Metabolic activation of nitrofurantoin--possible implications for carcinogenesis.

Although past investigations have indicated that nitrofurantoin is noncarcinogenic, the present studies demonstrate several features of the metabolism of the drug which are similar to those of other nitrofurans that are known carcinogens. Microsomal and soluble fractions from both rat liver and lung mediated the covalent binding of [¹⁴C]nitrofurantoin to tissue macromolecules in vitro. Oxygen strongly inhibited the binding in both the microsomal and soluble fractions, and carbon monoxide failed to inhibit binding in microsomal preparations, indicating nitrofurantoin was activated in both systems by nitroreduction and not by oxidation of the furan ring. An antibody against NADPH-cytochrome c reductase inhibited the microsomal nitroreduction and covalent binding of nitrofurantoin, while the addition of a flavin (FAD) markedly enhanced the covalent binding. Maximal rates of covalent binding were obtained in soluble fractions in the presence of NADH or hypoxanthine; covalent binding was inhibited in these fractions by allopurinol. an inhibitor of xanthine oxidase. Nitroreduction of nitrofurantoin was enhanced, but covalent binding was decreased, in liver microsomes from phenobarbital-pretreated rats. Phenobarbital did not alter nitroreduction or covalent binding of nitrofurantoin in lung microsomes or in soluble fractions from lung or liver. Reduced glutathione markedly decreased covalent binding of nitrofurantoin. in both the microsomal and the soluble fractions from liver and lung. but did not alter the rate of nitroreduction in any of the fractions. Radioactivity was covalently bound in several organs of rats given [¹⁴C]nitrofurantoin in vivo.     

Protection by diethyldithiocarbamate against carbon tetrachloride lethality in rats and against carbon tetrachloride-induced lipid peroxidation in vitro

Diethyldithiocarbamate provides excellent protection against carbon tetrachloride-induced lethality when administered 1 hr prior to the poison. Lipoperoxidation of liver microsome preparations and the pro-oxidant effect of carbon tetrachloride in vitro are inhibited by low concentrations of diethyldithiocarbamate. On the other hand, NADPH-cytochrome c reductase activity is not affected by addition of diethyldithiocarbamate. These results suggest that the action of the protective agent is not at the level of the flavoprotein of the microsomal electron transport system but elsewhere in the process of lipid peroxidation.     

Inhibition of rat liver microsomal lipid peroxidation by boldine.

The alkaloid boldine, found in the leaves and bark of boldo, was an effective inhibitor of rat liver microsomal lipid peroxidation under a variety of conditions. The following systems all displayed a similar sensitivity to boldine: non-enzymatic peroxidation initiated by ferrous ammonium sulfate; iron-dependent peroxidation produced by ferric-ATP with either NADPH or NADH as cofactor; organic hydroperoxide-catalyzed peroxidation; and carbon tetrachloride plus NADPH-dependent peroxidation. Boldine inhibited the excess oxygen uptake associated with microsomal lipid peroxidation. Thus, boldine was effective in inhibiting iron-dependent and iron-independent microsomal lipid peroxidation, with 50% inhibition occurring at a concentration of about 0.015 mM. Boldine did not appear to react efficiently with superoxide radical or hydrogen peroxide, but was effective in competing for hydroxyl radicals with chemical scavengers. Concentrations of boldine which produced nearly total inhibition of lipid peroxidation had no effect on microsomal mixed-function oxidase activity nor did boldine appear to direct electrons from NADPH-cytochrome P450 reductase away from cytochrome P450. Boldine completely protected microsomal mixed-function oxidase activity against inactivation produced by lipid peroxidation. The effectiveness of boldine as an anti-oxidant under various conditions, and its low toxicity, suggest that this alkaloid may be an attractive agent for further evaluation as a clinically useful anti-oxidant.     

Dissociation of microsomal oxygen reduction and lipid peroxidation with the electron acceptors, paraquat and menadione.

Paraquat, diquat and menadione, electron acceptors which interact with the microsomal electron transport chain, were used to investigate the relationship between microsomal lipid peroxidation and microsomal oxygen reduction. All three compounds stimulated hydrogen peroxide production and the rate of superoxide production by mouse liver microsomes. However, while paraquat and diquat stimulated microsomal lipid peroxidation (2-fold in liver microsomes and 6- to 10-fold in lung microsomes), menadione was a potent inhibitor. Superoxide dismutase and catalase had no effect on paraquat-stimulated lipid peroxidation. Diquat, at concentrations sufficient to stimulate Superoxide production, was unable to stimulate lipid peroxidation. Based on the above observations, a mechanism of paraquat- and diquat-initiated lipid peroxidation independent of superoxide and peroxide generation is proposed. The stimulatory effects of paraquat and diquat on lung microsomal lipid peroxidation are also discussed in relation to the lipid peroxidation hypothesis of paraquat lung toxicity.     

