Oxidative metabolism of butylated hydroxytoluene by hepatic and pulmonary microsomes from rats and mice

Metabolism of the antioxidant butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-4-methylphenol) has been studied with liver and lung microsomes from rats and mice. The structures of several previously reported metabolites were confirmed, the identities of four new metabolites were determined, pathways of oxidation were investigated, and quantitative data were obtained for several of the products. Two main metabolic processes occur, hydroxylation of alkyl substituents and oxidation of the aromatic pi electron system. The former leads to the 4-hydroxymethyl product (BHT-BzOH) and a primary alcohol resulting from hydroxylation of a t-butyl group (BHT-tBuOH). Additional metabolites were produced by oxidation of BHT-BzOH to the corresponding benzaldehyde and benzoic acid derivatives. Hydroxylation of BHT-tBuOH occurs at the benzylic methyl position, and the resulting diol is oxidized further to the hydroxybenzaldehyde derivative. Oxidation of the pi system leads to the quinol, 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone, the quinone, 2,6-di-t-butyl-4-benzoquinone, and the quinone methide, 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone. Derivatives of the quinol and quinone with a hydroxylated t-butyl group were also formed. Quantitative data demonstrate that BHT-BzOH is the principal metabolite in rat liver and lung microsomes. On the other hand, mice produce large amounts of both BHT-BzOH and BHT-tBuOH in these tissues. The metabolite profile was similar in rat liver and lung. Mouse lung, however, produced more quinone relative to other metabolites than mouse liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA cleavage by metabolites of butylated hydroxytoluene

The effect of butylated hydroxytoluene (BHT) and its metabolites on DNA cleavage in vitro was studied with supercoiled plasmid DNA, pUC18, by agarose gel electrophoresis. Among several BHT metabolites, 2,6-di -t-butyl-p-benzoquinone (BHT-quinone) caused cleavage of supercoiled DNA (form I) at a concentration as low as 1 × 10^–6 M. The relative amount of linear form (form III) was increased with increasing concentration of BHT-quinone. 2,6-Di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHT-peroxyquinol) and 3,5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) also cleaved DNA, but to a lesser extent than BHT-quinone. No DNA cleavage was detected by BHT, 2,6-di-t-butyl-4-hydroxymethyl phenol (BHT-OH), 3,5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH), 2,6-di-t-butyl-4-hydroxy-4-methyl-2,5-cyclohexadienone (BHT-quinol) or 2,6-di-t-butyl-4-methylene-2,5-cyclohexadienone (BHT-quinone methide). The DNA cleavage by BHT-quinone was inhibited by oxygen radical scavengers including Superoxide dismutase (SOD), catalase, polyethylene glycol,t-butyl alcohol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine, while it was enhanced by the addition of FeCl₂. The production of Superoxide radical in a solution of BHT-quinone was confirmed by cytochrome c reduction assay. Superoxide was not produced by BHT or other BHT metabolites except for BHT-quinone. These results suggest that BHT-quinone, one of the principal metabolites of BHT, cleaves DNA strands via its generation of oxygen radicals. Such modification of DNA observed in vitro may be relevant to genotoxicity by BHT after metabolic activation in vivo.     

Studies using structural analogs and inbred strain differences to support a role for quinone methide metabolites of butylated hydroxytoluene (BHT) in mouse lung tumor promotion

Chronic treatment of BALB and GRS mice with BHT (2,6-di-tert-butyl-4-methylphenol) following a single urethane injection increases lung tumor multiplicity, but this does not occur in CXB4 mice. Previous data suggest that promotion requires the conversion of BHT to a tert-butyl-hydroxylated metabolite (BHTOH) in lung and the subsequent oxidation of this species to an electrophilic quinone methide. To obtain additional evidence for the importance of quinone methide formation, structural analogs that form less reactive quinone methides were tested and found to lack promoting activity in BHT-responsive mice. The possibility that promotion-unresponsive strains are unable to form BHTOH was tested by substituting this compound for BHT in the promotion protocol using CXB4 mice. No promotion occurred, and in-vitro work demonstrated that CXB4 mice are, in fact, capable of producing BHTOH and its quinone methide, albeit in smaller quantities. Incubations with BALB lung microsomes and radiolabeled substrates confirmed that more covalent binding to protein occurs with BHTOH than with BHT and, in addition, BHTOH quinone methide is considerably more toxic to mouse lung epithelial cells than BHT quinone methide. These data are consistent with the hypothesis that a two-step oxidation process, i.e. hydroxylation and quinone methide formation, is required for the promotion of mouse lung tumors by BHT.     

