Biomimetic models for monooxygenases

The microsomal mixed function oxidase system contains the cytochrome P-450 oxidative drug metabolizing family of enzymes. The catalytic cycle of cytochrome P-450 is believed to involve the formation of an active iron-oxygen species which is responsible for oxygen transfer to the substrate. This assumption is supported by the fact that a number of peroxidative agents can replace NADPH, the reductase, and oxygen as co-reactants in most oxidative reactions of microsomal cytochrome P-450. We have found that a mixture of either ferrous or ferric ions with hydrogen peroxide (Fenton and Ruff reagents) can serve as biomimetic models for cytochrome P-450 in hydroxylation, exposidation, sulfoxidation, and N-demethylation of various drugs. The existance of an iron-oxo active species in both Fenton and Ruff type reactions has been postulated and provides reaction cycles similar to those of cytochrome p-450. Other model systems for the hepatic hydroxylation and epoxidation using transition metal complexes with porphyrin are also discussed. The present paper reviews the various biomimetic models of the heme cytochrome P-450 and emphasizes their simulation of hepatic drug metabolism and their potential medical and industrial applications.     

The human hepatic cytochromes P450 involved in drug metabolism.

The cytochromes P450 are a superfamily of hemoproteins that catalyze the metabolism of a large number of xenobiotics and endobiotics. The type and amount (i.e., the animal's phenotype) of the P450s expressed by the animal, primarily in the liver, thus determine the metabolic response of the animal to a chemical challenge. A majority of the characterized P450s involved in hepatic drug metabolism have been identified in experimental animals. However, recently at least 12 human drug-metabolizing P450s have been characterized at the molecular and/or enzyme level. The characterization of these P450s has made it possible to "phenotype" microsomal samples with respect to their relative levels of the various P450s and their metabolic capabilities. The purpose of this review is to compare and contrast the human P450s involved in drug metabolism with their related forms in the rat and other experimental species.     

Essential role of singlet oxygen species in cytochrome P450-dependent substrate oxygenation by rat liver microsomes

Previously, we reported that singlet oxygen (1O2) was involved in rat liver microsomal P450-dependent substrate oxygenations in such reactions as p-hydroxylation of aniline, O-deethylation of 7-ethoxycoumarin, omega- and (omega-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine. In order to confirm the generality of 1O2 involvement, we have further investigated which kinds of reactive oxygen species (ROS) are formed during P450-dependent substrate oxygenation in microsomes. We examined CYP2E1-dependent hydroxylation of p-nitrophenol in rat liver microsomes in the presence of some ROS scavengers, because CYP2E1 has been reported to predominantly generate ROS in the hepatic microsomes and to relate with the oxidative stress in the body. The addition of 1O2 quenchers, beta-carotene, suppressed the hydroxylation of p-nitrophenol. Furthermore, a nonspecific P450 inhibitor, SKF525A, and a ferric chelator, deferoxamine, both suppressed the hydroxylation. No other ROS scavengers such as superoxide dismutase (SOD), catalase, or mannitol altered the reaction. 1O2 was detectable during the reaction in the microsomes as measured by an electron spin resonance (ESR) spin-trapping method when 2,2,6,6-tetramethyl-4-piperidone (TMPD) was used as a spin-trapping reagent. The 1O2 was quenched by additions of beta-carotene, p-nitrophenol, and SKF525A. The reactivity of p-nitrophenol and 1O2 correlated linearly with its hydroxylation rate in the microsomes. On the basis of these results, we conclude that 1O2 contributes to the p-nitrophenol hydroxylation in rat liver microsomes, by adding a new example of 1O2 involvement in the CYP2E1-dependent substrate oxygenations.     

