A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl₄-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species.     

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.     

Inhibition of Cumene Hydroperoxide-Induced Lipid Peroxidation by a Novel Pyridoindole Antioxidant in Rat Liver Microsomes

Abstract: The ability of stobadine, a novel pyridoindole antioxidant, to inhibit lipid peroxidation induced by cumene hydroperoxide was investigated in rat liver microsomes. In the micromolar range stobadine effectively inhibited lipid peroxidation as measured by the formation of thiobarbituric acid reactive products. The peroxidation-related degradation of microsomal cytochrome P-450 was prevented by stobadine in the same pattern. Another line of evidence in support of the antioxidant action of stobadine was given by its inhibition of cumene hydroperoxide-induced oxygen consumption in microsomal incubations. Inhibition of lipid peroxidation was not a function of decreased bioactivation of cumene hydroperoxide, as stobadine did not affect the rate of cytochrome P-450 dependent cleavage of cumene hydroperoxide. Neither had stobadine any effect on cytochrome P-450 peroxidase function characterized by the rate of cumene hydroperox-ide-dependent oxidation of TMPD, and no direct spectral interaction with microsomal cytochrome P-450 was observed in the micromolar region. We suggest that it is the ability of stobadine to scavenge alkoxyl and peroxyl radicals that is predominantly responsible for the observed antioxidant effect.     

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoxide (LAHP) were compared. In the absence of added Fe2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloromercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation, the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe2+, whereas LAHP-dependent lipid peroxidation is autocatalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H2O2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.     

Halomethane hepatotoxicity: induction of lipid peroxidation and inactivation of cytochrome P-450 in rat liver microsomes under low oxygen partial pressures

Halomethane-induced lipid peroxidation and inactivation of cytochrome P-450 were studied in liver microsomes from phenobarbital-pretreated rats in the presence of NADPH at steady-state O2 partial pressures (PO2). As indicated by the formation of thiobarbituric acid-reactive material and the stimulation of O2 uptake, significant lipid peroxidation was induced by those halomethanes containing more than two Cl, Br, or I atoms. Lipid peroxidation decisively depended on the PO2 present, showing distinct maxima at PO2 between 1 and 10 mm Hg. Those halomethanes inducing lipid peroxidation also led to inactivation of microsomal cytochrome P-450, as indicated by a loss of cytochrome P-450 detectable as ferrous CO complex and an equimolar loss of microsomal heme. Under anaerobic conditions inactivation of cytochrome P-450 presumably resulted solely from an attack of halomethane radicals on its heme moiety. Under aerobic conditions lipid peroxidation made an additional contribution to the inactivation of cytochrome P-450. These results suggest that the reductive activation to free radicals, catalyzed by cytochrome P-450, and thus the induction of lipid peroxidation at low but physiological PO2 are characteristic not only of CCl4 but also of other polyhalogenated methanes, especially CBrCl3, CBr4, CHI3, CHBr3, and CHBr2Cl.     

Analysis of the effects of inhibitors on NADPH-dependent electron transport in rat liver microsomes.

Analysis of the effects of inhibitors on NADPH-oxidation in rat liver microsomes shows that the rate of total oxygen consumption parallels the rate of peroxidation of unsaturated fatty acids but not the rate of electron transfer within the chain. The addition of EDTA, 1 mM, to the incubation medium inhibits peroxidation and does not affect NADPH-dependent electron transfer. There are at least three sites for oxygen reduction within the NADPH-oxygenase complex. Oxygen reduction in the chain may occur both at the flavoprotein level and at the level of cytochrome P-450. In addition, Fe²⁺ ions bound with the functional groups of electron carriers and catalyzing the peroxidation of unsaturated fatty acids also participate in oxygen reduction. The fact that there are several points of activation of molecular oxygen within the NADPH-dependent electron transport chain precludes the evaluation of the effectiveness of action of inhibitors on cytochrome P-450 by measurement of the total oxygen consumption. NADH oxidation inhibited by EDTA is activated by addition of Ca²⁺.     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

Carbon tetrachloride-induced loss of microsomal glucose 6-phosphatase and cytochrome P-450 in vitro

Rat liver microsomes were incubated in the presence of NADPH and CCl4 under various conditions, and losses of glucose 6-phosphatase (G-6-Pase) and cytochrome P-450 were examined in terms of lipid peroxidation and CCl4 metabolism. Loss of G-6-Pase activity correlated well with the enhancement of lipid peroxidation. Loss of cytochrome P-450 was also dependent on the lipid peroxidation, under aerobic conditions. However, the cytochrome was destroyed under anaerobic conditions in which lipid peroxidation and loss of G-6-Pase were greatly suppressed. This anaerobic loss of cytochrome P-450 may be linked with the metabolism of CCl4 by this hemoprotein, as evidenced by the observation that CCl4 metabolism occurred only under anaerobic conditions and was inhibited by carbon monoxide accompanied by the suppression of the loss of cytochrome P-450.     