Formation of a glutathione conjugate from butylated hydroxytoluene by rat liver microsomes

Butylated hydroxytoluene (BHT) was converted to S-(3,5-di-tert-butyl-4-hydroxybenzyl)-glutathione (BHT-glutathione) by rat liver microsomes in the presence of NADPH, molecular oxygen, and glutathione. NADH was far less effective than NADPH and exhibited little synergistic effect when used together with NADPH. Cytochrome P-450 inhibitors, such as SKF 525-A, alpha-naphthoflavone, metyrapone, and carbon monoxide, significantly inhibited BHT-glutathione formation. Liver microsomes from phenobarbital-treated rats catalyzed the formation of BHT-glutathione at a rate that was nine times the rate of adduct formation by control microsomes. No stimulation of BHT-glutathione formation was observed with the addition of liver cytosol fraction to the microsomal incubation mixtures even at low glutathione concentrations. These results support the view that BHT is converted by the cytochrome P-450 monooxygenases to a chemically reactive metabolite, possibly BHT-quinone methide, which forms BHT-glutathione by nonenzymatic conjugation with glutathione.     

The role of 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide) in the metabolism of butylated hydroxytoluene

Male rats were fed 5.45 mmol/100 g diet butylated hydroxytoluene (BHT) or 2,6-di-tert-butyl-4-hydroxymethylphenol (BHT alcohol) in either a standard or purified diet for 1 wk, after which their livers were analysed for levels of unconjugated BHT metabolites and their blood clotting times were assayed. The BHT quinone methide, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, was only found in appreciable concentrations (6–9 μg/g liver) in the livers of rats given BHT. For rats fed the purified diet, BHT and BHT alcohol caused significant reductions of the prothrombin index to 23 and 70%, respectively, of the control value, though rats fed the standard diet were not similarly affected. Liver concentrations of BHT in rats fed BHT alcohol also varied according to diet, indicating that the metabolic pathway may be affected by diet. Biliary excretion of the quinone methide was observed in rats given 140 mg BHT alcohol ip.     

Enhancement of adenoma formation in mouse lung by butylated hydroxytoluene

Male mice, 6–8 weeks old, were injected ip with a single dose of urethan, an agent known to produce lung adenomata within 14–24 weeks after administration. When urethan was followed by weekly ip administrations of butylated hydroxytoluene (BHT), the number of tumors per lung found 14 to 24 weeks after urethan was increased. Increased numbers of tumors per lung were also found when the interval between urethan injection and the first BHT treatment was extended up to 6 weeks. When the total number of BHT injections was reduced from 13 to 4, tumor development was still enhanced. Repeated treatment with BHT, followed by urethan, had no effect and concomitant administration of urethan and BHT reduced the number of tumors found. BHT did not produce more tumors per lung in mouse strains resistant to adenoma formation. It is concluded that, in mouse lung, BHT has several properties of a promoting agent for urethan-initiated adenoma formation.     

Enhancement of tumor formation in mouse lung by dietary butylated hydroxytoluene

Male A/J mice were injected i.p. with a single dose of urethan and fed 0.75% butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) or ethoxyquin in the diet. All animals were killed 4 months after urethan and the number of lung tumors counted. Exposure to BHT, but not to BHA or ethoxyquin significantly enhanced formation of lung tumors if animals were given the BHT-containing diet once a week for 8 consecutive weeks or were kept on it continuously for 8 weeks. Prefeeding mice with BHT had no effect on tumor formation but prefeeding BHA reduced the number of tumors formed by urethan. It is concluded from this and previous work that in mouse lung BHT enhances tumor formation regardless of route of administration and over a 100-fold dose range.     

The acute toxicity of butylated hydroxytoluene and its metabolites in mice

The acute toxicity of butylated hydroxytoluene (BHT) and four of its metabolites, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien- 1-one(BHT-OOH), 2,6-di-tert-butyl-4-hydroxy-4-methyl-2,5-cyclohexadien- 1-one(BHT-OH), 2,6-di-tert-butyl-p-benzoquinone (DBQ), and 2,6-di-tert- butyl-4-[(methylthio)methyl] phenol (BHT-SCH₃) was studied in young male mice following intraperitoneal administration. The i.p. LD₅₀ values of BHT, BHT-OOH, BHT-OH, DBQ, and BHT-SCH₃ were 3550,190, above 1600, 2270, and 1840 $\text{mg}\text{kg}$, respectively. These results suggest that BHT-OOH probably is the most toxic metabolite of BHT.     

