized with RLV compared with the initial embryo cells to transforming effect of BP, which was described previously, is possibly associated with elevated expression of CYP1 isoforms.     

Gamma-glutamyltransferase induction by dexamethasone in cytochrome P-450-depleted rat liver

The effects of the cytochrome P-450 depletion by cobaltic protoporphyrin IX on the postnatal glucocorticoid-inducibility of the membrane-bound enzyme gamma-glutamyltransferase have been assessed in the rat liver. Dexamethasone-induced gamma-glutamyltransferase activity in 14-, 28- and 77-day-old rats was high, weak and absent, respectively, and inversely correlated with the physiological cytochrome P-450 activity. In the liver acinus, the enzyme was reexpressed by the zone 1 and zone 2 hepatocytes in suckling rats, substantially only by the zone 1-hepatocytes in just weaned rats. Following cytochrome P-450 depletion, gamma-glutamyltransferase induction by dexamethasone was more rapid, more intense and more extended in the liver, acinus, occurring also in the zone 3 hepatocytes in suckling rats, in the zone 2 and a few zone 3 hepatocytes in just weaned rats. Further, the enzyme induction occurred also in adult rats in the zone 1 and in some zone 2 cells. This shows that cytochrome P-450 modulates the extent of hepatic gamma-glutamyltransferase induction by dexamethasone in postnatal rat-hepatocytes. The phenomenon may be consequent on hormone biotransformation changes caused by the cytochrome P-450 depletion.     

Regional Distribution of Cytochrome P-450 in the Rat Brain: Spectral Quantitation and Contribution of P-450b,e and P-450c,d

The cytochrome P-450 (P-450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P-450b,e and P-450c,d to brain P-450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P-450b and P-450c, respectively). P-450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p-chloroamphetamine-coupled Sepharose. The yield of P-450 from whole brain was 90 +/- 19 pmol/g of tissue, which is approximately 1% of the level in liver microsomes from control rats. The amount of P-450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P-450 content, with yields of up to 400 pmol/g whereas the substantia nigra yielded only 16-20 pmol/g. Immunohistochemical studies with anti-P-450b and anti-P-450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti-P-450c. In the cerebellum, both anti-P-450b and anti-P-450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti-P-450b gave weak staining of certain astroglia, but with anti-P-450c, there was intense staining of neuronal somata.(ABSTRACT TRUNCATED AT 250 WORDS)