Evidence for singlet oxygen involvement in rat and human cytochrome P450-dependent substrate oxidations

Recently, we proposed that singlet oxygen ((1)O(2)) plays an essential role in microsomal cytochrome P450 (P450)-dependent p-hydroxylation of aniline and O-deethylation of 7-ethoxycoumarin. We then examined whether the role of (1)O(2) is general in the P450-dependent substrate oxidations. In the present study, we examined omega- and (omega-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine in rat liver microsomes. The addition of beta-carotene and NaN(3) significantly suppressed these reactions in a concentration-dependent manner, and (1)O(2) during the reactions was detected by ESR spin-trapping using 2,2,6,6-tetramethyl-4-piperidone (TMPD) as a (1)O(2)-spin trapping reagent, where the addition of (1)O(2) quenchers, SKF-525A as a P450 inhibitor, or p-nitroanisole decreased ESR signal intensities due to TMPD-(1)O(2) adduct. Next, we examined the effect of (1)O(2) quenchers on P450-dependent reactions in the human liver microsomes, and (1)O(2) was also indicated to be an active species in substrate hydroxylations and dealkylations such as nifedipine oxidation by CYP3A4. On the basis of the results, we concluded that (1)O(2) is an essentially important active oxygen species in both rat and human P450-dependent substrate oxidations.     

Oxidative inactivation of cytochrome P-450 1A (CYP1A) stimulated by 3,3',4,4'-tetrachlorobiphenyl: production of reactive oxygen by vertebrate CYP1As.

Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost fish scup) is inactivated by the aryl hydrocarbon receptor agonist 3,3',4,4'-tetrachlorobiphenyl (TCB). Here, the mechanism of CYP1A inactivation and its relationship to reactive oxygen species (ROS) formation were examined by using liver microsomes from scup and rat and expressed human CYP1As. In vitro inactivation of scup CYP1A activity 7-ethoxyresorufin O-deethylation by TCB was time dependent, NADPH dependent, oxygen dependent, and irreversible. TCB increased microsomal NADPH oxidation rates, and CYP1A inactivation was lessened by adding cytochrome c. CYP1A inactivation was accompanied by loss of spectral P-450, a variable loss of heme and a variable appearance of P-420. Rates of scup liver microsomal metabolism of TCB were < 0.5 pmol/min/mg, 25-fold less than the rate of P-450 loss. Non-heme iron chelators, antioxidant enzymes, and ROS scavengers had no influence on inactivation. Inactivation was accelerated by H(2)O(2) and azide but not by hydroxylamine or aminotriazole. TCB also inactivated rat liver microsomal CYP1A, apparently CYP1A1. Adding TCB to scup or rat liver microsomes containing induced levels of CYP1A, but not control microsomes, stimulated formation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-expressed human CYP1A1 but not CYP1A2. The results indicate that TCB uncouples the catalytic cycle of CYP1A, ostensibly CYP1A1, resulting in formation of ROS within the active site. These ROS may inactivate CYP1A or escape from the enzyme. ROS formed by CYP1A1 may contribute to the toxicity of planar halogenated aromatic hydrocarbons.     

Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells

Mitochondrial P450 type enzymes catalyze central steps in steroid biosynthesis, including cholesterol conversion to pregnenolone, 11beta and 18 hydroxylation in glucocorticoid and mineralocorticoid synthesis, C-27 hydroxylation of bile acids, and 1alpha and 24 hydroxylation of 25-OH-vitamin D. These monooxygenase reactions depend on electron transfer from NADPH via FAD adrenodoxin reductase and 2Fe-2S adrenodoxin. These systems can function as a futile NADPH oxidase, oxidizing NADPH in absence of substrate, and leak electrons via adrenodoxin and P450 to O(2), producing superoxide and other reactive oxygen species (ROS). The degree of uncoupling depends on the P450 and steroid substrate. Studies with purified proteins and overexpression in cultured cells show consistently that adrenodoxin, but not reductase, is responsible for ROS production that can lead to apoptosis. In the ovary and corpus luteum, antioxidant enzyme activities superoxide dismutase, catalase, and glutathione peroxidase parallel steroidogenesis. Antioxidant beta-carotene, alpha-tocopherol, and ascorbate can protect against oxidative damages of P450 systems. In testis Leydig cells, steroidogenesis is associated with aging of the steroidogenic capacity.     

Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and β-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from β-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.     