Potentiation by carbon tetrachloride of NADPH-dependent lipid peroxidation in lung microsomes.

NADPH-dependent lipid peroxidation occurs in rat lung microsomes in vitro. Expressed per wet weight of tissue we found that lung had only $\text{1}\text{100}$ the activity of liver. However, examination of the rate of malonyl dialdehyde production with different concentrations of NADPH revealed that the kinetics of lipid peroxidation in lung microsomes was indistinguishable from that of NADPH-dependent lipid peroxidation in liver microsomes. With lung microsomes supplemented with NADPH, lipid peroxidation was potentiated by CCl₄ and inhibited by EDTA, Mn²⁺, and cytochrome c.     

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.     

Alterations in mouse liver monooxygenases by benzothiadiazoles

Administration of 1,2,3-benzothiadiazoles to mice had a biphasic effect on liver microsomal monooxygenases. During the first 15 hr of treatment, an inhibition of the in vivo metabolism of hexobarbital, as well as of the in vitro hydroxylation of naphthalene and N-demethylation of aminopyrine, was observed. An apparent decrease in cytochrome P-450 and in the activity of the NADPH-cytochrome c reductase also occurred. The levels of cytochrome b₅ and NADH-cytochrome c reductase activity were only slightly affected. A shift to 452 nm in the carbon monoxide difference spectrum was obtained with dithionite-reduced microsomes and this was not modified by ferricyanide. After the initial inhibitory phase, an enhancement of drug-metabolizing activities in vivo and in vitro and in the levels of some components of the mixed function oxidase system was observed. The carbon monoxide difference spectra of dithionite-reduced microsomes returned to a maximal absorption at 450 nm. The stimulatory effect on monooxygenase activity, elicited by benzothiadiazoles, was prevented completely by actinomycin D and was accompanied by increases in liver weight, microsomal protein, and incorporation of labeled amino acids into microsomal protein, as well as by proliferation of smooth and rough endoplasmic reticulum. Acrylamide gel analysis of liver microsomes from mice, given a single dose of 6-chloro-1, 2,3-benzothiadiazole 48 hr prior to being killed, showed preferential induction of cytochrome P-450 apoproteins of 50,000, 52,000 and 53,000 molecular weight.     

Effect of delta9-tetrahydrocannabinol on rat liver microsomal lipid peroxidation.

In vivo ip administration of saline-Tween 80 suspensions of pure Δ⁹-tetrahydrocannabino (Δ⁹-THC) under both acute (10 and 50 mg/kg) and chronic (10 mg/kg/day for 21 days) conditions, to adult male albino rats inhibited liver microsomal lipid peroxidation. In vitroΔ⁹-THC (0.5–8 μg/mg protein) also markedly lowered NADPH- and ascorbate-induced microsomal lipid peroxidation. Δ⁹-THC was also effective in lowering CCl₄-induced lipid peroxidation in vitro. These results suggest that Δ⁹-THC exerts a stabilizing effect on hepatic microsomal membrane.     

K-region epoxides of polycyclic hydrocarbons: formation and further metabolism by rat-lung preparations

Epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene that have been identified as the K-region epoxides, 7-methylbenz[a]anthracene 5,6-oxide and benzo[a]pyrene 4,5-oxide, have been detected as microsomal metabolites using preparations from the lungs of rats that had been pretreated with the microsomal mixed function oxidase inducer, 3-methylcholanthrene. It was also possible, using lung microsomal preparations from uninduced animals, to demonstrate the formation of an epoxide identified as the K-region derivative, benz[a]anthracene 5,6-oxide, as a microsomal metabolite of benz[a]anthracene. The K-region epoxides of 7-methylbenz[a]anthracene and of benzo[a]pyrene could not always be detected as metabolites when lung microsomal preparations from uninduced rats were used. The activities of two other enzymes present in pulmonary tissue fractions that are involved in the further metabolism of polycyclic hydrocarbon epoxides have also been measured and the values compared with those obtained with rat-liver. When benz[a]anthracene 5,6-oxide was used as substrate, much lower levels of microsomal epoxide hydrase activity were found in lung than in liver, but soluble-supernatant fractions of rat-lung appeared to possess higher levels of glutathione S-epoxide transferase activity than were present in rat-liver.The significance of these results in relation to the metabolic activation of polycyclic hydrocarbons by epoxide formation and to the induction of tumours of the respiratory tract by members of this class of chemical carcinogens is discussed.     

