Metal working fluids: sub-chronic effects on pulmonary functions in B6C3F1 mice given vitamin E deficient and sufficient diets

Metal working fluids (MWFs) have been widely known to cause asthma and neoplasia of the larynx, pancreas, rectum, skin and urinary bladder (Textbook of Clinical Occupational and Environmental Medicine (1994) 814; Am. J. Ind. Med. 32 (1997) 240; Am. J. Ind. Med. 33 (1997) 282; Am. J. Ind. Med. 22 (1994) 185). Other non-neoplastic respiratory effects in industrial workers attributed to MWFs include increased rates of cough, phlegm production, wheeze, chronic bronchitis and chest tightness (Eur. J. Resir. Dis. 63(118) (1982), 79; J. Occup. Med. 24 (1982) 473; Am. J. Ind. Med. 32 (1997) 450). The epidemic and endemic nature of immune mediated lung morbidity commonly known as hypersensitivity pneumonitis in workers from several different industries using MWFs has been well documented (J. Allergy clin. Immunol. 91 (1993) 311; Chest 108 (1995) 636; MMWR45 (1996) 606; Am. J. Ind. Med. 32 (1997) 423). We studied morphological/functional and antioxidant outcomes in lungs after inhalation exposure of vitamin E deficient mice to MWF (27 mg m(-3) 17 weeks, 5 days a week, 6 h a day). Mice were given vitamin E deficient (<10 IU kg(-1) vitamin E) or basal diets (50 IU kg(-1) vitamin E) for 35 weeks. Inhalation exposure to MWF started after 18 weeks on diet. Microscopic observation of lungs from mice given vitamin E deficient or sufficient diets revealed no inflammation or morphological alteration after exposure to MWF. Mice given vitamin E deficient diet exhibited a significant decrease (P<0.05) in breathing rate, peak inspiratory/expiratory flow, minute ventilation, and tidal volume compared with sufficient controls. However, no differences were found after exposure to MWF in pulmonary function, with the exception of tidal volume which also significantly decreased (P<0.05). Exposure to MWF reduced vitamin E, protein thiol and ascorbate level in lungs. Exposure to MWF in combination with a vitamin E deficient diet resulted in significantly enhanced accumulation of peroxidative products compared with vitamin E deficient controls. This is the first report that describes the increase of oxidative stress in the lungs after MWF exposure.     

Role of hippocampal oxidative stress in memory deficits induced by sleep deprivation in mice

Numerous animal and clinical studies have described memory deficits following sleep deprivation. There is also evidence that the absence of sleep increases brain oxidative stress. The present study investigates the role of hippocampal oxidative stress in memory deficits induced by sleep deprivation in mice. Mice were sleep deprived for 72 h by the multiple platform method-groups of 4-6 animals were placed in water tanks, containing 12 platforms (3 cm in diameter) surrounded by water up to 1 cm beneath the surface. Mice kept in their home cage or placed onto larger platforms were used as control groups. The results showed that hippocampal oxidized/reduced glutathione ratio as well as lipid peroxidation of sleep-deprived mice was significantly increased compared to control groups. The same procedure of sleep deprivation led to a passive avoidance retention deficit. Both passive avoidance retention deficit and increased hippocampal lipid peroxidation were prevented by repeated treatment (15 consecutive days, i.p.) with the antioxidant agents melatonin (5 mg/kg), N-tert-butyl-alpha-phenylnitrone (200 mg/kg) or vitamin E (40 mg/kg). The results indicate an important role of hippocampal oxidative stress in passive avoidance memory deficits induced by sleep deprivation in mice.     

Antioxidant balance and free radical generation in vitamin e-deficient mice after dermal exposure to cumene hydroperoxide

Organic peroxides are widely used in the chemical industry as initiators of oxidation for the production of polymers and fiber-reinforced plastics, in the manufacture of polyester resin coatings, and pharmaceuticals. Free radical production is considered to be one of the key factors contributing to skin tumor promotion by organic peroxides. In vitro experiments have demonstrated metal-catalyzed formation of alkoxyl, alkyl, and aryl radicals in keratinocytes incubated with cumene hydroperoxide. The present study investigated in vivo free radical generation in lipid extracts of mouse skin exposed to cumene hydroperoxide. The electron spin resonance (ESR) spin-trapping technique was used to detect the formation of alpha-phenyl-N-tert-butylnitrone (PBN) radical adducts, following intradermal injection of 180 mg/kg PBN. It was found that 30 min after topical exposure, cumene hydroperoxide (12 mmol/kg) induced free radical generation in the skin of female Balb/c mice kept for 10 weeks on vitamin E-deficient diets. In contrast, hardly discernible radical adducts were detected when cumene hydroperoxide was applied to the skin of mice fed a vitamin E-sufficient diet. Importantly, total antioxidant reserve and levels of GSH, ascorbate, and vitamin E decreased 34%, 46.5%. 27%, and 98%, respectively, after mice were kept for 10 weeks on vitamin E-deficient diet. PBN adducts detected by ESR in vitamin E-deficient mice provide direct evidence for in vivo free radical generation in the skin after exposure to cumene hydroperoxide.     

