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.     

环磷酰胺对丁硫氨酸亚砜胺在Walker-256荷瘤大鼠体内的药代动力学的影响

目的　研究环磷酰胺 (CTX)对化疗增敏剂丁硫氨酸亚砜胺 (BSO)在Walker 2 5 6荷瘤大鼠体内的药代动力学的影响。方法　Walker 2 5 6荷瘤大鼠ipCTX 2 0mg·kg-1或生理盐水 4d后 ,ivBSO 2 0 0mg·kg-1。以邻 苯二甲醛柱前衍生反相HPLC为检测手段 ,测定血浆中BSO的浓度 ,以 3P87对实验数据进行拟合 ,计算药代动力学参数。结果　荷瘤大鼠静脉注射BSO 2 0 0mg·kg-1,体内的动力学过程为二室模型 ,T1/ 2α为 (11 1± 2 4 )min ,T1/ 2 β为 (6 5± 14 )min ,CLs为(12 8± 1 3)ml·min-1·kg-1,AUC为 (2 6 2± 2 6 )mg·L-1·h ;BSO在CTX治疗组荷瘤大鼠体内的动力学特征也是二室模型 ,T1/ 2α为 (8 2± 1 8)min ;T1/ 2 β为 (42± 3)min ;CLs为 (13 4± 1 9)ml·min-1·kg-1,AUC为 (2 5 2± 35 )mg·L-1·h。结论　CTX治疗组与对照组相比 ,用药组BSO的消除显著快于未经CTX治疗的大鼠 (P <0 0 5 ) ,其余各参数差异无显著性     

Nephrotoxicity of butylated hydroxytoluene in phenobarbital-pretreated male rats

A single large dose of butylated hydroxytoluene (BHT, 1000 mg/kg) in male Fischer 344 rats produced some renal damage, reduced accumulation of p-aminohippuric acid in renal slices, proteinuria and enzymuria, in addition to hepatic damage. Further, prior administration of phenobarbital (80 mg/kg, i. p., daily for 4 days) in the high-dose BHT-treated male rats produced renal damage accompanied by slight tubular necrosis. The renal damage was confirmed by biochemical and histological changes. These changes were dose dependent, with a maximum at 24 h after BHT administration, but had returned to the normal range by 48 h. Female rats, on the other hand, were less susceptible to BHT-induced renal and hepatic damage than male rats. The results indicate sex differences in BHT-induced renal or hepatic damage.     

Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine

Gender is a factor that influences susceptibility of individuals to drug-induced liver injury in experimental animals and humans. In this study, we investigated the mechanisms underlying resistance of female mice to acetaminophen (APAP)-induced hepatotoxicity. Overnight-fasted male and female CD-1 mice were administered APAP intraperitoneally. A minor increase in serum alanine aminotransferase levels was observed in female mice after APAP administration at a dose that causes severe hepatotoxicity in males. Hepatic glutathione (GSH) depleted rapidly in the both genders prior to development of hepatotoxicity, whereas its recovery was more rapid in female than in male mice. This was consistent with higher induction of hepatic glutamate-cysteine ligase (GCL) in females. Pretreatment of mice with L-buthionine sulfoximine (BSO), an inhibitor of GCL, exaggerated APAP hepatotoxicity only in female mice, resulting in much higher hepatotoxicity in female than in male mice. In addition, hepatic GSH was markedly depleted in BSO-pretreated female mice compared with male mice, which supports severe hepatotoxicity in BSO-pretreated females. APAP treatment highly induced multidrug resistance-associated protein 4 (Mrp4) only in female mice. The resulting high Mrp4 expression could thus contribute to decreased hepatic GSH levels via sinusoidal efflux when GCL is inhibited. In conclusion, resistance to APAP hepatotoxicity in female mice and its reversal by pretreatment with BSO could be attributed to sex differences in disposition of hepatic GSH, which may generally determine susceptibility to drug-induced liver injury.     