Propranolol as an inhibitor of some cytochrome P450-dependent monooxygenase activities in native and induced rat liver microsomes

The in vitro effect of propranolol (10(-3) M and 10(-4) M), a nonselective and extensively metabolized beta-adrenergic blocking agent, on rat liver drug metabolism in native and induced (with phenobarbital and beta-naphthoflavone [beta-NF]) microsomes was studied. The type of inhibition and the inhibitory constants of some cytochrome P450-dependent microsomal enzyme reactions (hexobarbital oxidation [HBO], ethylmorphine-N-demethylation [EMND], aniline hydroxylation [AH], ethoxycoumarin-O-deethylation [ECOD], ethoxyresorufin-O-dealkylation [EROD] and penthoxyresorufin-O-dealkylation [PROD]) were estimated. The results showed that propranolol competitively inhibited AH activity in native microsomes. The type of inhibition was changed from competitive to noncompetitive in all other enzyme activities studied. This inhibition was more pronounced after phenobarbital induction in PROD (Ki = 0.11 +/- 0.01 mM), ECOD (Ki = 0.40 +/- 0.09 mM) and EMND (Ki = 0.59 +/- 0.1 mM), and after beta-NF induction in AH (Ki = 0.28 +/- 0.05 mM) and in HBO (Ki = 0.35 +/- 0.1 mM) in native microsomes. It was assumed that the noncompetitive type of inhibition is due to the covalent binding of reactive metabolites derived from propranolol to hepatic microsomal proteins. The competitive type of inhibition of AH suggested a common P450 isoenzyme in the metabolism of propranolol and aniline. Thus, in this study, propranolol has been found to be not only a selective inhibitor of CYP2D6 isoenzyme-dependent reactions, but also a nonspecific inhibitor of other cytochrome P450 isoenzymes.     

Ethylbenzene induces microsomal oxygen free radical generation: antibody-directed characterization of the responsible cytochrome P450 enzymes

Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to its fluorescent product 2',7'-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H(2)O(2)) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to influence free radical production and that the increase in free radical production in EB treated rats is consistent with the EB-mediated elevation of CYP2B, CYP 2E1, and CYP3A2. Such alterations in free radical generation in response to hydrocarbon treatment may contribute to the toxicity of these compounds.     

Overexpression of cytochrome P450 NADPH reductase sensitises MDA 231 breast carcinoma cells to 5-fluorouracil: possible mechanisms involved.

Activity of cytochromes P450 is highly dependent on cytochrome P450 NADPH reductase (P450R), but this enzyme can also metabolise drugs on its own. MDA 231 breast adenocarcinoma cells transfected with human P450R (MDA R4) or an empty vector (MDA EV) were exposed to a series of commonly used chemotherapeutic drugs. Overexpression of P450R did not affect cell sensitivity to cisplatin, mitoxantrone, paclitaxel, docetaxel, vincristine or etoposide. However, MDA R4 cells showed increased sensitivity to mitomycin C (6.6-fold) and also to 5-fluorouracil (2.8-fold). In vitro toxicity assays where mitomycin C, 5-fluorouracil and vincristine were preincubated with microsomes expressing recombinant P450R showed that this effect was not a result of direct metabolism by P450R. Levels of NADPH were considerably decreased in MDA R4 as compared to MDA EV cells, while reactive oxygen species (ROS) production was increased in MDA R4 cells in basal conditions, showing no significant further increase after treatment with mitomycin C or 5-fluorouracil. P450R overexpression appears therefore to be detrimental to MDA 231 cells, depleting NADPH and increasing ROS levels; the increased oxidative stress observed in MDA R4 cells might explain the enhanced sensitivity to 5-fluorouracil. Expression of this enzyme in tumour cells might therefore modulate response to 5-fluorouracil.     

Enzymes involved in the metabolism of the carcinogen 2-nitroanisole: evidence for its oxidative detoxication by human cytochromes P450