Vitamin E deficiency enhances pulmonary inflammatory response and oxidative stress induced by single-walled carbon nanotubes in C57BL/6 mice

Exposure of mice to single-walled carbon nanotubes (SWCNTs) induces an unusually robust pulmonary inflammatory response with an early onset of fibrosis, which is accompanied by oxidative stress and antioxidant depletion. The role of specific components of the antioxidant protective system, specifically vitamin E, the major lipid-soluble antioxidant, in the SWCNT-induced reactions has not been characterized. We used C57BL/6 mice, maintained on vitamin E-sufficient or vitamin E-deficient diets, to explore and compare the pulmonary inflammatory reactions to aspired SWCNTs. The vitamin E-deficient diet caused a 90-fold depletion of alpha-tocopherol in the lung tissue and resulted in a significant decline of other antioxidants (GSH, ascorbate) as well as accumulation of lipid peroxidation products. A greater decrease of pulmonary antioxidants was detected in SWCNT-treated vitamin E-deficient mice as compared to controls. Lowered levels of antioxidants in vitamin E-deficient mice were associated with a higher sensitivity to SWCNT-induced acute inflammation (total number of inflammatory cells, number of polymorphonuclear leukocytes, released LDH, total protein content and levels of pro-inflammatory cytokines, TNF-alpha and IL-6) and enhanced profibrotic responses (elevation of TGF-beta and collagen deposition). Exposure to SWCNTs markedly shifted the ratio of cleaved to full-length extracellular superoxide dismutase (EC-SOD). Given that pulmonary levels of vitamin E can be manipulated through diet, its effects on SWCNT-induced inflammation may be of practical importance in optimizing protective strategies.     

DERMAL AND SYSTEMIC TOXICITY AFTER APPLICATION OF SEMISYNTHETIC METAL-WORKING FLUIDS IN B6C3F1 MICE

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 µl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 5% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5% MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Phenol-induced in vivo oxidative stress in skin: evidence for enhanced free radical generation, thiol oxidation, and antioxidant depletion

A variety of phenolic compounds are utilized in industry (e.g., for the production of phenol (PhOH)-formaldehyde resins, paints and lacquers, cosmetics, and pharmaceuticals). They can be toxic to skin, causing rash, dermal inflammation, contact dermatitis, depigmentation, and cancer promotion. The biochemical mechanisms for the dermal toxicity of phenolic compounds are not well understood. We hypothesized that topical PhOH exposure results in the generation of radicals, possibly via redox-cycling of phenoxyl radicals, which may be an important contributor to dermal toxicity via the stimulation of the induction and release of inflammatory mediators. To test this hypothesis, we (1) monitored in vivo the formation of PBN-spin-trapped radical adducts by ESR spectroscopy, (2) measured GSH, protein thiols, vitamin E, and total antioxidant reserves in the skin of B6C3F1 mice topically treated with PhOH, and (3) compared the responses with those produced by PhOH in mice with diminished levels of GSH. We found that dermal exposure to PhOH (3.5 mmol/kg, 100 microL on the shaved back, for 30 min) caused oxidation of GSH and protein thiols and decreased vitamin E and total antioxidant reserves in skin. The magnitude of the PhOH-induced generation of PBN-spin-trapped radical adducts in the skin of mice with diminished levels of GSH (pretreated with BCNU, an inhibitor of glutathione reductase, or BSO, an inhibitor of gamma-glutamylcysteine synthetase) was markedly higher compared to radical generation in mice treated with PhOH alone. Topical exposure to PhOH resulted in skin inflammation. Remarkably, this inflammatory response was accelerated in mice with a reduced level of GSH. Epidermal mouse cells exposed to phenolic compounds showed the induction of early inflammatory response mediators, such as prostaglandin E 2 and IL-1beta. Since dermal exposure to PhOH produced ESR-detectable PBN spin-trapped signals of lipid-derived radicals, we conclude that this PhOH-induced radical formation is involved in oxidative stress and dermal toxicity in vivo.     