Effect of buthionine sulfoximine on orthophenylphenol-induced hepato- and nephrotoxic potential in male rats

A single oral administration of orthophenylphenol (OPP, 1400 mg/kg; about half the LD₅₀) to male Fischer 344 rats produced an elevation of serum transaminase activity 24 h later. Pretreatment with l-buthionine-S,R-sulfoximine (BSO, 900 mg/kg) in the OPP-treated rats potentiated the hepatic and renal damage which was accompanied by necrosis. Six hours after the administration of OPP (700 or 1400 mg/kg), hepatic and renal glutathione (GSH) levels decreased with increasing dosage. Hepatic GSH depletion with OPP was enhanced with BSO pretreatment and the recovery of GSH in both organs was slow in the high-dose OPP group. These results suggest that hepatic and renal damage is associated with a serious and prolonged GSH depletion. When either phenyl-p-benzoquinone (PBQ) or phenylhydroquinone (PHQ), which are intermediates of OPP, was administered orally to rats at 700 or 1400 mg/kg, the mortality with the high dose of PBQ was 75% at 24 h. The serum transaminase activity and UN level increased with the low dose of PBQ, accompanied by necrotic hepatocytes. The toxic effects of PHQ on kidney or liver were less than those on PBQ. These observations suggest that the liver and kidney may be target organs for toxic actions of a large dose of OPP and its intermediate, PBQ.Part of this work was presented at IInd International ISSX Meeting Xenobiotic Metabolism and Disposition, May 16–20, 1988, Kobe, Japan     

Effects of butylated hydroxytoluene (BHT) on biliary excretion of xenobiotics and bile flow in rats

A significant enhancement in the biliary excretion of iv injected sulfobromophthalein (BSP), phenol- 3,6 -dibromphthalein disulfonate (DBSP), procaine amide ethobromide (PAEB) and ouabain was observed in rats maintained on diets containing 0.25% BHT for periods of 10 days. The enhanced biliary excretion of these drugs in BHT treated rats appears to be correlated with the increase in bile flow produced by BHT. The increased bile flow was due to an increase in canalicular bile production rather than a change in net ductular secretion or reabsorption of fluid since bile to plasma concentration ratios of erythritol were unchanged and no permeability change in the biliary tree was observed when mannitol was administered by retrograde intrabiliary injection. The increase in bile flow was not due to an enhanced excretion of bile salts into bile, because both the biliary bile acid concentration and total biliary excretion of bile acids were lower in BHT-treated rats than in control rats. It appears that the increase in bile flow produced by BHT is due to the osmotic choleresis related to the secretion of BHT and its metabolites into bile.     

Effects of buthionine sulfoximine and diethyl maleate on glutathione turnover in the channel catfish.

Despite the growing use of fish in toxicological studies, little is known regarding glutathione (GSH) metabolism and turnover in these aquatic species. Therefore, we examined GSH metabolism in the liver and gills of channel catfish (Ictalurus punctatus), a commonly employed aquatic toxicological model. Treatment of channel catfish with L-buthionine-S,R-sulfoximine (BSO, 400 or 1000 mg/kg, i.p.), an inhibitor of GSH biosynthesis, did not deplete hepatic GSH in channel catfish. In addition, hepatic GSH concentrations did not fluctuate in catfish starved for 3 days, indicating relatively slow turnover of hepatic GSH. However, hepatic GSH concentrations were reduced significantly (P less than 0.05) after 7 days of starvation. Administration of the thiol alkylating agent diethyl maleate (DEM, 0.6 mL/kg, i.p.) resulted in depletion of 85% of hepatic GSH at 6 hr post-DEM, with complete GSH recovery observed at 24 hr post-DEM. Co-administration of BSO and DEM (1000 mg/kg, 0.6 mL/kg, respectively) substantially depleted gill GSH and eliminated detectable liver GSH. Following BSO/DEM, GSH recovery in hepatic mitochondria occurred more rapidly than did liver cytosolic GSH. gamma-Glutamylcysteine synthetase (GCS) activities were comparable in the 10,000 g supernatants of catfish liver and gills (204 +/- 21 and 268 +/- 20 nmol/min/mg protein, respectively) whereas gamma-glutamyltranspeptidase (GGT) activity was not detected in the 600 g post-nuclear fraction of either liver or gills. In conclusion, i.p. administration of DEM was an effective means for achieving short-term hepatic GSH depletion in channel catfish, whereas co-administration of BSO and DEM elicited prolonged and extensive hepatic GSH depletion in this species. Like rodents, channel catfish maintained physiologically distinct hepatic mitochondrial and cytosolic GSH pools, and also regulated hepatic GSH levels by in situ hepatic GSH biosynthesis. However, unlike rodents, there was no evidence for a labile hepatic cytosolic GSH pool in channel catfish. These similarities and differences need to be considered when designing toxicological studies involving the GSH pathway in channel catfish and possibly other fish species.     