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Determining the capability of humans to metabolize 2-NA and understanding which human cytochrome P450 (P450) enzymes are involved in its activation and/or detoxification are important to assess an individual's susceptibility to this environmental carcinogen. We compared the ability of hepatic microsomal samples from different species including human to metabolize 2-NA. Comparison between experimental animals and human P450 enzymes is essential for the extrapolation of animal carcinogenicity data to assess human health risk. Human hepatic microsomes generated a pattern of 2-NA metabolites, reproducing that formed by hepatic microsomes of rats and rabbits. An O-demethylated metabolite of 2-NA (2-nitrophenol) and two ring-oxidized derivatives of this metabolite (2,6-dihydroxynitrobenzene and 2,X-dihydroxynitrobenzene) were produced. No nitroreductive metabolism leading to the formation of o-anisidine was evident with hepatic microsomes of any species. Likewise, no DNA binding of 2-NA metabolite(s) measured with either tritium-labeled 2-NA or the (32)P-postlabeling technique was detectable in microsomes. Therefore, hepatic microsomal P450 enzymes participate in the detoxication reactions of this environmental carcinogen. Using hepatic microsomes of rabbits pretreated with specific P450 inducers, microsomes from Baculovirus transfected insect cells expressing recombinant human P450 enzymes, purified P450 enzymes, and selective P450 inhibitors, we found that human recombinant P450 2E1, 1A1, and 2B6, as well as orthologous rodent P450 enzymes, are the most efficient enzymes metabolizing 2-NA. The role of specific P450 enzymes in the metabolism of 2-NA in human hepatic microsomes was investigated by correlating specific P450-dependent reactions with the levels of 2-NA metabolites formed by the same microsomes and by examining the effects of specific inhibitors of P450 enzymes on 2-NA metabolism. On the basis of these studies, we attribute most of the 2-NA oxidation metabolism in human microsomes to P450 2E1. These results, the first report on the metabolism of 2-NA by human P450 enzymes, clearly demonstrate that P450 2E1 is the major human enzyme oxidizing this carcinogen in human liver.     

Cytochrome P-450-dependent formation of reactive oxygen radicals: isozyme-specific inhibition of P-450-mediated reduction of oxygen and carbon tetrachloride

1. Ethanol-inducible P450 IIE1 exhibits a high rate of oxygen consumption and oxidase activity. The enzyme is selectively distributed in the liver centrilobular area, the acinar region specifically destroyed after treatment with P450 IIE1 substrates/inducers such as ethanol, carbon tetrachloride, chloroform, N-nitrosodimethylamine and paracetamol. 2. Twenty substrates and ligands for cytochrome P450 IIB4 and P450 IIE1 were evaluated for their ability to inhibit microsomal and reconstituted NADPH-dependent oxidase activity, and the P450 IIE1-catalysed reduction of carbon tetrachloride to chloroform. Type I ligands and substrates did not inhibit the processes whereas nitrogen-containing compounds such as octylamine, cimetidine, imidazole and tryptamine inhibited NADPH oxidation and H2O2 formation in microsomes from starved and acetone-treated rats by around 50%. 3. Tryptamine, octylamine, isoniazid and p-chloroamphetamine inhibited reconstituted P450 IIE1-dependent oxidase activity with half maximal effects at 14-170 microM. 4. Isoniazid, cimetidine and tryptamine inhibited the P450 IIE1-dependent reduction of carbon tetrachloride, whereas acetone was without effect. 5. The oxygen dependency of microsomal oxidase activity exhibited high-affinity and low-affinity phases, with partial saturation at 20 microM of O2. 6. It is concluded that microsomal oxidase activity takes place at physiological concentrations of O2 and that isozyme-specific type II ligands compete with oxygen or carbon tetrachloride for reduction by P-450 haem.     

CYP450氧化还原酶的药物基因组学研究进展

细胞色素P450氧化酶(cytochrome P450enzymes,CYP)的氧化还原反应是人体内重要的生理生化反应,参与许多内、外源化合物的代谢和激素类化合物的合成.CYP450氧化还原酶(cytochrome P450 oxidoreductase,POR)是所有肝微粒体内CYP酶的唯一电子供体.POR不仅可作为电子供体参与由CYP介导的药物代谢,而且可通过1-电子还原反应直接介导一些抗肿瘤前体药物的代谢和转化.可见,POR在药物代谢过程中发挥着极其重要的作用.众多研究证实,编码人POR的基因具有遗传多态性,对临床药物代谢乃至疗效有着显著影响,具有重要的临床意义.下面对近年来POR的药物基因组学最新研究进展作一综述.     

