Biological markers of oxidative stress induced by ethanol and iron overload in rat.

Studies on rats treated for 15 months with ethanol (10%, w/v, solution in drinking water) revealed that the stimulation of hepatic cytochrome P-450 monooxygenases activity was accompanied by enhanced microsomal malondialdehyde formation, a lipid peroxidation index and a decreased level of the antioxidant, alpha-tocopherol. The other components of the prooxidant/antioxidant system, diene conjugates and catalase, glutathione peroxidase and superoxide dismutase activities were unaffected. Oxidative stress in blood was shown by a significant decrease in the alpha-tocopherol level whereas lipid peroxidation and antioxidant enzyme activity remained unchanged. The prooxidative effect of ethanol was catalytically promoted by an iron overload (Fe-saccharate, 100 mg Fe3+/kg body wt. intraperitoneally, 2, 5 and 7 day before test) to simulate the effect of alcoholic hemochromatosis. Thus, the level of malondialdehyde and alpha-tocopherol in the serum may be recommended as biological markers of ethanol-provoked oxidative stress, which is especially useful in the evaluation of the combined effect of ethanol and other chemicals that affect the redistribution of active iron complexes.

[Induction of cytochrome P-450 monooxygenase after combined exposure of rats to xylene and ethanol]. / Indukcja cytochrom P-450 monooksygenaz pod wplywem kombinowanego narazenia szczurów na ksylen i etanol.

Increased biological risk following from combined exposure to xylene, an industrial solvent, and ethanol, the most likely additional factor to occupational exposure, may result from inductive effects of the chemicals on cyt. P-450 monooxygenase, where biotransformation of xylene and, in part of ethanol, takes place. Studies were carried out on rats: 1) preinduced with ethanol (10% solution in drinking water for 8 months) and for next 9 days jointly exposed to xylene vapour at concentration of 12,000 mg/m3; 2) preexposed to m-xylene vapours at concentration of 4,000 mg/m3 for 6 and 12 weeks or at concentration of 400 mg/m3 for 5 months and for next 3 days jointly administered ethanol (5 doses of 2.5 g/kg in 12 hours intervals). It has been found that ethanol and xylene in both models of combined exposure exert additive stimulatory effects on the activity of cyt. P-450 monooxygenases (aniline p-hydroxylase, microsomal ethanol oxidizing system, NADPH-cyt. c reductase, cytochrome P-450) and intenseness of the effects depends both on the level and duration of exposure to xylene/m-xylene and ethanol.

[Stimulation of microsomal lipid peroxidation as the effect of combined action of xylene and ethanol]. / Stymulacja peroksydacji lipidów mikrosomalnych jako efekt lacznego dzialania ksylenu i etanolu.

The aim of the study was to evaluate if in the case of combined exposure of rats to xylene and ethanol stimulation of lipid peroxidation in the liver microsomes (an index of interaction with lipids and derangements of integrity/fluidity of membranes) might occur. Experiments were carried out on male Wistar rats in the conditions of prolonged, inhalatory preexposure to m-xylene at concentration of 4000 mg/m3 for 6 and 12 weeks, and next joint 5-fold treatment with ethanol (2.5 g/kg oral doses in 12 hours intervals for 3 days). The degree of lipid peroxidation was assessed both in vivo and in vitro under chemical stimulation: enzymatically (NADPH, Fe2+) and nonenzymatically (ascorbic acid, Fe2+). The chemical stimulation permits to measure multiplied biological effects of chemicals acting in vivo. As a results of performed studies it was found that the highest increase of lipid peroxidation appeared in the case of prolonged, 12 weeks exposure to m-xylene (4000 mg/m3) and successively under subacute ethanol treatment and 6-week m-xylene exposure. Thus, it was evidenced that stimulation of lipid peroxidation depends on the duration of exposure to m-xylene. Stimulation of lipid peroxidation, revealed here, may arise from the processes of biotransformation of xylene in cyt. P-450 monooxygenase system where generated oxygen free radicals may attack the lipid components of microsomal membrane as well as from the mechanisms leading to decrease of antioxidant ability of the organism (decrease of glutathione-SH and vitamins E and C levels).