Cytotoxicity of butylated hydroxyanisole and butylated hydroxytoluene in isolated rat hepatocytes

The effects of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on isolated rat hepatocytes were investigated. Both antioxidants were observed to be cytotoxic in a concentration-dependent manner at concentrations ranging from 100 to 750 microM. At equimolar concentrations BHT was more cytotoxic than BHA. Their toxicity appeared to be independent of their metabolism to reactive intermediates since inhibitors of cytochrome P-450 (metyrapone, SKF 525-A and piperonyl butoxide) had no effect on the cytotoxicity and N-acetylcysteine was also without protective effect. In addition, deuterated BHT was equitoxic with BHT. Only low temperature incubation (4 degrees), which has previously been shown to inhibit the insertion of these compounds into biomembranes, was effective in inhibiting the cytotoxic effects. Using isolated rat liver mitochondria we observed that both BHA and BHT inhibited respiratory control primarily by stimulating state 4 respiration and thus acting as membrane uncouplers. BHA and BHT also effectively dissipated membrane potential across the mitochondrial membrane and caused the release of calcium and mitochondrial swelling. These mitochondrial effects were reflected by a rapid decrease in ATP levels in intact hepatocytes which preceded cell death. These results suggest that the observed cytotoxicity of BHA and BHT to hepatocytes is related to their effects on biomembranes and mitochondrial bioenergetics.     

Tissue-specific changes in glutathione and cysteine after buthionine sulfoximine treatment of rats and the potential for artifacts in thiol levels resulting from tissue preparation

L-Buthionine-S,R-sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthetase, is commonly used as an experimental tool for the specific depletion of glutathione. Since cysteine is a key precursor for glutathione biosynthesis, we investigated the possibility that BSO might also affect the free cysteine pool in rat liver and kidney tissues in vivo. Male CD(SD)BR rats (150-200 g) were injected ip with various doses of BSO (0.25-4.0 mmol/kg), and glutathione and cysteine were measured in liver and kidney using HPLC with electrochemical detection and/or spectroscopic techniques. No hepatotoxicity or nephrotoxicity was observed at the highest BSO dose (4.0 mmol/kg) used. BSO caused the expected decreases of hepatic and renal glutathione at all doses, although glutathione depletion was more rapid, was achieved at a lower BSO dose, and was more sustained in kidney than in liver. Hepatic cysteine levels nearly doubled 20 min after BSO treatment (1.0 mmol/kg, ip), but were not significantly different from control at later time points. In contrast, renal cysteine was significantly depleted from 20 min to 25 hr postinjection with a time course closely paralleling that of renal glutathione depletion. These changes are discussed in the context of models for inter- and intraorgan transport of glutathione and cysteine. We also provide evidence that an artifact, most likely the gamma-glutamyltranspeptidase (GGT)-initiated breakdown of glutathione, leads to a rapid postmortem increase of cysteine levels in liver and particularly in kidney of rats. Simultaneous decreases in GSH levels can be demonstrated in kidney. This artifact needs to be minimized in toxicological studies of glutathione and cysteine in kidney and other GGT-rich organs, as the measured levels of these thiols may not reflect the true concentrations occurring in vivo.     