Carbon tetrachloride-induced lipid peroxidation of rat liver microsomes in vitro.

Characteristics of carbon tetrachloride-induced lipid peroxidation of rat liver microsomes and effect on microsomal enzymes were studies in vitro. Microsomes isolated from well-perfused livers and washed with EDTA-containing medium exhibited low endogenous lipid peroxidation when incubated in a phosphate buffer (> 0.1 M) in the presence of NADPH, whereas carbon tetrachloride stimulated to a great extent the peroxidation under these conditions. The stimulation was dependent on the concentration of NADPH, neither NADH nor ascorbic acid being replaced. The stimulatory action by bromotrichloromethane was more marked than that by carbon tetrachloride, however chloroform had no stimulatory action. N,N-Diphenyl-p-phenylene diamine, diethyldithiocarbamate and disulfiram inhibited carbon tetrachloride-induced lipid peroxidation in low concentrations. Inhibitions by thiol compounds and EDTA were weaker. Ferricyanide, cytochrome c and vitamine K₃ inhibited the stimulation by carbon tetrachloride while no inhibition was seen with carbon monoxide. An increase in the degree of carbon tetrachloride-induced lipid peroxidation resulted in a coincidental decrease in microsomal cytochrome P-450 content accompanying a parallel loss in aminopyrine demethylase activity, while NADH-ferricyanide dehydrogenase and NAD(P)H-eytochrome c reductase activities, and cytochrome b₅ content remained unaffected. Similar results were obtained when microsomes were peroxidized with NADPH in combination with ferric chloride and pyrophosphate. Regarding the mechanism of hepatotoxic action of carbon tetrachloride, these results support the hypothesis of lipid peroxidation.     

Protective Role of Ascorbic Acid Against Asbestos Induced Toxicity in Rat Lung: In Vitro Study

ABSTRACT

Asbestos fibers adsorb cytochrome P-450 and P-448 proteins from rat lung micosomal fractions and liberate heme from cytochrome P-448 on prolonged incubation in vitro. further, fibers, decrease the activities of benzo(a)pyrene hydroxylase and glutathione-S-transferase in microsomal and cytosolic fractions respectively. Mineral fibers also stimulate both the enzymatic (NADPH-induced) and non-enzymatic (Fe²⁺ -induced) lipid peroxidation in microsomal fractions. Preincubation of microsomal and cytosolic fractions with a physiological concentration of ascorbic acid ameliorates, to a large extent, the changes induced by asbestos fibers.     

Evidence for carbon tetrachloride-induced lipid peroxidation in mouse liver

The involvement of lipid peroxidation in the mechanism of carbon tetrachloride-induced hepatotoxicity has been a point of controversy. Previous investigators have reported an absence of lipid peroxidative degradation products in mice after exposure to carbon tetrachloride and have used this evidence against the hypothesis that lipid peroxidation is an integral part of the events that cause tissue damage. We have compared the extent of lipid peroxidation caused by carbon tetrachloride between Sprague-Dawley rats and three strains of mice (A/J, BALB/cJ, and C57B1/6J) in in vitro and in vivo systems. Hepatic microsomes isolated from fasted mice of each strain produced more malondialdehyde (a degradation product of lipid peroxidation) per mg microsomal protein than those isolated from fasted rats at all times of incubation with CCl₄. In vivo lipid peroxidation was estimated by the lipid conjugated diene content in hepatic microsomes from the rat and three strains of mice. Increased conjugated diene formation was observed in microsomal lipids of these animals after intraperitoneal injection of CCl₄ (1 ifml/kg as a 20% solution in corn oil) when compared to animals given only corn oil, but no differences were found in the amount of conjugated dienes between mice and rats. Our observations show that the CCl₄-treated mouse undergoes hepatic lipid peroxidation at least as well as the rat, and indicate that lipid peroxidation cannot be excluded as a mechanism of carbon tetrachloride hepatotoxicity as has been claimed on the basis of its ineffectiveness in the mouse.     

Alteration of hepatic microsomal enzymes by griseofulvin treatment of mice.