Evidence for a role of tert-butyl hydroxylation in the induction of pneumotoxicity in mice by butylated hydroxytoluene

Previous studies have shown that BHT must be biotransformed, probably to a quinone methide, in order to cause pneumotoxicity in mice. When BHT is incubated with mouse hepatic or pulmonary microsomes, a major metabolite that is formed is the tert-butyl-hydroxylated derivative of BHT (BHT-BuOH). Herein we show that BHT-BuOH has a fourfold greater potency than BHT in increasing the lung wt/body wt ratio, decreases lung cytosolic Ca2+-dependent protease activity at 1/10 the dose required for BHT to do this, and causes pulmonary histopathology at 1/20 the dose of BHT. Lung damage occurs earlier and is repaired faster at lower concentrations of BHT-BuOH than of BHT, but the nature of the damage (type I cell death) and regenerative response (type II cell hyperplasia and differentiation) is apparently identical. Neither BHT-BuOH nor BHT cause damage to liver, kidney, or heart as assessed by light microscopy, so they are both specific pulmonary toxicants. We postulate that BHT-BuOH formation is an essential step in the conversion of BHT to the ultimate pneumotoxin, which might be the corresponding quinone methide.     

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。     

Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. II. Chronic feeding studies

The antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were fed in the diet to male F344 rats in two chronic feeding studies. In one study, feeding BHT for 76 wk at concentrations ranging from 100 to 6000 ppm produced no increase in neoplasms at any site. In a second study, feeding 12,000 ppm BHT for 110 wk had no neoplastic effect at any site, whereas feeding BHA at 12,000 ppm resulted in a small increase in squamous cell papillomas of the non-glandular squamous portion of the stomach.     

Inhibition by butylated hydroxytoluene and its oxidative metabolites of DMBA-induced mammary tumorigenesis and of mammary DMBA-DNA adduct formation in vivo in the female rat.

The phenolic food antioxidant butylated hydroxytoluene (BHT) has been reported to inhibit the initiation stage of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in the female rat. However, the mechanism for this antitumorigenic effect of BHT is unknown. The present studies were conducted to evaluate the relative effect of the parent chemical BHT and two of its major oxidative metabolites, 2,6-di-tert-butyl-4-hydroxymethylphenol (BHT-BzOH) and 2,6-di-tert-butyl-1,4-benzoquinone (BHT-quinone), on DMBA-induced rat mammary tumorigenesis and on the formation of rat mammary DMBA-DNA adducts in vivo. The ip administration of either BHT or BHT-quinone at 200 mg/kg body weight for 2 wk before until 1 wk after DMBA administration inhibited the development of mammary tumours as compared with controls. The extent of tumour inhibition by BHT (39%) was greater than that exhibited by BHT-quinone (25%). The administration of BHT-BzOH at 200 mg/kg body weight did not inhibit mammary tumorigenesis. Thus, the inhibition of DMBA-induced mammary tumorigenesis by BHT does not appear to be mediated by the oxidative BHT metabolites BHT-BzOH or BHT-quinone. In addition, there was a good quantitative correlation between the inhibition of mammary tumorigenesis by BHT and BHT-quinone and their respective abilities to decrease total binding in vivo of DMBA to mammary DNA. The inhibition of specific mammary DMBA-DNA adducts by BHT was not identical to the inhibition of adducts by BHT-quinone. However, the decrease in formation of the major mammary adduct derived from the anti-dihydrodiolepoxide of DMBA bound to deoxy-guanosine most closely correlated to the relative abilities of BHT and BHT-quinone to inhibit mammary tumorigenesis. When mammary adduct formation was examined in response to BHT dose, the administration of BHT at doses of 100 mg/kg body weight and 200 mg/kg body weight resulted in the inhibition of anti-derived but not syn-derived mammary DMBA-DNA adducts. Together, these studies suggest that in addition to the inhibition of total mammary DMBA-DNA adduct formation, the inhibition of mammary DNA adducts formed from the anti-dihydrodiolepoxide of DMBA also may be specifically important in the inhibitory effect of BHT on DMBA-induced mammary tumorigenesis.     