Effects of butylated hydroxyanisole on acetaminophen hepatotoxicity and glucuronidation in vivo

The present study examined the effects of butylated hydroxyanisole (BHA) on acetaminophen-induced hepatotoxicity and metabolism in vivo with emphasis on possible changes in the glucuronidation pathway. Female Swiss-Webster mice received BHA in the diet (1% w/w) for 12 days (600 to 800 mg/kg/day). BHA prevented acetaminophen hepatotoxicity (600 mg/kg, ip), based on serum alanine and aspartate aminotransferase activities and histopathological examination. The rate of elimination of acetaminophen from blood was 10-fold higher in BHA-fed mice (clearance, 49 ml/min/kg) than in controls (4.4 ml/min/kg). In general, the urinary metabolite excretion patterns in control and BHA-treated mice were the same. However, the rates of acetaminophen conjugation via the sulfation, glucuronidation, and mercapturic acid pathways were enhanced with the rate of glucuronide formation, the major biotransformation pathway of acetaminophen, increased sevenfold in BHA-treated mice (0.041 min-1) compared to controls (0.006 min-1). BHA increased hepatic UDP-glucuronosyltransferase activity twofold, as well as hepatic UDP-glucuronic acid concentrations. In addition, after acetaminophen administration, UDP-glucuronic acid in BHA-treated mice was depleted to a lesser extent and returned to control values more rapidly than in untreated animals. BHA had a similar but less pronounced effect on hepatic glutathione levels. The findings indicate that the rate of acetaminophen glucuronidation is increased in vivo during BHA feeding to mice. This effect appears to play a role in the enhanced excretion of acetaminophen as well as protection against acetaminophen-induced hepatotoxicity.     

Effect of the glutathione-depleting agents diethylmaleate, phorone and buthionine sulfoximine on biliary copper excretion in rats

The involvement of glutathione (GSH) in the biliary excretion of Cu was investigated in bile-cannulated inbred WAG/Rij and BN rats, pretreated with diethylmaleate (DEM), phorone or buthionine sulfoximine (BSO) and injected with Cu doses of 10 or 30 micrograms/100 g body wt. DEM reduced liver GSH to 27-56% and biliary GSH excretion to 18-38%; phorone reduced GSH in the liver to 55% and increased it in the bile (113%) followed by a slight decrease (79%); BSO reduced liver GSH to 50% and bile GSH to 20%. After injection of Cu to control rats a profile of biliary Cu excretion was found, composed of a slowly (SCuE) and a rapidly (RCuE) disappearing component, the latter only present after the dose of 30 micrograms Cu. DEM had no effect on SCuE after a 10 micrograms dose and a temporary effect on SCuE after a 30 micrograms dose in both WAG/Rij and BN rats. Phorone reduced SCuE after both Cu doses to 50%. Both agents abolished RCuE and reduced endogenous biliary Cu excretion to less than 50%. Release of injected Cu from plasma and uptake by the liver was inhibited by DEM and phorone in both rat strains; in BN rats basal plasma Cu level of DEM-treated rats was increased as well. BSO reduced SCuE after both Cu doses but had no influence on RCuE. Endogenous Cu excretion was reduced by BSO in BN rats but not in WAG/Rij rats. The results show that biliary Cu excretion proceeds by a pattern, the components of which can be affected differently by the various drugs. They also indicate that GSH is not directly involved in biliary Cu excretion but suggest that it may play a role in the metabolism of Cu in the liver.     