Rat lung and liver cytochrome P-450 isozymes involved in the hydroxylation of m-xylene

The primary metabolism of m-xylene in rat lung and liver microsomes was investigated. The ratio of side chain to aromatic hydroxylation was found to be approximately 1:1 in lung microsomes from untreated rats and in a reconstituted system containing the major cytochrome P-450 isozyme induced in rat liver by phenobarbital, cytochrome P-450-PB-B2, as compared to 8:1 in liver microsomes. Antibody inhibition studies showed the major importance of cytochrome P-450-PB-B2 for the formation of both primary m-xylene metabolites (3-methylbenzylalcohol and 2,4-dimethylphenol) in lung microsomes. Antibodies to the major cytochrome P-450 isozyme induced in rat liver by beta-naphthoflavone, P-450-BNF-B2, did not inhibit m-xylene metabolism in either liver or lung microsomes from beta-naphthoflavone treated rats although this isozyme efficiently catalyzed m-xylene hydroxylation in a reconstituted system. m-Xylene metabolism by purified P-450-BNF-B2 appeared to cause rapid inactivation of the enzyme.     

Effect of phenobarbital and 3-methylcholanthrene on the early oxidative stress component induced by lindane in rat liver.

1. Lindane administered to untreated rats or rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) increased liver lipid peroxidation, of the same magnitude in all groups. 2. PB pretreatment produced a 50% increase in lipid peroxidation (TBAR) by liver homogenates and microsomes, an effect accompanied by increases in cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH oxidase and microsomal superoxide anion production, MC pretreatment resulted in increases in liver cytochrome P-450 and NADPH oxidase only. 3. Pretreatment of rats with PB, but not MC or lindane, gave increases in glutathione peroxidase and reductase. 4. Pretreatment with PB, but not MC, increased liver GSH. Lindane decreased liver GSH to the same extent as PB plus lindane. 5. Biliary GSH, GSSG and bile flow were decreased by lindane to similar extents in all groups. 6. Lindane induced periportal necrosis with haemorrhagic foci in all groups. 7. Data presented indicate that the early lipid peroxidative response of liver to lindane was unchanged by PB- or MC-stimulated hepatic microsomal enzyme induction.     

Comparison of effects of acetaminophen on liver microsomal drug metabolism and lipid peroxidation in rats and mice.

Studies were conducted to determine the in vivo effect of acetaminophen (AAP) on the lipid peroxidation, drug metabolizing enzyme activity and microsomal electron transfer system of rat and mouse liver. AAP was found to inhibit ethylmorphine N-demethylase activity in the presence of NADPH and this inhibition of the enzyme was due to decrease in cytochrome P-450 content, but not due to change in lipid peroxidation in liver microsomes. Kinetical data showed that AAP administration had no effect on Km values of ethylmorphine N-demethylase, however, a decrease in the Vmax values was seen in rats and mice. There was no significant effect of AAP on both NADPH-cytochrome c reductase and the content of cytochrome b5 3 hours after this administration to rats and mice. On the other hand, AAP induced a significant decrease in NADH-ferricyanide reductase in mice, but not in rats. The greatest decrease in cytochrome P-450 observed among the components of the liver microsomal electron transfer system of rats and mice.     

Effect of linoleic acid hydroperoxide on liver microsomal enzymes in vitro.

