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 effect of indomethacin, prednisolone and Cis-4-hydroxyproline on pulmonary fibrosis produced by butylated hydroxytoluene and oxygen

The purpose of this study was to examine whether development of pulmonary fibrosis in mice could be influenced by indomethacin, prednisolone or a proline analog. Pulmonary fibrosis was produced in mice treated with butylated hydroxytoluene (BHT) 400 mg/kg and immediately exposed to 80% oxygen for 3 days. This treatment regimen resulted in 47% mortality. Surviving mice exhibited significant accumulations of pulmonary collagen as evidenced by increases in total lung hydroxyproline levels. The administration of indomethacin (4 mg/kg/day) on days 1–6 after BHT decreased mortality to 14% and diminished the accumulation of collagen in lung tissue. Indomethacin also enhanced survival when administered on days 1–3 after BHT/O₂ but had no effect on lung collagen levels. Treatment with indomethacin on days 4–6 after BHT had no beneficial effect. The administration of prednisolone (60 mg/kg/day) on days 1–3, 1–6 or 4–6 after BHT decreased mortality but had no effect on accumulation of lung collagen. Cis-4-hydroxyproline (400 mg/kg/day) also had no effect on pulmonary fibrosis but did enhance survival when given on days 1–3 after BHT. Administering prednisolone (60 mg/kg/day) on days 1–6 after BHT to mice left in room air produced significantly more pulmonary fibrosis than in BHT-treated mice given saline. These data support the use of the BHT/O₂ model of pulmonary fibrosis for screening potential antifibrotic agents. The possibility that corticosteroid treatment may enhance pulmonary fibrosis in a damaged lung is also demonstrated.     

Protein-deficient diets II. Toxicity of butylated hydroxytoluene

The acute toxicity of butylated hydroxytoluene (BHT) was determined in rats in relation to the level of protein in their diet. In a short-term study the effect of BHT on the activity of alanine and aspartate aminotransferases, fructosediphosphate aldolase and glucosephosphate isomerase in rat serum and liver homogenates was analysed. Rats were fed diets containing 24%, 8% and 4% of protein and 0.5% and 0.05% of BHT for 1, 4 and 12 weeks. The body weight gain, food consumption, liver weight, total level of serum protein, protein fractions and soluble liver protein have also been investigated.     

Effect of butylated hydroxytoluene on the lipid composition of rat liver

Hepatic lipids were studied in Sprague-Dawley male rats given butylated hydroxytoluene (BHT) at a level of 1.20% for 1 week. BHT significantly increased cholesterol esters and phospholipids but decreased triglycerides, non-esterified fatty acids and diglycerides. BHT also increased phosphatidylethylanolamine or decreased phosphatidylinositol and lysophosphatidylcholine. Fatty acid composition of each lipid class was also changed by BHT-feeding. The decrease in $\text{16:}\text{1}\text{16}\text{:0, 18:}\text{1}\text{18}\text{:0}\text{and}\text{20:}\text{4}\text{18}\text{:2}$ ratios of total lipids, non-esterified fatty acids or phospholipids of BHT-given rats suggests that BHT decreases the activity of fatty acid desaturase in the liver.     

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.     

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.     

Long-term studies on chemically induced liver enlargement in the rat I. Sustained induction of microsomal enzymes with absence of liver damage on feeding phenobarbitone or butylated hydroxytoluene

As part of a study on the relationships between liver enlargement, induction of drug-metabolising enzymes and certain forms of liver damage, sequential biochemical and morphological observations were made on the livers of female rats fed diets containing 0.4% butylated hydroxytoluene (BHT) or 0.25% phenobarbitone for periods of up to 80 weeks. Both compounds increased the relative liver weight and produced marked enhancement of the activities of ethylmorphine N-demthylase, aniline 4-hydroxylase, biphenyl 4-hydroxylase and NADPH-cytochrome c reductase and the contents of cytochromes P-450 and b₅ and microsomal protein. These changes were evident after 1 week of treatment and persisted until treatment was stopped after 80 weeks. The only morphological changes observed throughout the period of treatment were centrilobular cell enlargment and hypertrophy of the smooth endoplasmic reticulum (SER). Histochemical examination revealed a centrilobular depression of glucose-6-phosphatase but there was no disturbance of the normal pericanalicular distribution of lysosomes. All changes observed with either compound were reversible on cessation of treatment at week 80.These results support the concept that liver enlargement accompanied by induction of drug-metabolising enzymes represents an adaptive response and they provide a basis for the interpretation of pathological changes developing in the enlarged liver unaccompanied by drug-metabolising enzyme induction.     