Effects of dietary butylated hydroxytoluene on functional and biochemical properties of platelets and plasma preceding the occurrence of haemorrhage in rats

Measurements of platelet-particle concentration, platelet haematocrit and mean platelet volume showed no significant differences between control rats and rats given 1.2% butylated hydroxytoluene (BHT) in the diet for 1 wk, but the platelet distribution width was significantly smaller in the rats fed BHT. By optical measurement, epinephrine-induced platelet aggregation was found to be significantly decreased in both the platelet-rich plasma and washed platelets of rats given BHT. Phosphatidylethanolamine in the platelet lipid was significantly increased by BHT treatment, while in the total lipid of the platelets, linoleic acid and eicosatrienoic acid were increased and arachidonic acid was decreased. The arachidonic/linoleic ratio was decreased by about 50% in the platelets of BHT-treated rats. BHT ingestion also changed the lipid composition, the concentration of lipid hydroperoxides and the zinc content of the plasma. The relationship of the observed effects to haemostatic function is discussed.     

Reduced aggregation capacity of washed platelets and dysfibrinogenaemia in rats given butylated hydroxytoluene

Calcium-dependent aggregation of washed platelets and the fibrin monomer polymerization (FMP) ratio were determined in rats fed 1.20% butylated hydroxytoluene (BHT) for 7 days. Thrombin-induced aggregation was significantly reduced in rats given BHT, but calcium ionophore A23187-induced aggregation was not. The FMP ratio was significantly increased by BHT administration. These results provide additional evidence of defective platelet function and coagulation-factor abnormality (dysfibrinogenaemia) in rats fed BHT.     

Effect of N-acetyl-cysteine, D-penicillamine and buthionine sulfoximine on glutathione levels and CNS oxygen toxicity in rats

The effect of glutathione (GSH) synthesis modulators - L-buthionine sulfoximine (BSO), N-acetyl cysteine (NAC) and D-penicillamine (DPA) - on the susceptibility of rat CNS to O2 toxicity was investigated. The animals were given 5% sucrose or 40 mM solutions of BSO, NAC or DPA in 5% sucrose as drinking water for one week and sacrificed prior to or after exposure to 4.5 ATA O2. The GSH content in brain, liver, lung and blood, and the activity of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glucose-6-phosphate dehydrogenase (G-6-PD) and superoxide dismutase (SOD) in brain and lungs were measured. The brain GSH content and the enzyme activities were not changed by any of the drugs. BSO decreased the GSH content in all the other tissues; NAC and DPA treatments increased the GSH content in lungs, blood and/or liver. The CNS toxicity threshold as measured by the time of appearance of first electrical discharge (FED) on ECoG recording was not changed by NAC or DPA, but BSO brought about a significant delay in FED time. It is suggested that increased extracerebral GSH levels do not protect against CNS oxygen toxicity, and that BSO provides some protection, probably via a glutathione-independent mechanism.     

超高效液相色谱法测定辛伐他汀胶囊中叔丁基-4-羟基茴香醚与2,6-二叔丁基对甲酚的含量

目的建立超高效液相色谱法同时测定辛伐他汀胶囊中的抗氧剂叔丁基-4-羟基茴香醚(BHA)与2,6-二叔丁基对甲酚(BHT)。方法色谱柱为ACQUITY UPLCTM BEH C18(50mm×2.1mm,1.7μm)。以乙腈(A)-0.005mol.L-1醋酸铵(B)为流动相,梯度洗脱程序为:0min,60∶40;2min,60∶40;5min,90∶10;8min,90∶10;9min,60∶40;10min,60∶40。检测波长为280nm,流速为0.25mL.min-1,柱温为40℃。结果在该色谱条件下,BHA和BHT与维生素C峰均能良好分离。BHA的检出限为0.5ng;质量浓度在0.203 5～50.88μg.mL-1范围内与峰面积呈良好的线性关系,相关系数r=0.999 9;回收率为98.3%,RSD为1.0%。BHT的检出限为0.5ng;质量浓度在0.211 4～52.84μg.mL-1范围内与峰面积呈良好的线性关系,相关系数r=0.999 9;回收率为97.2%,RSD为0.5%。结论该方法快速、专属、灵敏度高,并且节能环保。     

