Michael A. Trush

Stimulation by adriamycin of rat heart and liver microsomal NADPH-dependent lipid peroxidation

Abstract Rat liver and heart microsomes catalyze the transfer of single electrons from NADPH to adriamycin forming semiquinone radicals which, in turn, activate molecular oxygen. This process stimulated lipid peroxidation 5- to 7-fold as measured by malonaldehyde formation. Adriamycinaugmented lipid peroxidation was linear with time to 60 min, optimal at 1.0 mg of microsomal protein/ml and pH 7.5, and was proportional to the adriamycin concentration up to 100 μM. An NADPH-generating system was superior to NADPH, and an oxygen atmosphere tripled the rate of peroxidation as compared to air. Nitrogen abolished adriamycin-stimulated peroxidation. Superoxide dismutase, reduced glutathione, α-tocopherol, EDTA, dioxopiperazinylpropane (ICRF-187), and dimethylurea were effective inhibitors of lipid peroxidation. This suggests that Superoxide anion and possibly hydroxyl radical may be formed by the oxidation of the adriamycin semiquinone radical and thus stimulate the peroxidation of microsomal unsaturated fatty acids. Although adriamycin failed to stimulate lipid peroxidation in heart microsomes from control animals, peroxidation was dramatically increased when adriamycin was added to cardiac microsomes from α-tocopherol-deficient rats. Lipid peroxidation in α-tocopheroldeficient liver microsomes was four times greater than in control microsomes with the NADPH-generating system, and adriamycin did not further increase that high rate of peroxidation; however, when NADPH was used as the source of electrons in place of the NADPH-generating system, adriamycin stimulated peroxidation more than 2-fold. These results suggest that microsomal lipid peroxidation may play a role in the cytotoxicity and cardiotoxicity of adriamycin.

Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin

Mitochondrial degeneration is a consistently prominent morphological alteration associated with adriamycin toxicity which may be the consequence of adriamycin-enhanced peroxidative damage to unsaturated mitochondrial membrane lipids. Using isolated rat liver mitochondria as an in vitro model system to study the effects of the anticancer drug adriamycin on lipid peroxidation, we found that NADH-dependent mitochondrial peroxidation--measured by the 2-thiobarbituric acid method--was stimulated by adriamycin as much as 4-fold. Marker enzyme analysis indicated that the mitochondria were substantially free of contaminating microsomes (less than 5%). Lipid peroxidation in mitochondria incubated in KCl-Tris-HCl buffer (pH 7.4) under an oxygen atmosphere was optimal at 1-2 mg of mitochondrial protein/ml and with NADH at 2.5 mM. Malonaldehyde production was linear with time to beyond 60 min, and the maximum enhancement of peroxidation was observed with adriamycin at 50-100 microM. Interestingly, in contrast to its stimulatory effect on NADH-supported mitochondrial peroxidation, adriamycin markedly diminished ascorbate-promoted lipid peroxidation in mitochondria. Superoxide dismutase, catalase, 1,3-dimethylurea, reduced glutathione, alpha-tocopherol and EDTA added to incubation mixtures inhibited endogenous and adriamycin-augmented NADH-dependent peroxidation of mitochondrial lipids, indicating that multiple species of reactive oxygen (superoxide anion radical, hydrogen peroxide and hydroxyl radical) and possibly trace amounts of endogenous ferric iron participated in the peroxidation reactions. In submitochondrial particles freed of endogenous defenses against oxyradicals, lipid peroxidation was increased 7-fold by adriamycin. These observations suggest that some of the effects of adriamycin on mitochondrial morphology and biochemical function may be mediated by adriamycin-enhanced reactive oxygen-dependent mitochondrial lipid peroxidation.

In vitro stimulation by paraquat of reactive oxygen-mediated lipid peroxidation in rat lung microsomes

Abstract Paraquat significantly stimulated lipid peroxidation in rat lung microsomes without the addition of exogenous iron. The ability of paraquat to stimulate this lipid peroxidation was dependent on the presence of adequate reducing equivalents (NADPH), aerobic conditions, and the duration of incubation, viz. optimal in vitro conditions. Even greater paraquat-mediated lipid peroxidation was observed if incubations were conducted under O2 or if vitamin E-deficient microsomes were utilized, factors which have previously been reported to increase the in vivo pulnonary toxicity of paraquat. Superoxide dismutase (3 μg/ml) significantly inhibited paraquat-stimulated lipid peroxidation in rat lung microsomes (73%), demonstrating a pivotal role for superoxide in this process. Thus, the redox cycling of paraquat and accompanying reactive oxygen generation was capable of mediating lipid peroxidation not only in mouse lung and rat liver microsomes but also in rat lung microsomes.

