Effect of strain differences and tumor presence on microsomal drug metabolism in the guinea pig: brief communication.

In vitro drug metabolism in the Hartley guinea pig was compared with that in two inbred guinea pig strains used as carriers for the line 10 hepatoma. We observed minor differences in enzyme specific activity among the three strains. Three weeks after intradermal inoculation of Strain 2 guinea pigs with line 10 hepatoma cells, cytochrome P450 levels and aminopyrine demethylase activity were significantly decreased. Seven to 10 days after inoculation with the ascites form of the tumor, the activities of aniline and biphenyl hydroxylases, p-aminobenzoic acid N-acetyltransferase, and dichloronitrobenzene glutathione S-aryltransferase, in addition to those of cytochrome P450 and aminopyrine N-demethylase, were probably also described.

Quantification of bleomycin pulmonary toxicity in mice by changes in lung hydroxyproline content and morphometric histopathology.

Bleomycin treatment produced dose-dependent changes in lung collagen content and in several measurable histopathological parameters. NIH/Swiss mice were treated twice weekly for 6 weeks with bleomycin, 0, 1, 20, or 40 mg/kg s.c. The two highest doses produced mortalities of 35 and 100%, respectively, as well as loss of body weight and increase in lung wet weight. Lung hydroxyproline content, an index of collagen, increased to 40 to 50% above control levels at 6 and 8 weeks after initiation of treatment with bleomycin 20 mg/kg. Morphometric analysis was applied to the following parameters at light microscopy: number of intraalveolar macrophages and leukocytes, total pulmonary cell count, alveolar wall thickness, and percentage of consolidation of lung parenchyma. The two highest doses produced increases in all of these parameters as compared to controls. The most marked changes occurred in the number of intraalveolar cells, which in the group given 20 mg/kg rose to 150, 190, and 210% of controls at 4, 6, and 8 weeks. The lowest dose of bleomycin, 1 mg/kg twice weekly for 6 weeks, evoked no pulmonary or other toxicity by the parameters examined. This model of chronic pulmonary toxicity may be useful in analog development, in testing potential antidotes, and in examining the effects of other factors that might modify the pulmonary toxicity of bleomycin.

Distribution and disposition of platinum following intravenous administration of cis-diamminedichloroplatinum(II) (NSC 119875) to dogs.

cis-Diamminedichloroplatinum(II) is an antineoplastic drug that is undergoing a renewed clinical interest as a drug for use in combination regimens. In order to increase the understanding of the pharmacokinetics of this drug, the plasma clearance and organ distribution of platinum were followed in female beagle dogs treated with a single i.v. dose of cis-diamminedichloroplatinum(II). Plasma levels of platinum were determined by flameless atomic absorption spectrometry and showed a distinctly biphasic clearance pattern with a rapid-phase half-time of considerably less than 1 hr and a slow-phase half-time of nearly 5 days. During the first 4 hr after treatment, plasma levels fell by 90% while 60 to 70% of the applied dose was recovered in the urine. Sixteen tissues plus plasma, bile, and urine were routinely analyzed for platinum content. An easily measurable plasma concentration of platinum was still detectable 12 days after treatment, with no significant change in plasma concentration between Days 4 and 12. Initial concentrations of platinum were highest in organs of excretion, gonads, spleen, and adrenals but remained significantly elevated only in kidney, liver, ovary, and uterus, where a tissue: plasma ratio of 3 to 4 was maintained for as long as 6 days posttreatment. The apparent in vitro binding of platinum to dog plasma and to bovine serum albumin was studied by ultrafiltration and increased progressively during 48 hr of incubation at 37 degrees.

In vitro studies on the metabolism and covalent binding of [14C]1,1-dichloroethylene by mouse liver, kidney and lung.