The effect of combined exposures to ethanol and xylene on rat hepatic microsomal monooxygenase activities.

Studies on rats treated for 8 months with ethanol (10% solution in drinking water) and simultaneously exposed to xylene vapour (12,000 mg/m3, 5 hr daily) for the last 9 days revealed that the chemicals exert additive stimulatory effect on hepatic microsomal monooxygenase: the activity of aniline p-hydroxylase increased by 380%, microsomal ethanol oxidizing system by 92%, NADPH-cyt. c reductase by 30% and the level of cytochrome P-450 by 70%. The changes were accompanied by a marked proliferation of smooth endoplasmic reticulum (a subcellular site of cytochrome P-450 monooxygenases in the hepatocytes) and an increased NADPH-Fe2+- and ascorbate-Fe2+-driven lipid peroxidation in microsomal membranes--a potential toxic mechanism. Interaction of ethanol and xylene with cytochrome P-450 monooxygenases may enhance metabolic capacity of the liver and in consequence modify biological/toxic effects of occupational exposure to solvents in the case of alcohol abuse.

Silica earth provoked lung fibrosis with stimulation of lysosomal enzymes and lipid peroxidation in rats.

The dynamics of the biological response of pulmonary tissue to silica dust (silica earth from Piotrowice, Poland, recommended as a domestic reference fibrogenic standard) was studied in rats after single-shot intratracheal instillation of a suspension of 20 mg of the dust for one, three, and seven months. Silica dust provoked pronounced pulmonary fibrosis as inferred from increased collagen content together with pathomorphological alteration (silicotic nodules). The lung burden of silica dust affected the lysosomal subfraction as manifested by an increase in its protein content with concomitant stimulation (release and presumably induction) of beta-glucuronidase and cathepsin D and a transient (up to three months) stimulation of lipid peroxidation. Stimulation of activity of lysosomal enzymes and lipid peroxidation mediated by silica dust may reflect destructive metabolic processes resulting in the development of pulmonary fibrosis as the sign of a pathological repair mechanism. The extent of the effects brought about by silica earth testify that it may be recommended as a reference standard for evaluating the potential health hazard from industrial exposure to dusts containing SiO2.

Enhanced lipid peroxidation and lysosomal enzyme activity in the lungs of rats with prolonged pulmonary deposition of crocidolite asbestos.

The interaction of UICC crocidolite asbestos with biological membranes in vivo was studied in rats after a single intratracheal dose of a suspension of 20 mg of fibres per rat. Development of lung fibrosis (increased level of hydroxyproline, a collagen index together with corresponding pathomorphological alteration) confirmed the penetration of crocidolite fibres into the lungs in the course of seven months exposure. The pulmonary deposition of crocidolite affected the lysosomal membranes of lung cells as manifested by (1) enhanced lipid peroxidation with (2) stimulation (release) of activity of beta-glucuronidase and cathepsin D. Enhanced lipid peroxidation and activity of beta-glucuronidase may contribute to the delayed, carcinogenic effects of crocidolite asbestos.

Combined effect of ethanol and carbon disulphide on cytochrome P-450 mono-oxygenase, lipid peroxidation and ultrastructure of the liver in chronically exposed rats.

