Effects of buthionine sulfoximine and cysteine on the hepatotoxicity of butylated hydroxytoluene in rats.

Pretreatment with buthionine sulfoximine (BSO; 900 mg/kg) induced the elevation of serum GOT and GPT activities in a non-toxic dose of butylated hydroxytoluene (BHT; 250-500 mg/kg) in rats. The elevation of serum enzyme activities was accompanied by a remarked depletion of the hepatic glutathione (GSH) concentration. In contrast, pretreatment with cysteine (100-200 mg/kg) inhibited the elevation of serum enzyme activities at a toxic dose of BHT (1000 mg/kg). The effects of BSO and cysteine on BHT-induced hepatotoxicity in rats are discussed.

Hepatotoxicity of butylated hydroxytoluene and its analogs in mice depleted of hepatic glutathione.

Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol, BHT) has been reported to be a lung toxicant. Mice treated with BHT (200-800 mg/kg, po) in combination with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BOS; 1 hr before and 2 hr after BHT, 4 mmol/kg per dose, ip) developed hepatotoxicity characterized by an increase in serum glutamic pyruvic transaminase (GPT) activity and centrilobular necrosis of hepatocytes. The hepatotoxic response was both time- and dose-dependent. BHT (up to 800 mg/kg) alone produced no evidence of liver injury. As judged by the observation of normal serum GPT, drug metabolism inhibitors such as SKF-525A, piperonyl butoxide, and carbon disulfide prevented the hepatotoxic effect of BHT given in combination with BSO. On the other hand, pretreatment with cedar wood oil resulted in increased hepatic injury in mice treated with both BHT and BSO. Pretreatment with phenobarbital also tended to increase hepatic injury as judged by changes in serum GPT. These results suggest that BHT is activated by a cytochrome-P-450-dependent metabolic reaction and that the hepatotoxic effect is caused by inadequate rates of detoxification of the reactive metabolite in mice depleted of hepatic GSH by BSO administration. The hepatotoxic potencies of BHT-related compounds also were examined in BSO-treated animals. For hepatotoxicity, the phenolic ring must have benzylic hydrogen atoms at the 4 position and an ortho-alkyl group(s) that moderately hinders the hydroxyl group. These structural requirements essentially are the same as those for the toxic potency in the lung (T. Mizutani, I. Ishida, K. Yamamoto, and K. Tajima (1982), 62, 273-281) and support the hypothesis that BHT-quinone methide plays a role in producing liver damage in mice with depressed hepatic GSH levels.

Protection by methylprednisolone against butylated hydroxytoluene-induced pulmonary damage and impairment of microsomal monooxygenase activities in the mouse: lack of effect on fibrosis.

The effects of the synthetic corticosteroid methylprednisolone (MP; 30 mg/kg, s.c. given twice daily for 3 days), on the pneumotoxic effects of a single dose of butylated hydroxytoluene (BHT; 400 mg/kg, i.p.) over a 10 day experimental period was investigated in male C57BL/6N mice. BHT alone caused time-dependent alveolar hypercellularity, inflammatory infiltration, alveolar septal thickening and hypercellularity of the bronchiolar epithelium, reaching a maximum by day 5 with some degree of recovery by day 10. The pulmonary monooxygenase activities reflected the degree of alveolar damage and Clara cell abnormality with time; reductions in monooxygenase activities occurred and minimum levels (7-15% of control) were reached by day 5 and again a trend towards recovery by day 10. MP administered 0, 24 and 48 hr after BHT treatment partially protected mice from these effects of BHT in a distinctly time-dependent fashion; the degree of protection decreased as the time between BHT challenge and MP treatment increased. Although MP alone did not morphologically affect Clara and alveolar cells, it increased, decreased or had no effect on the monooxygenase activities. About 25% of the mice that received BHT alone died by day 5 and 50% by day 10. MP completely blocked the lethal effects of BHT by day 5 and reduced the deaths to between 15% and 25% by day 10. Interestingly, MP did not protect against the BHT-induced pulmonary fibrosis, measured as total lung hydroxyproline content, irrespective of the time between BHT challenge and MP treatment. MP alone did not cause any deaths nor increase lung hydroxyproline content.

The effect of cyclosporin A on pulmonary fibrosis induced by butylated hydroxytoluene, bleomycin and beryllium sulfate.

Interstitial pulmonary fibrosis is characterized by an abnormal accumulation of fibroblasts with a resultant increase in lung collagen content. Previous research has implied a possible involvement of the T-lymphocyte in this process. We used cyclosporin A (Cy A), a known immunosuppressant, to deplete T-lymphocyte-dependent responses in animals following treatment with agents known to produce fibrosis; butylated hydroxytoluene (BHT), bleomycin and beryllium (Be). BHT-treated mice and bleomycin-treated rats showed significant reduction in total lung hydroxyproline content with Cy A (P less than 0.05). These results suggest a contribution of the T-lymphocyte in the overall process of fibrosis, but do not indicate its role as the sole causative agent.

Modification of butylated hydroxytoluene-induced pulmonary toxicity in mice by diethyl maleate, buthionine sulfoximine, and cysteine.

Treatment of mice with diethyl maleate (DEM) or buthionine sulfoximine (BSO) significantly enhanced the lung injury caused by butylated hydroxytoluene (BHT). Conversely, cysteine protected mice from the lung toxicity of BHT. BHT administration to mice produced a time-dependent reduction of glutathione (GSH) content in the lung, but not in the liver. These results support the concept that conjugation of 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide), a proposed reactive metabolite of BHT, with GSH is involved in the detoxification of BHT in mice.

Prevention of butylated hydroxytoluene-induced lung damage by diethyldithiocarbamate and carbon disulfide in mice.

Diethyldithiocarbamate (DTC) and carbon disulfide (CS2), at nearly equimolar doses (po), prevented mice from lung injury induced by butylated hydroxytoluene (BHT), as evidenced by suppression of increased lung weight and total DNA content as well as by histopathologic observations. CS2 pretreatment dose dependently decreased the amount of covalently bound [ring-14C]BHT to lung macromolecules in vivo. A slight, but significant, loss of lung GSH observed early after BHT administration was also prevented. The lung microsomal fraction exhibited NADPH-dependent covalent binding of BHT in vitro; this was inhibited completely by carbon monoxide and slightly by SKF-525A. This NADPH-dependent binding was suppressed in lung microsomes isolated from CS2-treated mice. CS2 also reduced various drug metabolizing enzyme activities and the cytochrome P-450 content of the lung microsomal fraction. These results support the metabolic activation hypothesis for BHT-induced lung damage, and the preventive action of CS2 and DTC may be due to an inhibition of this bioactivation step. Possible sites of the metabolic activation of BHT and its inhibition by CS2 are discussed.

Inhibition of butylated hydroxytoluene-induced mouse lung cell division by oxygen: time-effect and dose-effect relationships.

Mice were injected i.p. with 250 or 400 mg/kg of butylated hydroxytoluene (BHT). In vivo incorporation of thymidine into pulmonary DNA was measured on days 1-7 after BHT. 2, 3 and 4 days after BHT, DNA synthesis was inhibited by a 24-h exposure to 100% oxygen, whereas on days 5, 6 and 7 after BHT, oxygen failed to depress synthesis. A similar pattern was observed when incorporation of leucine into protein was measured: 2 and 4 days after BHT, oxygen decreased leucine incorporation, but had no effect 6 days after BHT or in animals not pretreated with BHT. It is concluded that the cells proliferating early after BHT, the type II alveolar cells, are more susceptible to the cytotoxic effects of oxygen than are interstitial and capillary endothelial cells.