Theodore E. Gram

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.

Effect of partial hepatectomy on the responsiveness of microsomal enzymes and cytochrome P-450 to phenobarbital or 3-methylcholanthrene

Abstract The effects of phenobarbital and 3-methylcholanthrene (3-MC) pretreatment on liver weight, microsomal protein, cytochrome P-450 content, microsomal aniline hydroxylase, hexobarbital hydroxylase and P -nitroanisole O -demethylase activities have been studied in control, sham-operated and partially hepatectomized rats. In unoperated rats, phenobarbital pretreatment significantly increased liver weight, microsomal protein, P-450 content and enzyme activities toward all three substrates. 3-MC pretreatment significantly increased liver weight, P-450 content and p -nitroanisole O -demethylation without influencing the other parameters. When administered to shamoperated or partially hepatectomized rats, phenobarbital and 3-MC caused substantially the same effects, though the magnitude of the effects was less than in unoperated animals. Thus, significant increases were seen in the various parameters after injection of phenobarbital or 3-MC into hepatectomized rats. It is concluded that the regenerating liver, like the fetal or newborn liver and certain rodent hepatomas, although exhibiting low levels of microsomal enzymes, has the capacity to respond to the enzyme inducers, phenobarbital and 3-MC. Differential comparison of the enzyme changes expressed in terms of microsomal protein and also in terms of microsomal P-450 content revealed that the activity of the enzymes catalyzing p -nitroanisole and aniline oxidations parallel the microsomal P-450 content more closely than does the enzyme catalyzing hexobarbital hydroxylation.

The effects of α-tocopherol on the toxicity, disposition, and metabolism of adriamycin in mice☆

Fourteen days after adriamycin treatment, 15 mg/kg ip, there was greater than 50% mortality in CDF1 mice pretreated with either olive oil or saline ip, but only 5% mortality in animals pretreated with a single dose of α-tocopherol. However, 60 days after adriamycin, mortality was 80±5% in all three groups. A single dose of α-tocopherol alone caused no deaths. Blood concentrations of [14C]adriamycin-derived radioactivity in mice pretreated with α-tocopherol of olive oil were significantly higher than those of saline controls between 3 and 15 min after injection. At 30 and 60 min blood concentrations of radioactivity did not differ in the three groups. Sixty minutes after adriamycin administration, concentrations of radioactivity in heart, kidney, muscle, and lung were significantly higher in the α-tocopherol and olive oil groups than in the saline controls. No differences in the metabolic profile of adriamycin were found in heart or liver, but renal concentration of adriamycin and several metabolites were higher in both the α-tocopherol and olive oil groups. In general, α-tocopherol pretreatment had only slight effect on the metabolism of adriamycin in vivo. Thus, the time-dependent efficacy of α-tocopherol in ameliorating the lethal toxicity of adriamycin in mice does not appear to result from acute changes in the distribution or metabolism of adriamycin. It appears that the effect of α-tocopherol is to delay rather than prevent the lethal toxicity of adriamycin.

Comparative alterations in extrahepatic drug metabolism by factors known to affect hepatic activity.

Abstract Various factors known to alter hepatic drug metabolism were examined for their effects on drug metabolism in certain extrahepatic organs, viz. lung and kidney. The prominent sex-related differences in drug metabolism in rat liver were not seen in either lung or kidney. Pretreatment of rats with phenobarbital produced the expected large increases in hepatic NADPH cytochrome c reductase, cytochrome P-450. aminopyrine demethylase and biphenyl hydroxylase activities without concomitant changes in any of these parameters in lung, and only scattered and smaller changes in kidney. 3-Methylcholanthrene (3-MC) pretreatment significantly increased cytochrome P-450 levels in all three organs. Pretreatment of rats with carbon tetrachloride (CCl4) produced consistent inhibition of mixedfunction oxidation in hepatic microsomes. but the extrahepatic effects were less predictable and were both organ- and enzyme-specific. An increase in renal UDP-glucuronyltransferase activity was observed after CCl4 treatment that paralleled a similar but larger increase observed in liver. Extrahepatic NADPH cytochrome c reductase and N-methyl-p-chloroaniline demethylase values were unaffected by CCl4. Lung and kidney responded in a like manner to liver to the additions in vitro of β-diethylaminoethyl diphenylpropylacetate (SKF-525A). Losses in enzyme activities in lung and kidney microsomes roughly paralleled those of liver when stored as pellets for up to 14 days at −70°. Two or 4 days of starvation produced substrate-specific changes in enzyme-specific activity in liver and kidney, with lung appearing resistant to the effect. When enzyme activity was expressed on a whole organ basis, however, lung cytochrome P-450 values decreased significantly and parameters from liver and kidney increased or decreased in a substrate-specific manner. It is concluded that some physiological and pharmacological factors that influence hepatic drug metabolism produce similar effects in lung and kidney, while other factors produce organ-specific effects.

