Extent and persistence of streptozotocin-induced DNA damage and cell proliferation in rat kidney as determined by in vivo alkaline elution and BrdUrd labeling assays.

The extent of DNA damage and cellular proliferation induced in rat kidneys by single doses of the diabetogenic alkylating agent streptozotocin (STZ) and the time course of repair of that damage were evaluated using an in vivo alkaline elution assay for DNA strand breaks and a bromodeoxyuridine (BrdUrd) labeling assay for cell replication. Male Sprague-Dawley rats were given iv injections of 0.25 to 60 mg/kg STZ and kidneys were harvested 3 hr later for alkaline elution. A dose of 2.5 mg/kg STZ was the lowest dose to induce detectable DNA strand breaks and extensive damage was produced by the commonly used diabetogenic dose of 60 mg/kg. To characterize the repair of the drug-induced DNA damage, kidneys were harvested from a 60 mg/kg group of animals 3 hr to 27 days after dosing. BrdUrd-labeled kidney sections were also evaluated to assess any cellular proliferative response associated with STZ administration. Significant DNA damage was detected up to 14 days after dosing with return to near background levels by 20 days. Similarly, treatment with 60 mg/kg STZ was associated with increases in BrdUrd labeling indices 4 and 9 days after treatment with resolution by 27 days. These results indicate that the cellular and molecular repair responses to a single diabetogenic dose of STZ are prolonged, requiring up to 3 weeks to complete. Thus, to avoid potential additive or synergistic effects on STZ-induced nephrotoxicity and/or genotoxicity, a delay in the start of experimental therapies in this model (other than insulin) should be considered.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase induce reductase accumulation and altered lamellar bodies in rat forestomach keratinocytes.

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and a potent hypocholesterolemic agent, induces a hyperplastic thickening of the rat forestomach mucosa after oral administration of its active form, a hydroxyacid. We studied the effects of lovastatin on the intracellular accumulation of HMG-CoA reductase immunostaining and the accompanying morphological changes in rat forestomach keratinocytes by immunofluorescence microscopy and transmission electron microscopy (TEM). Administration of lovastatin hydroxyacid induced increases in HMG-CoA reductase levels within forestomach keratinocytes that were dose and time dependent and reversible. The adjacent glandular stomach epithelium did not exhibit induction of reductase. A pharmacologically inactive epimer of lovastatin hydroxyacid did not increase keratinocyte reductase accumulation, and lovastatin lactone induced minimal forestomach reductase. TEM of forestomachs from rats given lovastatin hydroxyacid demonstrated profound alterations in epidermal lamellar bodies (organelles that transport lipids and steroids to the intercellular spaces of the stratum corneum). Treated cells lacked internal lipid lamellae and failed to secrete sheets of lipid material into the intercellular spaces of the stratum corneum. We hypothesize that sustained inhibition of HMG-CoA reductase in rat forestomach keratinocytes induces accumulation of HMG-CoA reductase and hyperplasia by inhibiting sterol synthesis, assembly of lamellar bodies, and formation of intercellular lipid sheets.

Studies on the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on the rodent forestomach.

A novel class of hypocholesterolaemic agents, HMG CoA reductase inhibitors, was shown to cause mucosal thickening in the rodent (mouse and rat) forestomach after subacute/subchronic oral administration. These changes were characterized histologically by acanthosis and hyperkeratosis of the squamous epithelium with submucosal oedema and occasionally cellular infiltration. This drug-induced hyperplastic response was both dose and time dependent, did not occur after subcutaneous administration, and was confined entirely to the rodent forestomach (not observed in any other area of the gastro-intestinal tract). The forestomach hyperplastic response correlated with the pharmacological potency of HMG CoA reductase inhibitors of similar structure (observed to varying degrees with all HMG CoA reductase inhibitors examined to date).

Preclinical evaluation of lovastatin.

Administration of lovastatin to animals at high dosage levels produces a broad spectrum of toxicity. This toxicity is expected based on the critical nature of the target enzyme (HMG CoA reductase) and the magnitude of the dosage levels used. The information reviewed in this paper demonstrates that these adverse findings in animals do not predict significant risk in humans. The reason for this derives from the fact that all the available evidence suggests that the adverse effects observed are produced by an exaggeration of the desired biochemical effect of the drug at high dosage levels. The presence of clear and high no-effect doses for these toxic effects along with the fact that most of the changes observed are clearly mechanism-based (directly attributable to inhibition of mevalonate synthesis) indicate that it is unlikely that similar changes will be observed at the therapeutic dosage levels in humans. This hypothesis is supported by the extensive human safety experience described by Tobert in the following report.

Ultrastructure of experimental cocaine hepatotoxicity.

Cocaine is a potent hepatotoxin in mice. It is converted in the liver by a minor oxidative pathway to the active metabolite, norcocaine nitroxide. Previous studies have shown evidence of a lipid peroxidative mechanism of toxicity, including increased conjugated diene absorption by hepatic microsomal lipids following a single 60 mg per kg i.p. dose of cocaine in DBA/2Ha mice. To explore this mechanism further, morphologic changes in the livers of DBA/2Ha mice were examined following the same dose of cocaine. The first ultrastructural change seen was dilatation of rough endoplasmic reticulum in centrilobular hepatocytes 1 hr following cocaine injection, coincident with the previously observed onset of increased conjugated diene absorption in microsomal lipids. During the previously observed period of peak conjugated diene absorption (2 to 4 hr), ultrastructural changes in centrilobular hepatocytes progressed. These included focal mitochondrial membrane disruption followed by more extensive mitochondrial swelling and disruption with increased swelling of rough endoplasmic reticulum. Changes in size, shape and concentration of histochemically labeled, morphometrically studied peroxisomes were also seen during this interval. Injury of centrilobular hepatocytes advanced to cell death in 6 to 8 hr. The time course and nature of these morphologic findings correlate with previously observed evidence of lipid peroxidation, supporting the hypothesis that this is the mechanism of cocaine hepatoxicity.