Feeding mice a diet containing 2.5% griseofulvin (GF) for 12 days caused an increase in liver weight to 9 per cent of the body weight with a proportional decrease in microsomal protein/g of liver wet weight. With respect to microsomal protein, the cytochrome P-450 content was 50 per cent and cytochrome b₅ was 200 per cent of that in control mouse liver microsomes. The amount of increase in cytochrome b₅ was approximately the same as the amount of decrease in cytochrome P-450. and there was no change in the total microsomal heme content. The microsomal content of NADH-cytochrome b₅ reductase, as measured by ferricyanide reduction, was unchanged, but in agreement with the elevated cytochrome b₅ content, NADH-cytochrome c reductase activity was doubled. While the cytochrome P-450 level was low in microsomes after GF feeding, the NADPH-cytochrome c reductase was significantly elevated. Since this enzyme is generally considered to be rate limiting for many mixed function oxidase reactions, its increase may explain the normal to slightly elevated rates of metabolism in vitro of several type I and type II substrates. Although cytochrome b₅, has been suggested as being rate limiting for input of a second electron to cytochrome P-450 linked mixed function oxidations, elevation of cytochrome b₅ in the microsomes did not change the extent of NADH-synergism of NADPH-supported aminopyrine demethylation. NADH-supported Δ,10-fatty acid desaturase activity, which requires cytochrome b₅, was elevated several-fold after GF feeding. In contrast, NADPH-supported lipid peroxidation showed a slower onset after GF treatment; the NADH-supported reaction, however, was slightly elevated.     

Effects of multiple administration of halothane on the mixed function oxidase system in liver microsomes. Difference between guinea pigs and rats

The effects of multiple administration of halothane on the mixed function oxidase system in liver microsomes were investigated and compared between guinea pigs and rats to clarify the difference in sensitivity to halothane. The mixed function oxidase system, except for NADPH-cytochrome P-450 recductase activity was increased in both animals. The inhibition in guinea pigs may have resulted from the microsomal membrane damage induced by the acceleration of lipid peroxidation.     

Inhibition by uric acid of free radicals that damage biological molecules

To clarify the antioxidative role of uric acid, its ability to scavenge carbon-centered and peroxyl radicals and its inhibitory effect on lipid peroxidation induced by various model systems were examined. Uric acid efficiently scavenged carbon-centered and peroxyl radicals derived from the hydrophilic free radical generator 2,2'-azobis-(2-amidinopropane)-dihydrochloride (AAPH). All damage to biological molecules, including protein, DNA and lipids induced by AAPH, was strongly prevented by uric acid. In contrast, alpha-tocopherol had little effect on damage to biological molecules. Lipid peroxidation by the lipophilic free radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) was little inhibited by uric acid, but not by alpha-tocopherol. Copper-induced lipid peroxidation was inhibited by uric acid and alpha-tocopherol. NADPH- and ADP-Fe(3+)-dependent microsomal lipid peroxidation was efficiently inhibited by alpha-tocopherol, but not by uric acid. Uric acid seems to scavenge free radicals in hydrophilic conditions to inhibit lipid peroxidation on the lipid-aqueous boundary, and the antioxidation is only little in lipophilic conditions.     

Defluorination of fluoroacetate in vitro by rat liver subcellular fractions.

An earlier report showing enhanced accumulation of ionic fluoride in bones of rats given fluoroacetate (FAc) suggested an in vivo defluorination of fluoroacetate. Rat liver an organ which shows minimal pathological and biochemical effects in FAc intoxication was found to possess defluorination activity in vitro. Subcellular fractionation of livers from male Sprague-Dawley rats was performed in 0.25 m sucrose and yielded the following fractions: whole homogenate, nuclear, mitochondrial, microsomal and 105,000 g supernatant. Defluorination activity was measured by incubating subcellular fractions and their boiled controls with FAc (1 hr, 37°C, pH 7.4, in 0.1 m Tris-HCl or 0.1% Triton X-100) and comparing the difference in ionic fluoride content at the end of the incubation. Defluorination activity based on protein content was consistently the highest in the 105,000 g supernatant fraction, the only fraction showing an activity increase over the original homogenate. The microsomal fraction had minimal activity. Defluorination activity in the 105,000 g supernatant as a function of time after fraction isolation at 4°C revealed a time-dependent reduction in activity. After 24 hr at 4°C, activity was almost completely lost. The time-dependent loss of activity at 4°C could be regained when glutathione (GSH) was added to this fraction at a final concentration of 5 mm. Furthermore, GSH significantly increased the defluorination activity in all subcellular fractions. A study of the optimum pH for defluorination activity in the 105,000 g supernatant fraction was performed in 0.1 m Tris-maleate buffer. Surprisingly, a complete loss of activity with this buffer resulted throughout the pH range studied (pH 5.6–8.0). Furthermore, defluorination activity of all subcellular rat liver fractions was completely inhibited by maleate and stimulated by glutathione. These results are consistent with the involvement of a sulfhydryl group in the defluorination of FAc in rat liver.     