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.

Effect of α-tocopherol upon lipid peroxidation and drug metabolism in hepatic microsomes☆

Abstract In an in vitro system consisting of rat liver 9000 g supernatant fraction and a TPNH-generating system, significant lipid peroxidation was observed during incubation at 37 °C; negligible peroxidation occurred under similar conditions with rabbit liver. With rat liver, the addition of hexobarbital or codeine slightly stimulated peroxidation, but the addition of aminopyrine, zoxazolamine, aniline, or 3,4-benzpyrene to the system markedly reduced or abolished it. Homogenization of the liver in the presence of α-tocopherol abolished lipid peroxidation when incubation was subsequently carried out either in the presence or absence of hexobarbital or codeine. When incubated with rat liver supernatant fraction, hexobarbital and codeine were metabolized linearly with time for relatively short periods; early plateaus in activity suggested enzymic inactivation. Although lipid peroxidation was abolished by α-tocopherol, no effect was observed on the time course curves of either hexobarbital or codeine metabolism, suggesting that peroxidation is not responsible for the inactivation of rat liver microsomal drug-metabolizing enzymes.

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.

The effects of ascorbic acid deficiency and repletion on pulmonary, renal, and hepatic drug metabolism in the guinea pig

Abstract The effects of ascorbic acid (AA) deficiency on microsomal and soluble (postmicrosomal supernatant) enzymes which catalyze drug metabolism were studied in the guinea pig liver, lung, and kidney, (i) Twenty-one days of AA depletion produced a 50–60% decrease in hepatic cytochrome P -450 levels, 20–30% decreases in renal levels, but no significant changes in pulmonary cytochrome P -450 content. Upon repletion of ascorbic acid, recovery to control levels occurred within 7 days. (ii) The decreases in hepatic cytochrome P -450 in scurvy were not accompanied by a corresponding increase in cytochrome P -420. (iii) Aminopyrine N -demethylation decreased by 40% in livers of deficient animals, and recovered within 3 days, but there were no corresponding changes in lungs and kidneys. (iv) There were no significant alterations of NADPH-cytochrome c reductase activity in scorbutic animals in any of the three organs. (v) Activity of “native” UDP-glucuronyl transferase was increased in liver microsomes after 21 days of deficiency, but this apparent increase was not observed when the enzyme was fully activated in vitro with UDP N -acetylglucosamine. “Native” UDP-glucuronyl transferase was increased in kidneys of deficient animals and unchanged in lungs. (vi) In the postmicrosomal supernatant, glutathione S -aryl transferase activity in deficient livers decreased tc 50% of control and did not fully recover after 14 days of ascorbic acid repletion. These changes were not seen in kidney and lung. (vii) Also in the postmicrosomal supernatant, p -aminobenzoic acid (PABA) N -acetyl transferase activity increased in the kidneys of deficient animals, but was unchanged in liver and lungs. (viii) Addition of ascorbic acid in vitro to hepatic microsomes prepared from scorbutic animals had no effect on activities of aminopyrine N -demethylase, NADPH-cytochrome c reductase, PABA N -acetyl transferase, and glutathione S -aryl transferase.