Studies on the in vitro interaction of mitomycin C, nitrofurantoin and paraquat with pulmonary microsomes: Stimulation of reactive oxygen-dependent lipid peroxidation☆

In vitro experiments were performed to evaluate the capacity of the redox cycling compounds mitomycin C (MC), nitrofurantoin (NF) and paraquat (PQ) to stimulate pulmonary microsomal lipid peroxidation. It was observed that the interaction of MC, NF or PQ with rat or mouse lung microsomes in the presence of an NADPH-generating system and an O2 atmosphere resulted in significant lipid peroxidation. All three compounds demonstrated similar concentration dependency, similar time courses and the ability to generate lipid peroxidation-associated chemiluminescence. The stimulation of lipid peroxidation by MC, NF or PQ was inhibited significantly by superoxide dismutase, glutathione, ascorbic acid, catalase and EDTA, agents which either scavenge reactive oxygen and/or prevent the generation of secondary reactive oxygen metabolites. In addition, the ability of MC or NF, but not PQ, to stimulate lipid peroxidation was reduced significantly following preincubation with microsomes and NADPH under N2 (15-20 min) prior to incubation under O2. During this period under N2. MC and NF underwent reductive metabolism of their quinone and nitro moieties respectively. Thus, it appears that under aerobic conditions the pulmonary microsomal-mediated redox cycling of MC, NF and PQ is accompanied by the stimulation of reactive oxygen-dependent lipid peroxidation.

A possible role for membrane lipid peroxidation in anthracycline nephrotoxicity.

Adriamycin causes both glomerular and tubular lesions in kidney, which can be severe enough to progress to irreversible renal failure. This drug-caused nephrotoxicity may result from the metabolic reductive activation of Adriamycin to a semiquinone free radical intermediate by oxidoreductive enzymes such as NADPH-cytochrome P-450 reductase and NADH-dehydrogenase. The drug semiquinone, in turn, autoxidizes and efficiently generates highly reactive and toxic oxyradicals. We report here that the reductive activation of Adriamycin markedly enhanced both NADPH- and NADH-dependent kidney microsomal membrane lipid peroxidation, measured as malonaldehyde by the thiobarbituric acid method. Adriamycin-enhanced kidney microsomal lipid peroxidation was diminished by the inclusion of the oxyradical scavengers, superoxide dismutase and 1,3-dimethylurea, and by the chelating agents, EDTA and diethylenetriamine-pentaacetic acid (DETPAC), implicating an obligatory role for reactive oxygen species and metal ions in the peroxidation mechanism. Furthermore, the inclusion of exogenous ferric and ferrous iron salts more than doubled Adriamycin-stimulated peroxidation. Lipid peroxidation was prevented by the sulfhydryl-reacting agent, p-chloromercuribenzenesulfonic acid, by omitting NAD(P)H, or by heat-inactivating the kidney microsomes, indicating the requirement for active pyridine-nucleotide linked enzymes. Several analogs of Adriamycin as well as mitomycin C, drugs which are capable of oxidation-reduction cycling, greatly increased NADPH-dependent kidney microsomal peroxidation. Carminomycin and 4-demethoxydaunorubicin were noteworthy in this respect because they were three to four times as potent as Adriamycin. In isolated kidney mitochondria, Adriamycin promoted a 12-fold increase in NADH-supported (NADH-dehydrogenase-dependent) peroxidation. These observations clearly indicate that anthracyclines enhance oxyradical-mediated membrane lipid peroxidation in vitro, and suggest that peroxidation-caused damage to kidney endoplasmic reticulum and mitochondrial membranes in vivo could contribute to the development of anthracycline-caused nephrotoxicity.

