Involvement of NADPH: cytochrome P450 reductase in the activation of indoloquinone EO9 to free radical and DNA damaging species.

Evidence suggests that DT-diaphorase is involved in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9 under aerobic conditions. Data also implicate a role for other enzymes including NADPH: cytochrome P450 reductase, especially in low DT-diaphorase tumour cells and under hypoxic conditions. Here, we used purified rat NADPH: cytochrome P450 reductase to provide additional evidence in support of a role for this enzyme in activation of EO9 to generate free radical and DNA-damaging species. Electron spin resonance spectrometry studies showed that NADPH: cytochrome P450 reductase reduced EO9 to a free radical species, including a drug radical (most likely the semiquinone) and reactive oxygen species. Plasmid DNA experiments showed that reduction of EO9 catalysed by NADPH: cytochrome P450 reductase results in single-strand breaks in DNA. The information obtained may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9 and may be useful in the design of future bioreductive drugs.     

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.     

Nuclear localization of NADPH:cytochrome c (P450) reductase enhances the cytotoxicity of mitomycin C to Chinese hamster ovary cells

Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence. Immunofluorescent imaging of transfected cell lines expressing the fusion protein confirmed the nuclear location of the enzyme. Regardless of the oxygenation state of the cell, mitomycin C (MC) cytotoxicity was enhanced in cells with overexpressed NPR localized to the nuclear compartment compared with cells overexpressing an endoplasmic reticulum localized enzyme. Enhanced cytotoxicity in cells treated under hypoxic conditions correlated with increases in genomic DNA alkylations, with more MC-DNA adducts being formed when the enzyme was expressed closer to its DNA target. No change was observed in the hypoxic/aerobic differential toxicity as a function of enzyme localization. These findings indicate that drug efficacy is increased when the subcellular site of drug activation corresponds to its site of action.     

Modulation of hepatic and renal metabolism and toxicity of trichloroethylene and perchloroethylene by alterations in status of cytochrome P450 and glutathione

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.     

Cytochrome P450 CYP1B1 protein expression: a novel mechanism of anticancer drug resistance

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line (P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.     

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.     

The enzymology of doxorubicin quinone reduction in tumour tissue.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.     

Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes.

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.     

Role of NADPH:cytochrome P450 reductase in the hypoxic accumulation and metabolism of BRU59-21, a technetium-99m-nitroimidazole for imaging tumor hypoxia

Nitroimidazoles labeled with technetium-99m are being investigated as non-invasive markers of tumor hypoxia. They are bioreductive compounds that require enzymatic reduction for retention in hypoxic cells, but little is known about the cellular factors affecting their accumulation in hypoxic cells. If the absolute accumulation of hypoxia markers is affected by enzyme levels, an inaccurate assessment of the hypoxic cell fraction in tumors may occur. BRU59-21, (99m)Tc-oxo[[3,3,9, 9-tetramethyl-6-[(2-nitro-1H-imidazol-1-yl)methyl]5-oxa-4, 8-diazadioximato]-(3-)-N,N',N",N"'] technetium (V), a technetium-99m-nitroimidazole that is being studied as a potential marker of tumor hypoxia, was used in the present study to evaluate the effect of NADPH:cytochrome P450 reductase (EC 1.6.2.4) levels on BRU59-21 accumulation and metabolism. Metabolism of BRU59-21 in hypoxic cellular lysates derived from Chinese hamster ovary cells overexpressing NADPH:cytochrome P450 reductase was 8-fold greater than in control cells. This effect required the presence of exogenous NADPH. The increased metabolism of BRU59-21 in lysates overexpressing NADPH:cytochrome P450 reductase was inhibited at 4 degrees and by the addition of NADPH:cytochrome P450 reductase inhibitors. The addition of inhibitors of other nitroreductase enzymes had no effect on BRU59-21 metabolism in these lysates. When the accumulation and metabolism of BRU59-21 were studied in stirred suspension cultures, it was found that cells overexpressing NADPH:cytochrome P450 reductase exhibited about a 3-fold increase in both the hypoxic metabolism and the accumulation of BRU59-21. These findings suggest that NADPH:cytochrome P450 reductase is an important enzyme in BRU59-21 metabolism in model systems of tumor hypoxia.     

Metabolic activation of 4-hydroxyanisole by isolated rat hepatocytes.