Interaction of metyrapone with adrenal microsomal cytochrome P450 in the guinea pig

Studies were carried out to compare the actions of metyrapone on adrenal mitochondrial and microsomal cytochrome P450-containing enzymes in the guinea pig and rat. As expected. addition of metyrapone to adrenal mitochondria inhibited 11β-hydroxylation in both species. the shape of the type II difference spectrum produced by metyrapone in mitochondria differed somewhat in rat (gDO.D._425?405nm) and guinea pig (gDO.D._425?390nm) and the magnitude of the speetrum was far greater in rat adrenal mitochondria, paralleling species differences in cytochrome P450 concentration (rat > guinea pig). In rat adrenal microsomes, metyrapone produced a small “reverse type I” spectral change (ΔO.D._420-385nm) but did not affect either 21-hydroxylation or the interaction of progesterone with eytochrome P450(as determined spectrally). In guinea pig adrenal microsomes, in contrast, metyrapone produced a large type II spectral change (ΔO.D._423-408nm) and inhibited both 21-hydroxylation and ethylmorphine demethylation, cytochrome P450-dependent reactions. The magnitudes of type I spectra produced by 17α-hydroxyprogesterone and ethylmorphine in guinea pig adrenal microsomes were significantly diminished by prior addition of metyrapone. The results indicate that metyrapone interacts with both microsomal and mitochondrial cytochrome P450 in the guinea pig and that its adrenal sites of action, therefore, are species dependent.     

Oxidative deboronation of the peptide boronic acid proteasome inhibitor bortezomib: contributions from reactive oxygen species in this novel cytochrome P450 reaction

Bortezomib (1) is a potent first-in-class dipeptidyl boronic acid proteasome inhibitor employed in the treatment of patients with relapsed multiple myeloma where the disease is refractory to conventional lines of therapy. The potency of 1 is owed primarily to the presence of the boronic acid moiety, one which is suited to establish a tetrahedral intermediate with the active site N-terminal threonine residue of the proteasome. Hence, deboronation of 1 represents a deactivation pathway for this chemotherapeutic agent. Deboronation of 1 affords a near equal mixture of diastereomeric carbinolamide metabolites (M1/M2) and represents the principal metabolic pathway observed in humans. In vitro results from human liver microsomes and human cDNA-expressed cytochrome P450 enzymes (P450) indicate a role for P450 in the deboronation of 1. Use of 18O-labeled oxygen under controlled atmospheres confirmed an oxidative mechanism in the P450-mediated deboronation of 1, as 18O was found incorporated in both M1 and M2. Chemically generated reactive oxygen species (ROS), such as those generated as byproducts during P450 catalysis, were also found to deboronate 1 resulting in the formation of M1 and M2. Known to undergo efficient redox cycling, P450 2E1 was found to catalyze the deboronation of 1 predominantly to the carbinolamide metabolites M1 and M2, as well as to a pair of peroxycarbinolamides, 2 and 3. The presence of superoxide dismutase (SOD) and catalase prevented the deboronation of 1, thus, supporting the involvement of ROS in the P450 2E1-catalyzed deboronation reaction. The presence of SOD and catalase also protected 1 against P450 3A4-catalyzed deboronation, albeit to a lesser extent. The remaining deboronation activity observed in the P450 3A4 reaction may suggest the involvement of the more conventional activated enzyme-oxidants previously described for P450. Our present findings indicate that the oxidase activity of P450 (i.e., formation of ROS) represents a mechanism of deboronation.     

Reconstitution of the interplay between cytochrome P450 and human glutathione S-transferases in clozapine metabolism in yeast

Clozapine, an often-prescribed antipsychotic drug, is implicated in severe adverse drug reactions (ADRs). Formation of reactive intermediates by cytochrome P450s (CYPs) has been proposed as a possible explanation for these ADRs. Moreover, a protective role for human glutathione S-transferases (hGSTs) was recently shown using purified enzymes. We investigated the interplay between CYP bioactivation and GST detoxification in a reconstituted cellular context using recombinant yeast expressing a bacterial CYP BM3 mutant (M11), mimicking the drug-metabolizing potential of human CYPs, combined with hGSTA1-1, M1-1 or P1-1. Clozapine and the N-desmethylclozapine metabolite caused comparable growth inhibition and reactive oxygen species (ROS) formation, whereas the clozapine-N-oxide metabolite was clearly less toxic. Clozapine metabolism by BM3 M11 and the hGSTs in yeast was confirmed by identification of stable clozapine metabolites and hGST isoform-specific glutathione-conjugates. Oxidative metabolism of clozapine by BM3 M11 increased ROS formation and growth inhibition. Co-expression of hGSTP1-1 protected yeast from BM3 M11 induced growth inhibition in presence of clozapine, whereas similar expression levels of hGSTA1-1 and hGSTM1-1 did not. ROS formation was not lowered by hGSTP1-1 co-expression and was unrelated to mitochondrial electron transport chain (mETC) activity. We present a novel cellular model to study the effect of CYP and GST interplay in drug toxicity.     