Chemical and biochemical characteristics of O-demethylation of chlorotrianisene in the rat

The effects of NADPH concentration and of two inhibitors of the microsomal mixed function oxidase system [2-diethylaminoethyl-2,2-diphenyl valerate hydrochloride (SKF 525-A) and metyrapone] on rat liver microsomal O-demethylation of the triphenylethylene estrogen chlorotrianisene (CTA) were studied. Comparative data were obtained using untreated and phenobarbital-pretreated rats of both sexes. In the presence of microsomes from males, O-demethylation was induced slightly by phenobarbital (PB), and it was inhibited substantially by SKF 525-A, particularly with uninduced microsomes. Metyrapone had little inhibitory effect. In the presence of microsomes from females, O-demethylation was neither induced by PB nor inhibited significantly by SKF 525-A or metyrapone. Incubation of CTA with male rat microsomes afforded, after purification, a mixture of monophenolic metabolites which consisted primarily of a 1:1 mixture of E- and Z-desmethylchlorotrianisene (DMCTA).     

Protective role of S-adenosyl-l-methionine against acetaminophen induced mortality and hepatotoxicity in mice

Acetaminophen toxicity is demonstrated to he related to the protein binding of a reactive metabolite formed by the action of a P₄₅₀ mixed function oxidase. Results of the present study indicate that S-adenosyl-l-methionine (SAMe) protects against mortality induced by high doses (710 mg/kg) of acetaminophen. The liver toxicity induced by acetaminophen also appears to be reduced by SAMe, as shown both by GOT and GPT plasma activities and by histologic observation of the liver. The results of experiments on in vivo and in vitro binding of the radioactivity from acetaminophen to liver microsomal proteins suggest that SAMe protection is related to its metabolism to thiol derivatives in the transmethylation-trans-sulfuration pathway.     

Carbon tetrachloride and bromotrichloromethane toxicity: Dual role of covalent binding of metabolic cleavage products and lipid peroxidation in depression of microsomal calcium sequestration

We have investigated the importance of covalent binding and lipid peroxidation on the depression of microsomal calcium sequestration associated with in vitro metabolism of ¹⁴CCl₄. Studies with CBrCl₃ are also reported. In aerobic systems, promethazine was used to block lipid peroxidation, measured as malondialdehyde (MDA) generation. Effects of low levels of lipid peroxidation were tested in Fe²⁺ -supplemented systems free of halogenated hydrocarbons. The results indicate that microsomal calcium sequestration can be depressed significantly by metabolism of either CCl₄ or CBrCl₃ in the absence of MDA generation, or by lipid peroxidation occurring in the absence of halogenated hydrocarbons.     

Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals

The antioxidant and anti-inflammatory activities of two transition metal complexes of bioflavonoid rutin, Fe(rut)Cl(3) and Cu(rut)Cl(2), were studied. It was found that Cu(rut)Cl(2) was a highly efficient in vitro and ex vivo free radical scavenger that sharply decreased (by 2-30 times compared to the parent rutin): oxygen radical production by xanthine oxidase, rat liver microsomes, and rat peritoneal macrophages; the formation of thiobarbituric acid-reactive products in microsomal lipid peroxidation; and the generation of oxygen radicals by broncho-alveolar cells from bleomycin-treated rats. The copper-rutin complex was also a superior inhibitor of inflammatory and fibrotic processes (characterized by such parameters as macrophage/neutrophil ratio, wet lung weight, total protein content, and hydroxyproline concentration) in the bleomycin-treated rats. The antioxidant activity of Fe(rut)Cl(3) was much lower and in some cases approached that of rutin. Fe(rut)Cl(3) also stimulated to some degree spontaneous oxygen radical production by macrophages. We suggested that the superior antioxidant and anti-inflammatory activity of the copper-rutin complex is a consequence of its acquiring the additional superoxide-dismuting copper center. The inhibitory activity of Fe(rut)Cl(3) was lower, probably due to the partial reduction into Fe(rut)Cl(2) in the presence of biological reductants; however, similarly to the copper-rutin complex, this complex efficiently suppressed lung edema.