A tyrosinase-directed therapeutic approach for treating malignant melanoma uses depigmenting phenolic prodrugs such as 4-hydroxyanisole (4-HA) for oxidation by melanoma tyrosinase to form cytotoxic o-quinones. However, in a recent clinical trial, both renal and hepatic toxicity were reported as side effects of 4-HA therapy. In the following, 4-HA (200 mg/kg i.p.) administered to mice caused a 7-fold increase in plasma transaminase toxicity, an indication of liver toxicity. Furthermore, 4-HA induced-cytotoxicity toward isolated hepatocytes was preceded by glutathione (GSH) depletion, which was prevented by cytochrome p450 inhibitors that also partly prevented cytotoxicity. The 4-HA metabolite formed by NADPH/microsomes and GSH was identified as a hydroquinone mono-glutathione conjugate. GSH-depleted hepatocytes were much more prone to cytotoxicity induced by 4-HA or its reactive metabolite hydroquinone (HQ). Dicumarol (an NAD(P)H/quinone oxidoreductase inhibitor) also potentiated 4-HA- or HQ-induced toxicity whereas sorbitol, an NADH-generating nutrient, prevented the cytotoxicity. Ethylenediamine (an o-quinone trap) did not prevent 4-HA-induced cytotoxicity, which suggests that the cytotoxicity was not caused by o-quinone as a result of 4-HA ring hydroxylation. Deferoxamine and the antioxidant pyrogallol/4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL) did not prevent 4-HA-induced cytotoxicity, therefore excluding oxidative stress as a cytotoxic mechanism for 4-HA. A negligible amount of formaldehyde was formed when 4-HA was incubated with rat microsomal/NADPH. These results suggest that the 4-HA cytotoxic mechanism involves alkylation of cellular proteins by 4-HA epoxide or p-quinone rather than involving oxidative stress.     

The nature of halogen substitution determines the mode of cytotoxicity of halopropanols

The cytochrome P450-dependent generation of reactive metabolites from 1,3-dichloropropanol and 1,3-dibromopropanol was assessed in a microsomal thiol depletion assay, while the toxicity of these compounds was assessed in rat hepatocyte cultures and in the 3T3 cell line. Thiol-depleting metabolites of both compounds were generated in the microsomal assay; however, only dibromopropanol extensively depleted glutathione when glutathione S-transferase was used as the enzyme source. The cytotoxicity of dichloropropanol was both cytochrome P450- and glutathione-dependent, whereas that of dibromopropanol was glutathione-dependent but largely independent of cytochrome P450. These results indicate that the mechanisms underlying the cytotoxicity of halopropanols are dependent on the nature of the halogen substitution and that microsomal and cellular assays for reactive metabolite generation may yield conflicting results.     

An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro.

The formation of chemically reactive metabolites from carbamazepine (CBZ) in the presence of mouse and human liver microsomes has been investigated using cytotoxicity and irreversible binding of radiolabelled compound as quantitative end-points. For comparison, the formation of the stable CBZ-10,11-epoxide (CBZ-10,11-E) has been measured. The formation of the cytotoxic, protein-reactive and stable metabolites of CBZ was increased by induction of the cytochrome P450 enzymes by phenobarbitone and reduced by co-incubation in vitro with ketoconazole (10-250 microM), suggesting that the formation of these metabolites is cytochrome P450 dependent. All human livers tested (N = 6) bioactivated CBZ to a protein-reactive metabolite, the mean covalent binding increasing from 0.08 +/- 0.01% (without NADPH) to 0.27 +/- 0.09% (with NADPH; P less than or equal to 0.05). The formation of the chemically reactive metabolites was reduced by a subphysiological concentration of reduced glutathione (GSH) (500 microM), while ascorbic acid (100 microM) had no effect. Neither compound affected the formation of CBZ-10,11-E. Microsomal epoxide hydrolase (mEH), but not cytosolic epoxide hydrolase, caused a concentration-dependent inhibition of cytotoxicity reaching a maximum of 60% at 100 U of mEH. Covalent binding was also reduced by 60% by 100 U mEH. The separated T- and B-lymphocytes showed no difference in sensitivity when incubated with CBZ and mouse microsomes. The study demonstrates that the balance between activation of CBZ by the cytochrome P450 enzymes to a chemically reactive arene oxide metabolite and its detoxification by mEH and GSH may contribute to individual susceptibility to CBZ idiosyncratic toxicity.     

