Cellular Cytochrome P-450 Research Articles

Effect of polyaromatic hydrocarbons on cellular cytochrome P450 1A induction

Effect of polyaromatic hydrocarbons on cellular cytochrome P450 1A induction

3D cell clusters combined with a bioreactor system to enhance the drug metabolism activities of C3A hepatoma cell lines.

Since clinical drugs need to be approved for their liver metabolism efficiency before commercialization, a powerful in vitro drug-screening platform is imperative and indispensable for the clinical medicine and pharmaceutical industries. An essential issue in the development of drug screening platforms is choosing cell candidates that mimic and perform cell/tissue functions of normal hepatic tissues in vivo. In this study, we developed a self-designed bioreactor system to provide and mimic an appropriate environment for systematic cell expansion, micro-tissue formation, and increased cellular cytochrome P450 (CYP) enzymatic activities. Since CYP3A4 is the most plentiful and crucial enzyme in drug metabolism among liver CYP superfamily members, we demonstrated that micro-tissue formation under three-dimensional dynamic conditions could enhance cellular CYP3A4 enzymatic activity, maintain cell viability, and preserve adhesive abilities. Furthermore, Ca-alginate scaffolds used in this study can be completely removed by a non-toxic chelating reagent (EDTA solution), and the functional micro-tissues can be collected by slow-speed centrifugation. In conclusion, these micro-tissues are advantageous and show great potential in in vitro drug metabolizing assays.

Induction of estradiol 2-hydroxylase activity in MCF-7 human breast cancer cells by pesticides and carcinogens

The induction of 17 β-estradiol (E2) 2-hydroxylase activity was investigated in MCF-7 human breast cancer cells using 2-[ 3H]E2 as the substrate in a radiometric assay. Treatment of MCF-7 cells with 10 μM indole-3-carbinol (I3C) for 48 h caused a 3.5-fold induction of E2 2-hydroxylase activity, whereas, I3C at concentrations as high as 100 μM did not induce CYP1A1 mRNA levels or immunoreactive protein. Thus, the induction of E2 2-hydroxylase activity using the radiometric assay was not dependent on induction of CYP1A1. E2 2-hydroxylase activity was also increased by I3C within 2 h after treatment suggesting in situ interactions with the cellular cytochrome P450 system. The time-dependent effects of various chlorinated pesticides, antiestrogens and mammary carcinogens on E2 2-hydroxylase activity were also investigated. p,p′-DDE, atrazine and the mammary carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) significantly decreased E2 2-hydroxylase activity after 2 h; whereas, only the latter two compounds decreased activity after 48 h. Both 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and the mammary carcinogen benzo[a]pyrene (BaP) induced E2 2-hydroxylase in MCF-7 cells after incubation for 48 h and this was also paralleled by induction of CYP1A1 protein. The antiestrogens ICI 164 384 and ICI 182 780 decreased E2 2-hydroxylase activity in MCF-7 cells after incubation for 48 h, whereas tamoxifen and 4-hydroxytamoxifen were inactive. The results indicate that chemical-induced modulation of E2 2-hydroxylase activity in MCF-7 cells is complex and does not predict their activity as mammary carcinogens.

Corticosterone 6 beta-hydroxylase in A6 epithelia: a steroid-inducible cytochrome P-450