Proliferative response and renewal of hepatic function following cocaine administration in mice.

Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). The time course of hepatic damage was monitored by assaying microsomal cytochrome P450 content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of 3H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. The LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P450 content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.

Biotransformation of norcocaine to norcocaine nitroxide by rat brain microsomes.

In the mid 1970's, norcocaine was identified as a metabolite of cocaine in rat brain tissue. We extend these studies by demonstrating that rat brain FAD-containing monooxygenase metabolizes norcocaine to N-hydroxynorcocaine. This hydroxylamine is then further oxidized to the nitroxyl free radical norcocaine nitroxide by rat brain cytochrome P-450. Brain microsomal reduction of norcocaine nitroxide leads to the generation of superoxide. Finally, incubation of rat brain microsomes with either N-hydroxynorcocaine or norcocaine nitroxide leads to significant lipid peroxidation as monitored by spin-trapping techniques.

N-demethylation of cocaine to norcocaine. Evidence for participation by cytochrome P-450 and FAD-containing monooxygenase.

Experiments were conducted to determine which microsomal enzymes are involved in the in vitro hepatic oxidative N-demethylation of cocaine to norcocaine, the first step in the biotransformation of cocaine to its ultimate hepatotoxic metabolite. Cocaine was found to undergo conversion to norcocaine by two alternate pathways, one involving only cytochrome P-450 and the other requiring both cytochrome P-450 and FAD-containing monooxygenase. In the first pathway, cocaine was directly N-demethylated to norcocaine by cytochrome P-450; this reaction was enhanced by phenobarbital induction and was inhibited by both n-octylamine and metyrapone. The second route was found to be a two-step reaction involving cocaine N-oxide as an intermediate. In this pathway, cocaine is first oxidized to cocaine N-oxide by FAD-containing monooxygenase, followed by a cytochrome P-450-catalyzed N-demethylation to norcocaine. This latter step was enhanced by phenobarbital treatment and inhibited by n-octylamine. Cocaine N-oxide also exhibited a Type I binding spectrum with mouse hepatic microsomes. In addition, a model system consisting of ferrous sulfate was found to catalyze the N-demethylation of cocaine N-oxide. On the basis of these experiments, it is concluded that cytochrome P-450 and FAD-containing monooxygenase participate in the initial oxidation of cocaine to norcocaine. We also propose a mechanism to account for the conversion of cocaine N-oxide to norcocaine.

Evidence of enhanced in vivo lipid peroxidation after acute cocaine administration.

An acute intraperitoneal dose (60 mg/kg) of cocaine to DBA/2Ha male mice results in enhanced lipid peroxidation in vivo, as measured by an increase in conjugated diene absorption in hepatic microsomal lipids. The initiation of this lipid peroxidation is an early consequence of cocaine administration; as early as 1 h after cocaine, peroxidized lipids are significantly greater in treated animals than in controls. This cocaine-induced lipid peroxidation remains at a maximal level from 2 to 4 h and returns approximately to control levels by 8 h. The metabolites of cocaine also produce lipid peroxidation in vitro. Liver microsomes from phenobarbital-treated DBA/2Ha male mice, incubated aerobically in the presence of NADPH, cocaine or the cocaine oxidative metabolites, norcocaine and norcocaine nitroxide, induced lipid peroxidation as measured by an increase in the production of thiobarbituric acid (TBA)-reactive products. The extent of lipid peroxidation is greater for the oxidative metabolites of cocaine than for cocaine itself.

Initiation of in vitro lipid peroxidation by N-hydroxynorcocaine and norcocaine nitroxide.

Norcocaine nitroxide and N-hydroxynorcocaine were found to stimulate hepatic microsomal lipid peroxidation in vitro, as measured by spin-trapping techniques using the spin trap alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone. It was determined that either norcocaine nitroxide or N-hydroxynorcocaine markedly enhanced the rate of spin trapping of lipid peroxyl radicals when added to hepatic microsomal preparations. Glutathione, in the presence of dialyzed cytosol, inhibited the formation of lipid peroxyl spin-trapped adducts. This finding suggests that cytosolic glutathione-dependent enzymes perhaps including glutathione peroxidase play an important role in the prevention of norcocaine nitroxide-or N-hydroxynorcocaine-mediated lipid peroxidation.

Androgenic suppression of mouse hepatic FAD-containing monooxygenase activity.

Sex-related differences in the activity of hepatic FAD-containing monooxygenase (FAD-M) were found in C3H/St mice. Adult female mice had enzyme activities nearly two-fold greater than male mice and these differences, which were absent in sexually immature mice, became apparent at the onset of puberty. The sex differences in hepatic FAD-M appeared to be mediated through the suppressive effect of testosterone; castration of male mice enhanced enzyme activity, while androgenic replacement returned activities to control levels. Testosterone's suppressive effect was found to be relatively specific for hepatic FAD-M. Treatment of castrated male mice with both the anti-androgen flutamide and testosterone returned enzyme activity to control levels, suggesting that testosterone's regulation of hepatic microsomal FAD-M is receptor-mediated. Female gonadectomy had no effect on this enzyme's activity.