Bronchiolar epithelial damage and impairment of pulmonary microsomal monooxygenase activity in mice by naphthalene

Abstract Injection of a single dose of naphthalene into C57BL/6J mice (225 mg/kg, ip) produced a significant (30–70%) and prolonged (8–15 days) impairment in pulmonary microsomal monooxygenase activities without altering these activities in liver microsomes. The time course of naphthalene-induced morphologic damage to bronchiolar epithelium paralleled compromises in pulmonary monooxygenase activity. No concomitant alterations in hepatic morphology were observed. Five microsomal enzymes were studied: benzphetamine N -demethylase, aryl hydrocarbon hydroxylase, NADPH cytochrome c reductase, 7-ethoxyresorufin O -deethylase (a cytochrome P -448-dependent enzyme), and styrene epoxide hydrolase (a cytochrome P -450-independent enzyme). In general, the time course of the inhibition of these pulmonary enzymes was similar but the magnitude of the inhibition varied somewhat. Maximum inhibition of enzyme activity occurred about 3 days after naphthalene administration; 7-ethoxyresorufin O -deethylase activity was reduced to about 30% of control values whereas benzphetamine N -demethylase declined to about 70% of control. The remaining enzymes clustered midway between these extremes at about 50% of control values. Inhibited activities remained at relatively constant levels between Days 3 and 8 and by Day 15, there was a clear trend returning toward controls. Despite this trend, three of the six pulmonary enzyme activities examined remained significantly below control levels 15 days after a single dose of the hydrocarbon. Histologically, the pulmonary nonciliated bronchiolar epithelial (Clara) cell was the primary target of naphthalene toxicity. At early time points and at low magnifications, it appeared as if the entire bronchiolar epithelium was undergoing necrosis and sloughing into the lumen. However, higher magnifications revealed residual ciliated epithelium. The distribution of Clara cell damage appeared to vary considerably. One could find bronchioles that appeared completely denuded of epithelium and others in the same section whose Clara cells had been spared or, alternatively, were in the process of regeneration. The results are discussed in relation to recent work which has shown selective covalent binding of naphthalene to pulmonary Clara cells.

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.

Clara cell damage and inhibition of pulmonary mixed-function oxidase activity by naphthalene

Abstract The administration of naphthalene has been shown previously to elicit selective damage and necrosis of the Clara cells of mouse lung. Under identical conditions, we have found naphthalene administration to produce selective depression of pulmonary monooxygenase activities without accompanying changes in hepatic monooxygenases. Similarly, morphologic studies revealed dose-dependent alterations of Clara cells lining pulmonary bronchioles but no remarkable changes in livers of the same animals. The underlying biochemical basis for these organ-specific effects of naphthalene is presently obscure.

Selective damage to nonciliated bronchiolar epithelial cells in relation to impairment of pulmonary monooxygenase activities by 1,1-dichloroethylene in mice

A single ip dose of 1,1-dichloroethylene (DCE) to mice (125 mg/kg) caused a reduction within 24 hr in cytochrome P-450 and related monooxygenases in lung microsomes, with no corresponding changes in liver and kidney microsomes. Light microscopy revealed that at 24 hr, DCE caused a highly selective and complete loss of the bronchiolar nonciliated (Clara) cells at all levels of the tracheobronchial tree. Electron microscopy showed that at this time, the bronchiolar luminal surface was covered by flattened, elongated ciliated cells. Within 24 hr total microsomal cytochrome P-450 and NADPH cytochrome c reductase were maximally reduced to about 50% of control and cytochrome P-450-dependent enzyme activities decreased to about 60% of control. By contrast, coumarin 7-hydroxylase was reduced to approximately 10% of control within 4 days. Since pulmonary coumarin 7-hydroxylase has been shown to reside almost exclusively in the Clara cells, this finding is in agreement with the observed extensive necrosis of the Clara cells. The return of lung microsomal P-450-linked enzyme activities took between 3 and 6 weeks and was paralleled by a corresponding slow reappearance of the bronchiolar Clara cells.

Lung-selective impairment of cytochrome p-450-dependent monooxygenases and cellular injury by 1,1-dichloroethylene in mice.

The acute toxic effects of 1,1-dichloroethylene (DCE; 125 mg/kg, i.p.) on mouse lung, liver and kidney were investigated 24 hr after its administration. DCE caused a reduction of cytochrome P-450 levels and related monooxygenases in lung microsomes with no corresponding changes in liver and kidney. Examination of the lung tissue by light microscopy revealed necrosis restricted to the Clara cells. In contrast, liver and kidney were relatively unaffected by DCE treatment, as indicated by lack of changes in microsomal monooxygenase activities and morphology.