Dermal and systemic toxicity after application of semisynthetic metal-working fluids in B6C3F1 mice

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 microl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 55% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5%, MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Effect of dietary fat-soluble vitamins A and E and proanthocyanidin-rich extract from grape seeds on oxidative DNA damage in rats.

This study reports the effect of the fat-soluble vitamin A or vitamin E and grape seed proanthocyanidin extract (GSPE) on oxidative DNA damage estimated by 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) contents in urine and leukocyte of rats. Little is known about the antioxidant potency of dietary anthocyanidins and consequently, the aim of this study was to establish whether anthocyanidins could act as putative antioxidant micronutrients. Seven groups of male Sprague-Dawley rats were fed during 47 days with the following diets: a basic diet, two deficient vitamin A or E diets, two supplemented vitamin A or E diets and two supplemented diets enriched with two doses of grape seed proanthocyanidin extract. At the end of the diet intervention period, 24h, urine and blood were collected. The levels of 8-oxodG in leukocytes rats were significantly lower in the supplemented vitamin A, E and GSPE diet groups with respect to the control group. However, consumption of alpha-tocopherol, vitamin A or GSPE had no effect on the excretion of the oxidised nucleoside 8-oxodG. These results suggest that a vitamin E and A and GSPE enriched-diets have a protective effect on oxidative DNA damage limited to rat leukocytes.     

Photoprotective actions of topically applied vitamin E

Topical application of vitamin E has been shown to decrease the incidence of ultraviolet (UV)-induced skin cancer in mice. Vitamin E provides protection against UV-induced skin photodamage through a combination of antioxidant and UV absorptive properties. Topical application of alpha-tocopherol on mouse skin inhibits the formation of cyclobutane pyrimidine photoproducts. However, topically applied alpha-tocopherol is rapidly depleted by UVB radiation in a dose-dependent manner. The photooxidative fate of the alpha-tocopherol depends on the local environment of the vitamin E. alpha-Tocopherol quinone and alpha-tocopherol quinone epoxides are principal photoproducts of vitamin E that has penetrated into the epidermal layer of the skin, whereas tocopherol dimers and trimers are formed from alpha-tocopherol in a bulk phase at the skin surface. Dimer and trimer products may participate in prevention of UV-induced photodamage.     

Lipid peroxidation and antioxidant systems in the liver injury produced by glutathione depleting agents

The mechanisms of the liver damage produced by three glutathione (GSH) depleting agents, bromobenzene, allyl alcohol and diethylmaleate, was investigated. The change in the antioxidant systems represented by alpha-tocopherol (vitamin E) and ascorbic acid were studied under conditions of severe GSH depletion. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. The hepatic level of vitamin E was decreased whenever extensive lipid peroxidation developed. In the case of bromobenzene intoxication, vitamin E decreased before the onset of lipid peroxidation. Changes in levels of the ascorbic and dehydroascorbic acid indicated a redox cycling of vitamin C with the oxidative stress induced by all the three agents. Such a change of the redox state of vitamin C (increase of the oxidized over the reduced form) may be an index of oxidative stress preceding lipid peroxidation in the case of bromobenzene. In the other cases, such a change is likely to be a consequence of lipid peroxidation. Experiments carried out with vitamin E deficient or supplemented diets indicated that the pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. Animals fed a vitamin E supplemented diet had an hepatic vitamin E level double that obtained with a commercial pellet diet. In such animals, bromobenzene and allyl alcohol had only limited toxicity and diethylmaleate none in spite of comparable hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of the toxicity by GSH depleting agents.     

Fructose 1,6-diphosphate alleviates UV-induced oxidative skin damage in hairless mice

Reactive oxygen species (ROS) are involved in the deleterious effects of UV light on skin. The antioxidant defense system is considered to be crucial for protecting skin from ROS. Recently, we showed that fructose 1,6-diphosphate (FDP), a glycolytic metabolite, reduced oxidative stress in UVB-irradiated keratinocytes. This study set out to determine whether topically applied FDP could exert protective effects against UV-induced skin damage in hairless mice. An in vitro skin permeation study using Franz-type diffusion cells showed that the amount of [14C]-FDP that diffused through the skin increased in a time-dependent manner, and about 3.5% of the applied FDP penetrated the skin after 24 h. Topical application of FDP (1%) preserved the endogenous antioxidant capacity of skin such as catalase and glutathione, which were significantly reduced after UVB irradiation without FDP. FDP also reversed the loss of catalase protein and prevented the accumulation of carbonylated proteins induced by UVB irradiation. These results provide evidence that topically administered FDP could penetrate into the skin and attenuate UVB-induced oxidative skin damage in hairless mice.     