The metabolism and covalent binding of 1,1-dichloro[1,2-14C]ethylene (DCE) to subcellular fractions of liver, kidney and lung of C57BL/6N mice have been investigated in vitro. Covalent binding was NADPH- and cytochrome P-450-dependent. The microsomal fraction bound more radiolabel than any other subcellular fraction, and the levels of covalent binding in cell fractions correlated well with their cytochrome P-450 content. Covalent binding by mouse liver and lung microsomes also reflected their cytochrome P-450 content. However, although mouse kidney microsomes contained twice as much total cytochrome P-450 as the lung, no detectable covalent binding of DCE-derived radioactivity occurred in kidney. Omission of NADPH, heat inactivation of microsomes, carbon monoxide, addition of SKF-525A, piperonyl butoxide or reduced glutathione (GSH), all inhibited (40-90%) covalent binding of radiolabel to liver and lung microsomes. The absence of O2 (incubation under N2) did not greatly affect the metabolism and covalent binding. Pretreatment of mice with various inducers, phenobarbital (PB), beta-naphthoflavone (beta-NF), pregnenolone 16 alpha-carbonitrile (PCN) and 3-methylcholanthrene (3-MC), evoked increases in total liver microsomal cytochrome P-450 content (2-fold) and corresponding increases in covalent binding (3-fold). However, microsomes from PCN-treated mice showed only a 50% increase in DCE binding. Kidney microsomes from control, PB-, and beta-NF-pretreated mice were incapable of covalent binding of radiolabel but those from PCN- and 3-MC-pretreated mice showed levels of binding similar to untreated mouse lung microsomes. It is proposed that the nephrotoxicity of DCE may be due to translocation of reactive metabolites from the liver to the kidney.

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice.

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.

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.

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.

Studies on the distribution and covalent binding of 1,1-dichloroethylene in the mouse. Effect of various pretreatments on covalent binding in vivo.

The distribution and covalent binding of a single dose of [1,2-14C] 1,1-dichloroethylene (DCE; 125 mg/kg, i.p.) was studied in male C57Bl/6N mice. Total radioactivity was distributed in whole homogenates of all tissues studied, with peak levels occurring within 6 hr. Covalent binding of radioactive material peaked at 6-12 hr in all tissues, and highest levels were found in kidney, liver, and lung with smaller amounts in skeletal muscle, heart, spleen, and gut. Covalent binding in kidney, liver, and lung fell to 50% of peak levels in about 4 days. Between 12 hr and 4 days after DCE administration, 70-100% of total radioactivity present in homogenates of kidney, liver, and lung was covalently bound. The three tissues showed a similar spread in total radioactivity in subcellular fractions 24 hr after exposure to DCE; most of the radioactivity was covalently bound (60-100%) and distributed fairly uniformly with a slight tendency to concentrate in the mitochondrial fraction. Phenobarbital (PB) and 3-methylcholanthrene (3-MC) pretreatments increased the covalent binding in the liver and lung but had no effect in the kidney. Piperonyl butoxide and SKF-525A decreased the covalent binding in liver and lung, but the latter increased binding in the kidney while the former decreased it. Diethylmaleate administration increased the covalent binding (2- to 3-fold) in all three tissues as well as increasing lethal toxicity. These results are consistent with the view that DCE is metabolized to some reactive intermediate(s) which may be detoxified by conjugation with glutathione.

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.

Sex-dependent differences in the effects of portacaval anastomosis on hepatic monooxygenases in rats.

Previous work revealed that portacaval anastomosis (PCA) in rats results in hepatic atrophy and marked decreases in components of the microsomal monooxygenase system such as cytochrome P-450. In the present study, the effects of PCA on hepatic monooxygenase activity were studied in more detail. We report that PCA, in general, produces effects resembling those of castration. Thus, in male rats, PCA depressed the activity of highly sex-dependent enzymes such as ethylmorphine and aminopyrine demethylases. Similar effects were produced by castration, and the combination of PCA and castration produced the same effect as either treatment alone. In male rats, non-sex-dependent enzymes such as aniline hydroxylase and NADPH-cytochrome c reductase were unaffected by either PCA or castration. By contrast, in female rats, neither PCA nor castration significantly affected microsomal monooxygenase activities. In male rats, PCA was accompanied by a 75% reduction in serum testosterone levels and a 6-fold increase in total estrogen levels. We conclude that these effects of PCA in male rats were due, in large measure, to a demasculinizing effect.