Rat liver microsomes incubated with linoleic acid hydroperoxide (LAHPO) lost cytochrome P-450 specifically among the enzymes of microsomal electron transport systems. The loss of cytochrome P-450 content and glucose-6-phosphatase activity by LAHPO was accompanied by an increase in malondialdehyde (MDA) production. Turbidity of microsomal suspensions was decreased with increasing MDA production, but not proportionately. Diethyldithiocarbamate (DTC), N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol inhibited almost completely the LAHPO-induced MDA production of microsomes, however no perfect protection against the loss of cytochrome P-450 content and glucose-6-phosphatase activity was observed. The decrease of microsomal turbidity by LAHPO was little affected in the presence of DTC. Purified cytochrome P-450 was destroyed by LAHPO, with minimal protection by the compounds described above. These results suggest the possibility that the loss of microsomal enzyme activities during lipid peroxidation may be attributed largely to a direct attack on enzyme proteins by lipid peroxides rather than indirectly to a structural damage of microsomal membranes resulting from peroxidative breakdown of membrane lipids.     

Reinduction of the cytochrome P-450 system of the liver in rats exposed to polychlorinated biphenyls during starvation

Contents of cytochrome P-450 and b5, rates of oxidation of aniline, amidopyrine and dimethylaniline as well as activities of NADP-H- and ascorbate-dependent systems of lipid peroxidation (LPO) in rat liver microsomes five months after single administration of the mixture of polychlorinated diphenyls (PCD) significantly exceeded the control level. Starvation of the animals for 120 hours led to an additional increase of cytochrome P-450 content and LPO activation. The rat liver monooxygenase system retained the ability to respond to the inducing action of the mixture of PCD (500 mg/kg) during starvation.     

Combined effect of ethanol and carbon disulphide on cytochrome P-450 mono-oxygenase, lipid peroxidation and ultrastructure of the liver in chronically exposed rats

A 5-month treatment of rats with ethanol (10% solution in drinking water) stimulated aniline p-hydroxylase and the microsomal ethanol oxidizing system (MEOS) by 140 and 70%, respectively, cytochrome P-450 by 22% and accompanied by lipid peroxidation by 40% in microsomes. It also caused smooth endoplasmic reticulum (SER) proliferation and rough endoplasmic reticulum (RER) degranulation in hepatocytes. Repeated inhalatory exposure of rats to 1.5 g/m3 of CS2, 5 h daily, 5 days a week for 5 months decreased aniline p-hydroxylase and MEOS by 70 and 55% respectively, doubled hexobarbital sleeping time and depressed cytochrome P-450 by 30% and its conversion to cytochrome P-420; these effects were accompanied by the appearance of cytochrome P-420, the intensification of lipid peroxidation in microsomes and some degranulation of RER in hepatocytes. Combined exposure of rats to ethanol and CS2 resulted in a significant potentiation of the inhibitory effects of CS2 on cytochrome P-450 mono-oxygenase and MEOS but with enhancement of CS2 effects on the liver microsomal mono-oxygenase, but CS2 decreased the effect of ethanol on SER proliferation. The interaction both on the biochemical and the morphological level can be explained with the ethanol-stimulated biotransformation of CS2 to reactive electrophilic derivative(s), the subsequent destruction of cytochrome P-450 to cytochrome P-420 and the intensification of lipid peroxidation.     

Effect of phenobarbital and 3-methylcholanthrene on the early oxidative stress component induced by lindane in rat liver

1. Lindane administered to untreated rats or rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC) increased liver lipid peroxidation, of the same magnitude in all groups.

2. PB pretreatment produced a 50% increase in lipid peroxidation (TBAR) by liver homogenates and microsomes, an effect accompanied by increases in cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH oxidase and microsomal superoxide anion production, MC pretreatment resulted in increases in liver cytochrome P-450 and NADPH oxidase only.

3. Pretreatment of rats with PB, but not MC or lindane, gave increases in glutathione peroxidase and reductase.

4. Pretreatment with PB, but not MC, increased liver GSH. Lindane decreased liver GSH to the same extent as PB plus lindane.

5. Biliary GSH, GSSG and bile flow were decreased by lindane to similar extents in all groups.

6. Lindane induced periportal necrosis with haemorrhagic foci in all groups.

7. Data presented indicate that the early lipid peroxidative response of liver to lindane was unchanged by PB- or MC-stimulated hepatic microsomal enzyme induction.     

Effects of motorcycle exhaust inhalation exposure on cytochrome P-450 2B1, antioxidant enzymes, and lipid peroxidation in rat liver and lung

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2 h daily for 4 wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7-ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl(3) -ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.     