Effects of acute alcohol consumption and vitamin E co-treatment on oxidative stress parameters in rats tongue

The aim of this study was to evaluate the effects of acute alcohol consumption and vitamin E co-treatment upon oxidative stress parameters in rats tongue. Thirty-eight, Wistar rats were separated into five groups (alcohol, alcohol/vitamin E, control, Tween, vitamin E). Alcohol and alcohol vitamin E groups had the standard diet, and 40% alcohol on drinking water. Other groups were fed with the same standard diet and water ad libitum. Vitamin E was given by gavage to vitamin E and alcohol/vitamin E rats twice a week. Alcohol and control groups were subjected to saline gavage and Tween group to 5% Tween 80 solution, the vitamin E vehicle. At day 14, the animals were anesthetized and specimens were obtained from tongue. Lipid peroxidation (TBARS), protein oxidative damage, catalase (CAT) and superoxide dismutase (SOD) activities were quantified. Alcohol group decreased TBARS in relation to control group and alcohol vitamin-treated animals decreased TBARS when compared to Tween and vitamin E groups. SOD activity was lower and CAT activity was higher in animals treated with both alcohol and vitamin E. These results suggest that short-term alcohol consumption decreases lipid peroxidation levels. Alternatively, alcohol/vitamin E group increased CAT, showing the toxicity of this association.     

Preventive effects of fenofibrate on insulin resistance, hyperglycaemia, visceral fat accumulation in NIH mice induced by small-dose streptozotocin and lard.

High-fat diets and oxidative damage may contribute to the development of type 2 diabetes. Hypolipidaemic drugs and antioxidants were supposed to prevent the development of the disease. In this study, we investigated preventive effects of fenofibrate (200 mg kg(-1)), vitamin C (30 mg kg(-1)), combination of both in mice induced by streptozotocin (35 mg kg(-1)) and soluble lard (15 ml kg(-1)). The results showed the mice demonstrated hyperglycaemia and hypercholesterolaemia, visceral fat accumulation, and a slight increase in liver glycogen/triglyceride and oxidative stress within 60 days of treatment. Fenofibrate enhanced insulin sensitivity, improved glycaemic control, lowered serum triglycerides, reduced body and visceral fat weights, and decreased liver glycogen/lipid levels but showed hepatotoxicity in the mice. Vitamin C neither itself prevented nor enhanced preventive effects of fenofibrate on glucose and lipid metabolism but partly attenuated the hepatotoxicity of fenofibrate. These results suggest that fenofibrate inhibit development of type 2 diabetes induced by high-fat diets and oxidative stress. However, here, vitamin C just can serve as an adjunct to fenofibrate therapy against its hepatotoxicity. In the future study, we should investigate if higher dosage of vitamin C or other antioxidants would enhance preventive effects of fenofibrate in type 2 diabetes.     

Alteration of convulsive threshold and conditioned avoidance response in mice fed diets containing contraceptive steroids

Female CD1 mice given the contraceptive steroids mestranol and norethynodrel (1:10) in the diet (0.0033%) for 4 months had a growth reduction of 20% when compared with mice fed a normal diet, and had lower convulsive thresholds when tested with the vitamin B6 antagonists 2,4-dimethyl-5-methyl-hydroxypyrimidine and thiosemicarbazide. In conditioned avoidance response (CAR) tests, mice fed the steroid-containing diets showed a decreased acquisition performance during all six sessions; however, mice fed the same diet supplemented with vitamin B6 (0.04%) performed as well during the last three sessions of the CAR tests as mice fed the normal diet.     

Evaluation of dichloroacetate treatment in a murine model of hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease severely affecting liver and kidney and is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). Administration of 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) improves the HT1 phenotype but some patients do not respond to NTBC therapy. The objective of the present study was to evaluate whether administration of dichloroacetate, an inhibitor of maleyl acetoacetate isomerase (MAAI) to FAH-knockout mice could prevent acute pathological injury caused by NTBC withdrawal. DCA (0.5 and 5g/L) was given in combination with a standard diet or with a tyrosine-restricted diet. With the low-tyrosine diet body weight loss and most of hepatic and renal injuries were prevented regardless the DCA dose. The administration of DCA with a standard diet did not prevent damage nor the oxidative stress response nor the AFP induction seen in FAH-knockout mice. DCA was shown to inhibit hepatic MAAI activity to 86% (0.5g/L) and 94% (5g/L) of untreated wild-type mice. Interestingly, FAH(-/-) mice deprived of NTBC (NTBC-OFF) and NTBC-treated FAH-knockout mice had similar low hepatic MAAI activity levels, corresponding to 10-20% of control. Thus the failure of DCA treatment in FAH(-/-) mice seems to be attributed to the residual MAAI activity, high enough to lead to FAA accumulation and HT1 phenotype.     