A comparison of the pulmonary toxicity and chemotherapeutic activity of bleomycin-BAPP to bleomycin and pepleomycin.

The pulmonary toxicity and antitumor activity of a new bleomycin analog butylamino-3-propylamino-3-propylamine (Blm-BAPP) was investigated and compared with bleomycin and pepleomycin. Blm-BAPP was significantly more pulmonary toxic than bleomycin and had no greater activity against B16 melanoma than either bleomycin or pepleomycin. Although pepleomycin was as equitoxic as bleomycin in producing pulmonary fibrosis, doses of pepleomycin greater than 5 mg/kg were more lethal than bleomycin. Not only did the three drugs function similarly in vivo, but they behaved similarly in two in vitro test systems: microsome-catalyzed drug-mediated DNA deoxyribose cleavage and binding to DNA.

Sex differences in the uptake and retention of imipramine and desmethylimipramine in the rat lung.

14C-Imipramine was administered to male and female Sprague-Dawley rats and animals were sacrificed at 4, 8, 12, and 20 hours later. At all time points, total radioactivity in female lung was several-fold higher than in males. In addition, female lungs had a higher concentration of desmethylimipramine (DMI) as compared to imipramine than did male rat lungs. This was reflected by a higher conversion of imipramine to DMI by hepatic and pulmonary microsomes from female rats. Finally male rats cleared both imipramine and DMI from their lungs at a slower rate than did female rats.

Interaction of the oncolytic drug, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with the mixed-function oxidase system in rats.

Effects of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) on the hepatic mixed-function oxidase system in male rats were studied both in vivo and in vitro. A single dose of CCNU (40 mg/kg) caused a significant reduction in hepatic mixed-function oxidase activities within 3 days after administration. The depression was prolonged for cytochrome P-450, total haem and the metabolism of several type I substrates lasting up to 10 weeks after a single dose. By contrast, aniline hydroxylase, cytochrome b5 and NADPH-cytochrome c reductase activities returned to near control levels after week two. Microsomal enzymes in the kidneys of treated animals however, were unaltered. Serum glutamic pyruvic and glutamic oxaloacetic transaminase and bilirubin levels, indicators of hepatotoxicity, were greatly elevated 3 days after CCNU treatment. These parameters fell rapidly but were still above control levels to the end of the 10-week study. When added in vitro, CCNU reduced apparent cytochrome P-450 content and the metabolism of type I substrates in microsomes from untreated, phenobarbital (PB) and 3-methylcholanthrene (3-MC)-pretreated rats. Total haem and NADPH-cytochrome c reductase were not affected whereas aniline hydroxylase activity was activated. CCNU interacted with hepatic microsomes to produce a type I difference spectrum.

The utility of changes in cardiac weight as an index of drug-induced cardiotoxicity.

The effects of toxic doses of various drugs and of food or water deprivation upon heart weights of mice were evaluated over a four day period to test the validity of the hypothesis that changes in cardiac weights are indicators of cardiotoxicity. Drugs included in the study were actinomycin-D, methotrexate, 5-fluorouracil, adriamycin, daunomycin, N-dimethyladriamycin, N-trifluoroacetyladriamycin-14-valerate, isoproterenol, atropine, and acetylsalicylic acid. Additional groups of mice served as vehicle controls, or were deprived of food or water for the duration of the experiment to control for the anorexia and dehydration accompanying treatment with antineoplastic drugs. Body weights were taken at the start of the experiment (day 0), day 2, and day 4 (just prior to sacrifice). Heart ventricle wet weights were determined immediately, and dry weights after thorough desiccation of the samples. Statistical evaluation of the weights revealed that there were no ventricular weight changes unique to any particular drug, and that decreases in heart weights correlated well with decreases in body weights, thereby reflecting the general toxicities of the drugs, including inanition, and not any specific cardiotoxicities.