A 5-month treatment of rats with ethanol (10% solution in drinking water) stimulated aniline p-hydroxylase and the microsomal ethanol oxidizing system (MEOS) by 140 and 70%, respectively, cytochrome P-450 by 22% and accompanied by lipid peroxidation by 40% in microsomes. It also caused smooth endoplasmic reticulum (SER) proliferation and rough endoplasmic reticulum (RER) degranulation in hepatocytes. Repeated inhalatory exposure of rats to 1.5 g/m3 of CS2, 5 h daily, 5 days a week for 5 months decreased aniline p-hydroxylase and MEOS by 70 and 55% respectively, doubled hexobarbital sleeping time and depressed cytochrome P-450 by 30% and its conversion to cytochrome P-420; these effects were accompanied by the appearance of cytochrome P-420, the intensification of lipid peroxidation in microsomes and some degranulation of RER in hepatocytes. Combined exposure of rats to ethanol and CS2 resulted in a significant potentiation of the inhibitory effects of CS2 on cytochrome P-450 mono-oxygenase and MEOS but with enhancement of CS2 effects on the liver microsomal mono-oxygenase, but CS2 decreased the effect of ethanol on SER proliferation. The interaction both on the biochemical and the morphological level can be explained with the ethanol-stimulated biotransformation of CS2 to reactive electrophilic derivative(s), the subsequent destruction of cytochrome P-450 to cytochrome P-420 and the intensification of lipid peroxidation.

Stimulation of lipid peroxidation and heme oxygenase activity with inhibition of cytochrome P-450 monooxygenase in the liver of rats repeatedly exposed to cadmium.

Exposure of rats to 0.1 and 0.5 mg Cd/kg subcutaneously (s.c.) thrice weekly for 5 weeks resulted in an accumulation of cadmium in the liver in concentrations of 40 and 95 micrograms/g tissue, respectively, and a microsomal burden of Cd amounting to approx. 2-3% of the retained cadmium. The cytoplasm contained about 80% of the cadmium. At an exposure dose of 0.1 mg Cd/kg, stimulation of lipid peroxidation by 22% and inhibition of ALA synthetase by 16% in the liver were observed. The higher exposure of 0.5 mg Cd/kg caused an inhibition of microsomal monooxygenase with depression of cytochrome P-450 and cytochrome b5 by 20% (over 2-fold prolongation of hexobarbital sleeping time and statistically significant decrease of activity of aniline p-hydroxylase). The loss of cytochrome P-450 probably was due to an intensified lipid peroxidation and induction of heme oxygenase (30% and 60% over control, respectively). Sequestration of cadmium by cytoplasm (metallothionein) does not protect microsomes against cadmium accumulation and specific biochemical disturbances.

Protective effect of methionine against vinyl chloride-mediated depression of non-protein sulphydryls and cytochrome p-450.

Potentiality of a protective effect of methionine against hepatotoxic action of vinyl chloride (VC) was assessed on rats, induced with phenobarbital and thereafter exposed to 50 000 ppm VC for 5 h. This extremely high dosage of VC warranted the appearance of drastic symptoms of liver injury in the course of 20 h postexposure. DL-Methionine in a dose of 1 g/kg given by gavage prior to the VC-exposure counteracted both the BC-provoked depletion of non-protein sulphydryls and destruction of cytochrome P-450 in the liver. In the methionine-pretreated rats the phenobarbital-induced synthesis of microsomal proteins in the livers was maintained during the post-exposure period, contrary to the non-pretreated animals. It may be justified to apply methionine as a prophylactic agent in cases of occupational exposure to VC.

Urinary excretion of thiodiglycollic acid and hepatic content of free thiols in rats at different levels of exposure to vinyl chloride.

In order to assess the process of oxidation and conjugation involved in biotransformation of vinyl chloride (VC), rats were exposed to 50, 200, 500, 1000 and 20,000 ppm inhaled VC. The rate of urinary excretion of thiodiglycollic acid (TDGA) after exposure to each investigated concentration of VC depends on the activity of microsomal monooxygenase. In general, higher levels of TDGA in urine were reflected by a more significant depression of non-protein sulfhydryl content in the liver of rats, whereas no changes were observed in those with inhibited activity of microsomal monooxygenase and depressed urinary levels of TDGA. The significance of alcohol dehydrogenase in the metabolism of low concentrations of VC has not been confirmed. Metabolism of VC in the range of 50--2 000 ppm is mediated by microsomal monooxygenase followed by conjugation with thiols.