Antitumor activity of methoxymorpholinyl doxorubicin: potentiation by cytochrome P450 3A metabolism

Methoxymorpholinyl doxorubicin (MMDX) is a novel liver cytochrome P450 (P450)-activated anticancer prodrug whose toxicity toward cultured tumor cells can be potentiated up to 100-fold by incubation with liver microsomes and NADPH. In the present study, a panel of human liver microsomes activated MMDX with potentiation ratios directly correlated to the CYP3A-dependent testosterone 6beta-hydroxylase activity of each liver sample. Microsome-activated MMDX exhibited nanomolar IC(50) values in growth-inhibition assays of human tumor cell lines representing multiple tissues of origin: lung (A549 cells), brain (U251 cells), colon (LS180 cells), and breast (MCF-7 cells). Analysis of individual cDNA-expressed CYP3A enzymes revealed that rat CYP3A1 and human CYP3A4 activated MMDX more efficiently than rat CYP3A2 and that human P450s 3A5 and 3A7 displayed little or no activity. MMDX cytotoxicity was substantially increased in Chinese hamster ovary cells after stable expression of CYP3A4 in combination with P450 reductase. CYP3A activation of MMDX abolished the parent drug's residual cross-resistance in a doxorubicin-resistant MCF-7 cell line that overexpresses P-glycoprotein. CYP3A-activated MMDX displayed a comparatively high intrinsic stability, with a t(1/2) of approximately 5.5 h at 37 degrees C. MMDX was rapidly activated by CYP3A at low ( approximately 1-5 nM) prodrug concentrations, with 100% tumor cell kill obtained after as short as a 2-h exposure to the activated metabolite. These findings demonstrate that MMDX can be activated by CYP3A metabolism to a potent, long-lived, and cell-permeable cytotoxic metabolite and suggest that this anthracycline prodrug may be used in combination with CYP3A4 in a P450 prodrug activation-based gene therapy for cancer treatment.     

Cytochrome P450 monooxygenases in crustaceans

1. The hepatopancreas is the major site of cytochrome P450-dependent xenobiotic monooxygenation in crustacean species, but the presence of monooxygenase inhibitors in hepatopancreas microsomes and cytosol from many decapod species has impeded in vitro studies. Cytochrome P450 and monooxygenase activities have been reported in other crustacean organs including the antennal gland (green gland) and stomach. 2. NADPH cytochrome c reductase activity is often very low (typically less than 10 nmol cytochrome c reduced/min per mg microsomal protein) in hepatopancreas microsomes from crustacean species. NADPH cytochrome P450 reductase activity has not yet been detected in crustacean hepatopancreas microsomes. 3. The cytochrome P450 present in hepatopancreas of several crab species and the spiny lobster has been resolved into several fractions by chromatography on DEAE-cellulose. One form of cytochrome P450 from spiny lobster has been purified to 12 +/- 2 nmol/mg protein. 4. Reconstitution studies with spiny lobster hepatopancreas P450 have shown that the vertebrate sex steroids, progesterone and testosterone, are excellent substrates, whereas ecdysone--the crustacean molting hormone--is not a substrate. Activity was found with several xenobiotic substrates including benzphetamine, aminopyrine, benzo(a)pyrene, ethyl-, benzyl- and pentyl-phenoxazones and ethoxycoumarin. Highest activities (greater than 50 nmol/min per nmol P450) were found for N-demethylation of benzphetamine and aminopyrine. 5. The ability of agents which induce vertebrate cytochrome P450 to induce cytochrome P450 in crustaceans is still unclear. Some studies indicate that polycyclic aromatic hydrocarbons, but not phenobarbital-type inducers, could induce cytochrome P450 in crustaceans, whereas other studies showed no effect of either inducer type. Crustaceans are not as sensitive as fish to induction of P450 and monooxygenase activity.     

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.     

The relative importance of NADPH: cytochrome c (P450) reductase for determining the sensitivity of human tumour cells to the indolequinone EO9 and related analogues lacking functionality at the C-2 and C-3 positions

Analogues of EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2[1H-indole-4-7-dione]prop-2-e n-1-ol) which lack functionality at either the C-2 or C-3 position were synthesised. The aim was to establish the importance of each group towards toxicity and to give an indication as to whether substitution at either position altered activation and toxicity after metabolism by cellular NADPH: cytochrome c (P450) reductase (P450R). MDA231 breast cancer cells were transfected with the cDNA for human P450R and stable clones were isolated. These high P450R-expressing clones were used to determine the aerobic and hypoxic toxicity of EO9 and the two analogues that lacked functionality at either C-2 or C-3. The results showed that P450R was strongly implicated in the bioactivation of EO9 and its analogues under both of these conditions. This data also showed that the C-3 functionality was primarily implicated in hypoxic toxicity.     

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.     