Inhibition of drug oxidation and stimulation of NADPH oxidase in vitro by doxorubicin and triferric-doxorubicin

The electrophilic properties of the quinone-hydroquinone configuration of anthracycline antibiotics suggests a possible influence on cytochrome P-450-mediated mono-oxygenase reactions. Both doxorubicin and triferric-doxorubicin (a derivative in which the quinone groups are blocked with iron) showed a similar dose-dependent inhibition of liver microsomal drug metabolism. A doxorubicin concentration-related stimulation of NADPH oxidase activity was found to be linear but that for triferric-doxorubicin was asymptotic. Neither inhibitor affected the activity of cytochrome c reductase, cytochrome b5 reductase or cytochrome P-450 reductase. However, doxorubicin did potentiate the inhibitory effect of aniline on cytochrome P-450 reductase and on ethylmorphine metabolism. It is concluded that these anthracyclines inhibit drug metabolism in vitro not by their electron-withdrawing potential but in a manner more similar to that described for type II compounds.     

Metabolism related toxicity of diclofenac in yeast as model system

Diclofenac is a widely used drug that can cause serious hepatotoxicity, which has been linked to metabolism by cytochrome P450s (P450). To investigate the role of oxidative metabolites in diclofenac toxicity, a model for P450-related toxicity was set up in Saccharomyces cerevisiae. We expressed a drug-metabolizing mutant of cytochrome P450 BM3 (BM3 M11) in yeast. Importantly, BM3 M11 yielded similar oxidative metabolite profiles of diclofenac as human P450s. It was found that yeast strains expressing BM3 M11 grew significantly slower when exposed to diclofenac than strains without BM3 M11. Furthermore, the amount of reactive oxygen species (ROS) after incubation with diclofenac was higher in strains expressing BM3 M11 than in strains without this enzyme, confirming that P450 activity increases diclofenac toxicity. Interestingly, 4′- and 5-hydroxydiclofenac had no effect on cell growth or ROS formation in cells expressing BM3 M11, although hydroxydiclofenac-derived quinone imines were identified in these strains by detection of their glutathione conjugates. This suggests that 4′- and 5-hydroxydiclofenac, as well as their quinone imines, are not involved in toxicity in yeast. Rather, the P450-related toxicity of diclofenac is caused by primary metabolites such as arene oxides resulting in hydroxydiclofenac or radical species formed during decarboxylation.     

The effects of adriamycin in vitro and in vivo on hepatic microsomal drug-metabolizing enzymes: role of microsomal lipid peroxidation

The quinone-containing anticancer drug adriamycin augments the reduction of dioxygen to reactive oxygen species and thereby stimulates (sixfold) NADPH-dependent microsomal lipid peroxidation. In vitro the extensive adriamycin-promoted peroxidation depleted (85%) rat liver microsomal cytochrome P-450, severely inhibited cytochrome P-450-dependent monooxygenation (70%), and glucose-6-phosphatase activity (80%), and activated (450%) UDP-glucuronyltransferase activity. When lipid peroxidation was blocked by EDTA, adriamycin selectively decreased cytochrome P-450 and aminopyrine N-demethylase activity; NADPH-cytochrome c reductase, UDP-glucuronyltransferase, and glucose-6-phosphatase activities were unchanged. Washing and resedimenting peroxidized microsomes to remove adriamycin and soluble lipid peroxidation products failed to restore enzyme activities to control values. Adriamycin administered subacutely (5 mg/kg × three doses) to rats significantly descreased hepatic microsomal cytochrome P-450 content and reduced aminopyrine N-demethylase and NADPH-cytochrome c reductase activities compared to saline-treated controls. Microsomal lipid peroxidation was increased following the above adriamycin treatment. Thus, these data suggested that adriamycin was capable of impairing hepatic drug metabolism in vitro by stimulating membrane lipid peroxidation in a manner similar to carbon tetrachloride; the mechanism by which adriamycin treatment in vivo decreased the activity of the drug monooxygenase system remains unclear.