Monooxygenase activity and ultrastructural changes of liver in the course of chronic exposure of rats to vinyl chloride.

The activity of microsomal cytochrome P-450 monooxygenase and ultrastructure of the liver have been studied in rats exposed dynamically to 50, 500, and 20,000 ppm of vinyl chloride (VC) over 10 months. After 1 and 3 months of exposure to 500 and 20,000 ppm of VC, the level of cytochrome P-450 was slightly lower than in the control animals and upon continuation of exposure it was restored to the original level accompanied by slight increase of activity of aniline p-hydroxylase. Liver enlargement, developed in the course of the exposure, was accompanied by ultrastructural alterations beginning in the 3rd month of exposure to all concentrations of VC. Development of hepatic alterations (hypertrophy of smooth and rough endoplasmic reticulum, swelling of mitochondria, accumulation of lipid droplets, focal cytoplasmic degradation) is discussed with regard to the activity of microsomal monooxygenase system in metabolizing VC to toxic metabolites.

Stimulatory effects of chlordiazepoxide, diazepam and oxazepam on the drug-metabolizing enzymes in microsomes.

5 days' exposure of rats to daily doses of 400 mg/kg body wt. of chlordiazepoxide, diazepam and oxazepam stimulated the microsomal metabolism in the liver, as evidenced by acceleration of both p-hydroxylation of aniline and hydroxylation of benzene. The effect was accompanied by an increased concentration of liver microsomal protein and by the development of tolerance to the drugs. Similar effects were found after exposure of rats to lower doses of the drugs. The metabolism of aniline in vivo in rats treated with chlordiazepoxide was accelerated; this was correlated with development of tolerance to these drugs. It is suggested that both the stimulation of microsomal metabolism and the development of tolerance are associated with the induction of microsomal drug-metabolizing enzymes.

The rate of aniline metabolism in vivo in rats exposed to aniline and drugs.

1. The rates of aniline metabolism have been studied in vitro using rat liver homogenates, and in vivo by determination of the unchanged aniline remaining in the cadaver. 2. Metabolism of aniline in vivo is stimulated by phenobarbital and 3,4-benzpyrene, and inhibited by SKF 525-A. 3. Cyclobarbital and phenacetin stimulate aniline metabolism both in vitro and in vivo. 4. Pre-treatment with aniline impaired the metabolism of aniline in vivo but increased the in vitro metabolism to p-aminophenol. Pre-treatment of rats with phenobarbital and aniline did not accelerate metabolism of aniline in vivo but the stimulating effect of phenobarbital on protein synthesis in microsomes was maintained. In contrast, pre-treatment with 3,4-benzpyrene and aniline stimulated metabolism of aniline in vivo. The possible mechanism of changes in aniline metabolism due to previous exposure to aniline is discussed.

Effect of repeated exposure to aniline, nitrobenzene, and benzene on liver microsomal metabolism in the rat.

Exposure of rats to aniline at daily doses of 50 mg/kg of body weight over a month stimulated the microsomal metabolism as manifested by (1) acceleration of p-hydroxylation of anilin and N-demethylation of aminopyrine in 9-000 times g postmitochondrial supernatant of the liver, (2) shortening the sleeping time after hexobarbital, and (3) reduction of the antipyretic effect of phenacetin. In the rats exposed to nitrobenzene in a similar manner to aniline, nitroreduction of nitrobenzene and p-hydroxylation of aniline remained unaffected; the antipyretic effect of phenacetin was decreased, whereas hexobarbital sleeping time remained unchanged. Exposure of rats to benzene (50 mg/kg of body weight daily for a month) had no effect on the rate of hydroxylation of benzene and N-demethylation of aminopyrine. In benzene-exposed rats hexobarbital sleeping time was prolonged whereas the antipyretic effect of phenacetin was unaffected. Microsomal metabolism of aniline, nitrobenzene, and benzene was stimulated and inhibited when the rats were pretreated with phenobarbital and SKF 525-A, respectively.