Inhibition of hepatic microsomal lipid peroxidation by endogenous glycogen in the rat

The effect of endogenous glycogen on lipid peroxidation was examined in hepatic microsomes from rats. Microsomes were prepared to retain endogenous hepatic glycogen (Pg+) or to minimize it (Pg-). The indices of lipid peroxidation examined included the rate of NADPH-dependent formation of malondialdehyde (MDA) and the concomitant destruction of cytochrome P-450 and decline in the linearity of benzphetamine N-demethylase activity in microsomes. Cytochrome P-450 was destroyed during benzphetamine N-demethylation in microsomes with the loss being more extensive in Pg- than in Pg+. The destruction of cytochrome P-450 and the concomitant loss in linearity of benzphetamine N-demethylation in Pg- were prevented by added EDTA. Added linoleic acid hydroperoxide (LAHP) also caused a time-dependent loss of cytochrome P-450 in microsomes with the rate being greater in Pg- than in Pg+. The results show that glycogen inhibits hepatic microsomal lipid peroxidation and suggest that variations in glycogen content may contribute to disparities in in vitro oxidative activities between different microsomal samples. Such disparities may be minimized by the removal of glycogen during the preparation of microsomes and then supplementing the incubation mixtures with EDTA. The in vivo relevance of the observed antioxidant effect of glycogen is discussed in terms of the possible modulation by the polysaccharide of hepatotoxicity by agents whose effects may be mediated by lipid peroxidation.     

Immunochemical analysis of a cytochrome P-450IA1 homologue in human lung microsomes

The monoclonal antibody MAb 1-7-1, which specifically binds to cytochromes P-450IA1 and P-450IA2 in 3-methylcholanthrene-induced rat liver microsomes, was used to identify a cytochrome P-450IA1 homologue in human lung microsomes. Although MAb 1-7-1 had similar affinity constants for human and rat microsomes, the amount bound to human lung microsomes was severalfold lower than that bound to microsomes from untreated rat or rabbit lung and much lower than the amount bound to 3-methylcholanthrene-induced rat lung or liver microsomes. The amount bound to untreated baboon lung microsomes was similar to that bound to human lung microsomes. Three cytochrome P-450IA1-catalyzed activities, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin, O-deethylase, and aryl hydrocarbon hydroxylase, were measurable in human lung microsomes, but the cytochrome P-450IA2-dependent activity acetanilide 4-hydroxylase was not. MAb 1-7-1 inhibited, and its binding correlated strongly with, 7-ethoxyresorufin O-deethylase activity (r = 0.92, p less than 0.01) in human lung microsomes. 7-Ethoxyresorufin O-deethylase activities in human lung were similar to those measured in untreated baboon lung but considerably lower than those present in untreated rabbit lung, untreated or 3-methylcholanthrene-induced rat lung and liver, or human liver. We conclude that MAb 1-7-1 recognizes a cytochrome P-450IA1 homologue in human lung and that no cytochrome P-450IA2 homologue is detected. Cytochrome P-450IA1 is expressed in human lung at relatively low levels, similar to those observed in untreated primate (baboon) lung. The majority of the 19 human lung samples examined do not exhibit a permanent polycyclic aromatic hydrocarbon-induced state with respect to this isozyme.     

Rat lung and liver microsomal cytochrome P-450 isozymes involved in the hydroxylation of n-hexane

The primary metabolism of n-hexane in rat lung and liver microsomes was investigated. In liver microsomes from untreated animals the formation of each of the metabolites, 1-, 2- and 3-hexanol, was best described kinetically by a two-enzyme system, whereas for lung microsomes a one-enzyme system was indicated for each metabolite. Cytochrome P-450-PB-B, the major cytochrome P-450 isozyme induced in rat liver by phenobarbital, appeared to be responsible for the formation of 2- and 3-hexanol in lung microsomes from untreated rats as judged by antibody inhibition studies. The presence of this isozyme was confirmed by immunoblotting. In contrast, formation of 1-hexanol in rat lung was catalyzed by a cytochrome P-450 isozyme different from the major isozymes induced by either phenobarbital or beta-naphthoflavone. Similarly, formation of 2,5-hexanediol from 2-hexanol was catalyzed by a P-450 isozyme different from cytochrome P-450-PB-B and present in liver but not in lung microsomes. Furthermore, alcohol dehydrogenase activity with hexanols or hexanediol as the substrate was found exclusively in liver cytosol. These results suggest that inhaled n-hexane must be transported to the liver either intact or in the form of 2-hexanol before the neurotoxic metabolite 2,5-hexanedione can be formed.     

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.