Formation and reactivity of alternative quinone methides from butylated hydroxytoluene: possible explanation for species-specific pneumotoxicity

Previous work has shown that butylated hydroxytoluene [2,6-di-tert-butyl-4-methylphenol (BHT)] undergoes pi-oxidation in liver microsomes to form the quinone methide 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (QM). This electrophilic species binds covalently to glutathione and protein thiols and is believed to initiate pulmonary toxicity in mice. In the present investigation, we identified another quinone methide metabolite of BHT, 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylene-2,5-cyclohexadienone (QM-OH), formed subsequent to the microsomal hydroxylation of BHT at a tert-butyl group. Mouse liver and lung microsomes generate the two quinone methides, and evidence was obtained that both metabolites also are formed in vivo. In contrast, rat microsomes produce QM almost exclusively, with only traces of QM-OH formed in liver and none in lung. Studies of the chemical reactivities of the two quinone methides with GSH demonstrated that QM-OH reacts about 6-fold faster than QM. Infrared spectra, 1H NMR spectra, and electrochemical measurements all support the proposal that the enhanced electrophilicity of QM-OH is due to intramolecular hydrogen bonding of the ring oxygen with the side-chain hydroxyl. The results provide evidence, therefore, that the previous metabolic scheme for bioactivation of BHT to a pulmonary toxin should be amended to include tert-butyl hydroxylation and subsequent pi-oxidation to the activated electrophile QM-OH. This scheme is consistent with published data concerning BHT-induced pulmonary toxicity and provides an explanation for the species specificity of this effect.     

Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used antioxidant food additives. They have been extensively studied for potential toxicities. This review details experimental studies of genotoxicity and carcinogenicity which bear on cancer hazard assessment of exposure to humans. We conclude that BHA and BHT pose no cancer hazard and, to the contrary, may be anticarcinogenic at current levels of food additive use.     

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.     

Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. I. Genetic and cellular effects

The cellular effects of the antioxidants butylated hydroxyanisole and butylated hydroxytoluene were studied in a battery of in vitro tests. No evidence of genotoxicity was obtained for either compound in the hepatocyte primary culture/DNA repair test, the Salmonella/microsome mutagenesis test, the adult rat liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase test, or for butylated hydroxyanisole in the Chinese hamster ovary cell/sister chromatid exchange test. Both compounds inhibited intercellular molecular exchange between cultured liver cells, an effect that has been observed for many agents with neoplasm-promoting activity.     

HPLC测定2,6-二叔丁基对甲酚的含量及有关物质

目的建立测定2,6-二叔丁基对甲酚含量及有关物质的方法。方法采用DiamonsilTMC18ODS柱(250 mm×4.6 mm,5μm);流动相为乙腈-乙醇(65∶35),流速0.6 ml.min-1,紫外检测波长278 nm,柱温30℃。结果在本色谱条件下,2,6-二叔丁基对甲酚与有关物质及溶剂峰分离度符合要求,在300~700μg.ml-1范围内线性良好(r=0.9998)。结论测定方法简便、准确、灵敏度高,方法可靠,可作为质量控制方法。     

Oxidation of raloxifene to quinoids: potential toxic pathways via a diquinone methide and o-quinones

Raloxifene was approved in 1997 by the FDA for the treatment of osteoporosis in postmenopausal women, and it is currently in clinical trials for the chemoprevention of breast cancer. Before widespread use as a chemopreventive agent in healthy women, the potential cytotoxic mechanisms of raloxifene should be investigated. In the current study, raloxifene was incubated with GSH and either rat or human liver microsomes, and the metabolites and GSH conjugates were characterized using liquid chromatography-tandem mass spectrometry. Raloxifene was converted to raloxifene diquinone methide GSH conjugates, raloxifene o-quinone GSH conjugates, and raloxifene catechols. For comparison, three raloxifene catechols were synthesized and characterized. In particular, 7-hydroxyraloxifene was found to oxidize to the 6,7-o-quinone. As compared with raloxifene diquinone methide, which has a half-life of less than 1 s in phosphate buffer, the half-life of raloxifene 6,7-o-quinone was much longer at t(1/2) = 69 +/- 2.5 min. The stability offered by raloxifene 6,7-o-quinone implies that it may be more toxic than raloxifene diquinone methide. Cytotoxicity studies in the human breast cancer cell lines S30 and MDA-MB-231 showed that 7-hydroxyraloxifene was more toxic than raloxifene in both cell lines. These results suggest that raloxifene could be metabolized to electrophilic and redox active quinoids, which have the potential to cause toxicity in vivo.