We found microsomal corticosterone 6 beta-hydroxylase (6 beta-OHase) from cultured A6 kidney epithelial cells to be a cytochrome P-450 enzyme with both similarities to and differences from the rat liver steroid 6 beta-OHase P-450p. Enzyme activity was inhibited by CO, alpha-naphthoflavone, metyrapone, and clotrimazole, well-known inhibitors of P-450 enzymes, and increased by known inducers of P-450 enzymes, including dilantin, phenobarbital sodium, and corticosteroids. Moreover, some additional, relatively specific inducers of P-450p (troleandomycin and pregnenolone-16 alpha-carbonitrile) also induced the A6 6 beta-OHase, whereas inducers of other forms of P-450 (aroclor, spironolactone, and isosafrole) appeared to repress the A6 enzyme. The time course of increase in enzyme activity and increased cellular cytochrome P-450 content were consistent with increased levels of enzyme protein. Induction of 6 beta-OHase by the substrate (corticosterone), the metabolite (6 beta-OH-corticosterone), dexamethasone, and aldosterone was biphasic as a function of inducer concentration, with approximate 50% effective concentration (EC50) values of 10(-8)-10(-9) M and 10(-5)-10(-6) M for the respective components of induction. Cortisol also induced the enzyme at 10(-8)-10(-6) M; however, its metabolite 6 beta-OH-cortisol was ineffective or decreased activity at higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Function and regulation of cytochrome P-450 in alkane-assimilating yeast

Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO2=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO2 of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.

Cobalt-protoporphyrin suppresses thyroid and testicular hormone concentrations in rat serum: a novel action of this synthetic heme analogue.

Treatment of rats with Co-protoporphyrin, a synthetic metalloporphyrin which produces a marked and sustained induction of hepatic heme oxygenase and decline in cellular cytochrome P-450 content, caused a significant reduction in serum testosterone, serum thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels. These endocrine changes were not accompanied by reciprocal elevations in either serum luteinizing hormone (LH) or thyroid-stimulating hormone (TSH) concentrations. Seminal vesicles from Co-protoporphyrin-treated animals were atrophic. The pituitary response to luteinizing hormone-releasing hormone (LHRH) in Co-protoporphyrin-treated animals was moderately attenuated (approximately equal to 50%) but the response to thyrotropin-releasing hormone (TRH) was normal. These findings suggest that at least part of the effects of the metalloporphyrin are mediated at the level of the hypothalamus, such that suppression of serum levels of testosterone, T4 and T3 fails to elicit compensatory pituitary outputs of the respective trophic hormones. It is not known whether these effects are due to direct actions of Co-protoporphyrin on the endocrine system or whether they are secondary consequences of other metabolic derangements, such as those related to the pronounced cellular depletion of heme and cytochrome P-450 caused by the synthetic metalloporphyrin.

Carbon tetrachloride and trichloroethylene toxicities to rat hepatocytes in primary monolayer culture: Its relationship to the level of cytochrome P-450

The effects of carbon tetrachloride (CCl 4) and trichloroethylene (TCE) on the synthesis and the secretion of triacylglycerols (TGs) in primary cultured rat hepatocytes were investigated in relation to the level of cellular cytochrome P-450 (P-450). Pretreatment of rats with phenobarbital (PB) and plating the hepatocytes in the presence of metyrapone attained a marked preservation of P-450 during the preparation of monolayer. CC1 4 was able to cause the accumulation of cellular TG in the hepatocytes when the content of P-450 was retained at the level equivalent to that in the liver in vivo.

Spectral properties of substrate-cytochrome P-450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout liver cells

A method for the isolation of intact viable rainbow trout liver cells in high numbers is described. The technique involves perfusion of collagenase through the liver. A major part of the cytochrome P-450 in isolated liver cells was present in the oxidized non-substrate bound form. It was observed that 7-ethoxycoumarin was rapidly taken up by the liver cells and bound to cellular cytochrome P-450. The substrate binding spectrum for isolated trout liver cells was slightly modified compared with that obtained with trout liver microsomes. The microsomal affinity of 7-ethoxycoumarin, calculated as the apparent spectral dissociation constant ( k s), was elevated 11-fold after fish were treated with β-napthoflavone, indicating a qualitative alteration in the nature of the constititive cytochrome P-450. The metabolism of 7-ethoxycoumarin in isolated liver cells was found to be af a comparable rate to that obtained in liver microsomes. Pretreatment of fish with Clophen A50 or β-naphthoflavone significantly increased the content of cytochrome P-450 and elevated the rate of 7-ethoxycoumarin deethylation in isolated liver cells. Furthermore, the rate of conjugation of 7-hydroxycoumarin was significantly elevated in liver cells isolated from β-naphthoflavone treated fish when compared with the control rate. In isolated liver cells, 90% of the 7-hydroxycoumarin formed from deethylation of 7-ethoxycoumarin was further metabolized to conjugated products. However, in β-naphthoflavone of Clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactions in the cell.