Carcinogenicity study on butylated hydroxytoluene (BHT) in Wistar rats exposed in utero

Groups of 60, 40, 40 and 60 F0 Wistar rats of each sex were fed a semi-synthetic diet containing butylated hydroxytoluene (BHT) in concentrations to provide intakes of 0, 25, 100 or 500 mg/kg body weight/day, respectively. The F0 rats were mated and groups of 100, 80, 80 or 100 F1 rats of each sex were formed from 40, 29, 30 and 44 litters, respectively. After weaning, the highest dose (500 mg BHT/kg/day) was lowered to 250 mg/kg/day for the F1 rats. The numbers of litters of ten or more pups at birth decreased with increasing BHT dose. At weaning, treated F1 rats had lower body weights than the controls, the extent of the reduction being dose related; the effect, which persisted throughout the study, was most pronounced in the males. The survival of BHT-treated F1 rats of both sexes was significantly better than that of the controls. No significant changes attributable to BHT treatment were found in the haematological parameters. F1 females on the highest dose showed an increase in serum cholesterol and phospholipids, and serum triglycerides were reduced in this group in both sexes. Dose-related increases in the numbers of hepatocellular adenomas and carcinomas were statistically significant (at P less than 0.05 or lower) in male F1 rats when all groups together were tested for heterogeneity or analysis for trend. The increase in hepatocellular adenomas and carcinomas in treated female F1 rats was only statistically significant for adenomas (at P less than 0.05) in the analysis for trend. All hepatocellular tumours were detected when the F1 rats were more than 2 yr old. Tumours were found in many other organs of some of the treated rats, but their incidence was not significantly different from that in controls. The role of BHT in the development of hepatocellular tumours requires further elucidation.     

Minimal ovarian upregulation of glutamate cysteine ligase expression in response to suppression of glutathione by buthionine sulfoximine

The antioxidant tripeptide glutathione (GSH) protects ovarian follicles against oxidative damage that may lead to apoptotic death. The rate-limiting step in synthesis of GSH is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic subunit (GCLC), and a modifier subunit (GCLM). We hypothesized that GSH depletion in vivo or in vitro with buthionine sulfoximine (BSO), a specific inhibitor of GCL activity, would increase ovarian and granulosa cell GCL subunit expression. Ovarian glutathione levels are lowest on proestrous morning and increase to their highest levels on estrus and metestrus. Therefore, we treated rats on proestrous morning or on proestrous morning and again 12h later to prevent the normal increase in ovarian glutathione between proestrus and estrus. Ovarian Gclc and Gclm mRNA levels and GCLC protein levels increased transiently by 1.4-1.5-fold at 8 h, but not at 12 or 24 h, after a single dose of BSO administered to adult rats on the morning of proestrus. GCLC protein levels were also modestly increased 1.4-fold at 12 h after a second dose of BSO. GCLM protein levels increased 1.4-fold at 24 h after a single dose of BSO, but not at other time points. BSO treatment did not significantly alter ovarian GCL enzymatic activity or the intraovarian localization of either GCL subunit mRNA. Treatment of a human granulosa cell line or primary rat granulosa cells with BSO suppressed intracellular GSH; however, there was no compensatory upregulation of GCL subunit protein or mRNA levels. These results demonstrate that ovarian follicles and granulosa cells are minimally able to respond to acute GSH depletion by upregulating expression of GCL.     

高效液相色谱法测定丁磺氨酸及其胶囊的含量

目的建立丁磺氨酸(BSO)及其胶囊的含量测定方法。方法以2,4-二硝基氟苯为柱前衍生化试剂,反相高效液相色谱法,十八烷基硅烷键合硅胶为填充剂,乙腈-0.05mol·L-1醋酸钠(1585,pH6.3)为流动相,检测波长为360nm。结果测定胶囊的平均回收率为99.7％(RSD=0.41％,n=5)。结论本实验建立的HPLC可用于测定BSO及其胶囊的含量