Oxidative stress mediated idiosyncratic drug toxicity

The following describes a novel screening method for "new chemical entities" (NCEs), suitable for ADMET studies, that measures ability to form prooxidant radicals on metabolism and their ability to induce oxidative stress in intact cells. The accelerated molecular cytotoxic mechanism screening (ACMS) techniques used with isolated rat hepatocytes showed that cytotoxicity is usually initiated as a result of macromolecular covalent binding or macromolecular oxidative stress. While P450 is likely responsible for drug metabolic activation in the liver, intestine, lung, and in other nonhepatic tissues, where P450 levels are low, peroxidases including prostaglandin synthetase peroxidase can catalyze xenobiotic one-electron oxidation to form prooxidant free radicals that may cause toxicity or carcinogenesis. Inflammation markedly activates H2O2, generating NADPH oxidase and peroxidase of certain immune cells when they infiltrate tissues including the liver. Myeloperoxidase and NADPH oxidase in the Kupffer cells (resident macrophages of the liver) also become activated during inflammation. The addition of noncytotoxic concentrations of peroxidase/H2O2 to the hepatocyte incubate markedly increased drug cytotoxicity and prooxidant radical formation as shown by glutathione or lipid oxidation. Many drugs that have hepato- or gastrointestinal (GI) toxicity problems or were withdrawn from the market for safety problems, e.g., troglitazone, tolcapone, mefenamic acid, diclofenac, and phenylbutazone, were markedly more toxic and prooxidant in this inflammation model system, whereas other drugs, e.g., entacapone, were not toxic in this inflammation model. Some of the idiosyncratic hepatotoxicity responsible for recent drug withdrawals may therefore result from commonplace sporadic inflammatory episodes during drug therapy.     

Application of the DataChip/MetaChip technology for the evaluation of ajoene toxicity in vitro

The DataChip is a universal platform for three-dimensional (3D) cell cultures on a micropillar chip, which can be applicable to a variety of human cells to simulate organ-specific toxicity. In addition, the MetaChip is developed for various combinations of drug metabolizing enzymes that can be spotted into the microwell chip and incubated with 3D human cells to simulate systematic compound metabolism in the human liver on a microscale format. Ajoenes have been known for various therapeutics activities, including anticancer effects, but there was limited information available in regard to their metabolism and cytotoxicity. In the present work, the metabolism-mediated toxicity of ajoenes was evaluated on a DataChip/MetaChip platform. In detail, we tested cytotoxicity of E- and Z-ajoene on 3D cultured Hep3B human hepatoma cells coupled with mixtures of drug metabolizing enzymes. Metabolic profiles of ajoenes were assessed with 23 representative drug metabolizing enzymes on the MetaChip. As a result, cytotoxicity of E-ajoene was significantly augmented in the presence of cytochrome P450 (CYP) isoforms, such as CYP2E1 and CYP3A5. Both E- and Z-ajoene were drastically detoxified in the presence of Phase II enzymes, including major UGTs, SULTs, NATs, and GSTs. Interestingly, All Mix, an artificial human liver microsome containing representative P450 mixture and phase II enzyme mixture, attenuated P450-induced cytotoxicity of ajoenes. Conclusively, we were able to confirm the metabolism-medicated toxicity of ajoenes on the chip.     

The effect of the anthrapyrazole antitumour agent CI941 on rat liver microsome and cytochrome P-450 reductase mediated free radical processes. Inhibition of doxorubicin activation in vitro

The anthrapyrazole CI941 is one of a new series of DNA complexing drugs which displays high level broad spectrum antitumour activity in mice. In view of the proposed role of drug free radical formation, superoxide generation and lipid peroxidation in anthracycline and anthraquinone induced toxicities, the redox biochemistry of CI941 has been investigated. Studies have been performed in vitro using rat liver microsomes and purified cytochrome P-450 reductase. In addition, the ability of CI941 to undergo chemical reduction has been examined. Pulse radiolysis of CI941 demonstrated that the drug can undergo chemical reduction with a one electron reduction potential of E1(7) = -538 +/- 10 mV. However, electron spin resonance (ESR) spectroscopy studies using either NADPH fortified microsomes or cytochrome P-450 reductase, failed to detect a drug free radical signal. Unlike doxorubicin, CI941 (150 microM) inhibited basal rate microsomal NADPH consumption by 45%. Furthermore, CI941 (50-200 microM) antagonised doxorubicin stimulated (1.8-fold) NADPH oxidation by over 50%. CI941 also antagonised the formation of a doxorubicin free radical ESR signal in a concentration dependent manner. CI941 induced minimal superoxide generation in the presence of either microsomes or cytochrome P-450 reductase and inhibited doxorubicin induced (50 microM) superoxide formation by up to 80% (50-200 microM CI941). Importantly, CI941 inhibits both basal rate and doxorubicin (100 microM) stimulated lipid peroxidation (52% inhibition at 5 microM CI941). These data suggest that CI941 is unlikely to induce free radical mediated tissue damage in vivo. On the contrary, CI941 may have a protective role if used in combination with doxorubicin.