Glutathione depletion: its effects on other antioxidant systems and hepatocellular damage.

1. The mechanisms of the liver damage produced by three glutathione (GSH)-depleting agents, bromobenzene, allyl alcohol and diethyl maleate, were investigated. 2. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH. 3. Changes in antioxidant systems by alpha-tocopherol (vitamin E) and ascorbic acid were studied. A decrease in the hepatic level of vitamin E, and a change in the redox state of vitamin C (increase in oxidized over reduced form) were evident whenever extensive lipid peroxidation developed. However, in the case of bromobenzene intoxication these alterations preceded lipid peroxidation, and may be an index of oxidative stress leading to subsequent membrane damage. 4. Experiments carried out with vitamin E-deficient or supplemented diets indicated that pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E-deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. In animals fed a vitamin E-supplemented diet, bromobenzene and allyl alcohol had only limited toxicity, and diethyl maleate none, in spite of similar hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of toxicity by GSH-depleting agents.     

High levels of apoptosis induced by total body irradiation in mice fed a low protein-low vitamin E diet.

Apoptosis in response to total body irradiation (TBI) in mice fed four kinds of diets was assessed. Male mice were fed a low protein-low vitamin E diet, a low protein-basal vitamin E diet, a basal protein-low vitamin E diet or a basal protein-basal vitamin E diet for 2 weeks, then received TBI at a dose of 0, 1 or 4 Gy, and were sacrificed 4 h after TBI. Apoptosis was assessed according to the appearance of DNA fragmentation patterns [DNA laddering and positive in situ end-labeling by terminal transferase (TUNEL) assay] and apoptotic index (AI). Electrophoresis of DNA from the spleen in the groups that did not receive TBI showed no cleavage, whereas that of DNA in the groups with 4 Gy of TBI displayed a ladder pattern, indicative of internucleosomal cleavage characteristic of apoptosis. In the groups with 4 Gy of TBI, apoptosis was enhanced in the low protein-low vitamin E group.     

Contact dermatoses in metal workers

We studied 150 metal workers occupationally exposed to metals and metalworking fluids (MWFs) to determine the prevalence and nature of contact dermatitis. 150 office workers were used as non-exposed control group. Questionnaires were administered to evaluate occupational and non-occupational exposure. All subjects underwent a dermatological examination and patch-testing with standard allergen series and MWFs used in the plant. Twenty-eight metal workers (18.6%) presented minor skin disorders involving the hands (vs. only 2% of the controls), ten (6.6%) had major disorders (similar to the figure for the control group - 5.4%), and 112 (74.8%) had no lesions, as opposed to 92.6% of the control group. Positive patch tests were found in ten metal workers: eight had major skin disorders (six to nickel, cobalt and chromium, one to nickel and cobalt, one to nickel) and the remaining two were asymptomatic (one positive for nickel and chromium, one for nickel). Among the controls there were three cases of positivity, all among asymptomatic subjects. Patch tests with MWFs were negative. The prevalence of dermatoses among the metal workers was significantly higher than that of controls (p<0.01), and all cases of allergy in this group were provoked by metals themselves.     

Glutathione depletion: Its effects on other antioxidant systems and hepatocellular damage

1. The mechanisms of the liver damage produced by three glutathione (GSH)-depleting agents, bromobenzene, allyl alcohol and diethyl maleate, were investigated.

2. With each toxin liver necrosis was accompanied by lipid peroxidation that developed only after severe depletion of GSH.

3. Changes in antioxidant systems by α-tocopherol (vitamin E) and ascorbic acid were studied. A decrease in the hepatic level of vitamin E, and a change in the redox state of vitamin C (increase in oxidized over reduced form) were evident whenever extensive lipid peroxidation developed. However, in the case of bromobenzene intoxication these alterations preceded lipid peroxidation, and may be an index of oxidative stress leading to subsequent membrane damage.

4. Experiments carried out with vitamin E-deficient or supplemented diets indicated that pathological phenomena occurring as a consequence of GSH depletion depend on hepatic levels of vitamin E. In vitamin E-deficient animals, lipid peroxidation and liver necrosis appeared earlier than in animals fed the control diet. In animals fed a vitamin E-supplemented diet, bromobenzene and allyl alcohol had only limited toxicity, and diethyl maleate none, in spite of similar hepatic GSH depletion. Thus, vitamin E may largely modulate the expression of toxicity by GSH-depleting agents.