Control of heme and cytochrome P-450 metabolism by inorganic metals, organometals and synthetic metalloporphyrins.

The heme-cytochrome P-450 complexes represent sensitive metabolic systems for examining the biological impact of metals on important cellular functions. Many metals, both in the inorganic form and bound to organic moieties, potently induce heme oxygenase, the rate limiting enzyme of heme degradation. The resulting increase in the rate of heme breakdown is reflected in a marked depression of cellular cytochrome P-450 content and impairment of the oxidative metabolism of natural and foreign chemicals dependent on this hemeprotein. Organometal complexes do not mimic in all their aspects the actions of the inorganic elements which they contain. For example, organotins, in contrast to inorganic tin, produce a prolonged induction response of heme oxygenase in the liver but not in the kidney. Co-protoporphyrin is a much more potent inducer of heme oxygenase in liver than is inorganic cobalt; and Sn-protoporphyrin inhibits heme oxygenase activity nearly completely, whereas inorganic tin is a powerful inducer of the renal enzyme. Contrasting effects on heme metabolism exist as well within the metalloporphyrin species as demonstrated by the effects in vivo of Co-protoporphyrin and Sn-protoporphyrin on heme oxygenase activity; the former induces the enzyme whereas the latter potently inhibits it. In vitro, however, both compounds competitively inhibit heme oxidation activity. These differences, among others which characterize metal actions in vivo and in vitro attest to the importance of pharmacokinetic, adaptive and other host factors in defining the responses of the heme-cytochrome P-450 systems to the impact of metals in the whole animal.

The metabolism of aflatoxin B1 by hepatocytes isolated from rats following the in vivo administration of some xenobiotics

Isolated rat hepatocytes, an intact cellular system capable of performing phase I and phase II metabolism, have been used to investigate metabolism of aflatoxin B1. These cells were found to metabolise [14C]aflatoxin B1 to aflatoxins M1 and Q1, and to radiolabelled polar material, presumably conjugates, as analysed by h.p.l.c., t.l.c. and radioactive determination. In vivo administration of the mixed function oxidase inducers, phenobarbitone and 3-methylcholanthrene, resulted in enhanced hepatocyte phase I (microsomal) metabolism of aflatoxin B1. In contrast to metabolism of AFB1 by in vitro subcellular systems increased production of polar material (conjugated metabolites) derived from [14C]aflatoxin B1 was also detected in hepatocytes isolated from these pretreated animals. Formation of aflatoxin Q1 by isolated hepatocytes appeared to be mediated by cytochrome P450-linked enzymes whereas cytochrome P448-linked enzymes were apparently involved in aflatoxin M1 production. Chronic feeding of aflatoxin B1 to rats enhanced hepatocyte production of conjugated material only and did not elevate cellular cytochrome P450 levels, thus suggesting that aflatoxin B1 is not an inducer of its own primary metabolism.

Spectral studies on the rapid uptake and subsequent binding of drugs to cytochrome P-450 in isolated rat liver cells

Summary The rate of formation of the type I spectral change upon drug addition to a suspension of isolated rat liver cells was used to study factors that influence drug uptake by the hepatocytes. Although considerably slower than in liver homogenates and microsomes, drug combination with cellular cytochrome P-450 was still rapid and occurred within a few seconds. The effects of varying temperature and concentration and lipid solubility of the drugs studied as well as the lack of effect of preincubation of the cells with rotenone on the rate of formation of the type I spectral change, lead us to suggest that drug uptake into the hepatocytes occurs by a non-energy requiring diffusion process.