Author index volume 15 (1980)

Butylated hydroxytoluene (BHT) 500 mg/kg body wt/day was given in the diet to F₀-rats from 6 weeks of age to weaning of the F₁-generation (growth period to age 19 week, gestation period and lactation period) and subsequent to F₁-animals until 21 days of age. Body weight of F₀-animals was recorded, and the influence of the F₁-generation was investigated applying crossfostering. Mortality, litter size, weight gain and some developmental events as well as test for auditory and visual function and locomotor coordination in the F₁-generation were recorded.The applied dose of BHT exerted a significant adverse effect on body weight in both F₀ and F₁-animals and on several developmental paramenters in F₁-animals. The effects arose during the lactation period.     

Effect of cobaltous chloride on butylated hydroxytoluene-induced hepatic necrosis in rats

Although a single dose of butylated hydroxytoluene (BHT); 1000 mg/kg to rats induced a hepatic injury accompanying centrilobular necrosis, the pretreatment with cobaltous chloride, an inhibitor of cytochrome P-450 synthesis, could inhibit the damage. The marked elevations of serum transaminase activities and bile acid content induced by BHT were diminished to nearly the control level by pretreatment with cobaltous chloride. The protective effect of cobaltous chloride on BHT-induced hepatotoxicity is discussed.     

Topical butylated hydroxytoluene treatment of genital herpes simplex virus infections of guinea pigs

The effect of topical treatment with butylated hydroxytoluene (BHT) was evaluated in primary and recurrent genital herpes simplex virus type 2 (HSV-2) infection of guinea pigs. In the first experiment, treatment with placebo, 5%, 10%, or 15% BHT was initiated 48 h after viral inoculation and continued 4 times daily for 15 days. During primary infection no differences in maximum lesion severity or titers of virus in lesions were observed, however, lesion duration was reduced in BHT-treated animals resulting in a significantly smaller lesion score-day area under the curve. In a second experiment using U.S.P. mineral oil as an additional placebo, BHT placebo and 15% BHT in a double blind trial, similar results were obtained. Treatment of the recurrent infection in either experiment failed to alter the number of recurrent episodes or days with lesions.     

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。     

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。     

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.     

Enhancing effect of butylated hydroxytoluene on the development of liver altered foci and neoplasms induced by N-2-fluorenylacetamide in rats

The enhancement of hepatocarcinogenesis by butylated hydroxytoluene (BHT) in comparison with that by phenobarbital (PB) was studied by quantifying their effects on N-2-fluorenylacetamide (FAA)-induced preneoplastic and neoplastic rat-liver lesions. Hepatocellular altered foci identified by iron exclusion and gamma-glutamyltranspeptidase (GGT) activity were induced by feeding 0.02% FAA for 8 wk. Subsequently, BHT was fed at concentrations of 300, 1000, 3000 or 6000 ppm for up to 22 wk after cessation of FAA exposure; PB was fed at concentrations of 316 or 500 ppm for comparison. The lower doses of BHT (300, 1000 and 3000 ppm) did not exert a significant effect on either foci development or the final yield of neoplasms. At 6000 ppm, BHT increased the number of foci, the area occupied by GGT-positive preneoplastic and neoplastic lesions and the neoplasm incidence, as did 316 and 500 ppm PB. Comparison of the effects of BHT and PB at equimolar concentrations revealed that BHT was a much weaker enhancer of liver carcinogenesis. Apparently, the effective dose range of BHT as an enhancer is rather restricted. On the basis of available evidence that BHT is nongenotoxic and exerts epigenetic effects, we conclude that BHT is a weak promoter of liver carcinogenesis.     

Effect of butylated hydroxytoluene pretreatment on the excretion, tissue distribution and DNA binding of [14C]aflatoxin B1 in the rat

The effect of butylated hydroxytoluene (BHT) pretreatment (0.5% in the diet for 10 days) on the excretion, tissue distribution and DNA binding of orally administered [14C]aflatoxin B1 (AFB1) was determined in male Fischer F344 rats. The amount of radioactivity excreted in the urine and faeces by 24 hr was higher in BHT-treated rats than in controls. Treatment with BHT enhanced the excretion of water-soluble metabolites in the urine and in the large intestines plus faeces at the earlier sampling times. The amount of radioactivity bound to hepatic nuclear DNA was six times less in the BHT-pretreated rats than in controls 6 hr after administration of the isotope. The half-lives of [14C]DNA in the rat liver were 30 and 46 hr for control and BHT-pretreated rats, respectively. These results indicate that BHT pretreatment may protect the animal from the carcinogenic effects of AFB1 by enhancing the detoxification and excretion of the mycotoxin.     

The effects of butylated hydroxytoluene on the in vitro metabolism, DNA-binding and mutagenicity of aflatoxin B1 in the rat

Butylated hydroxytoluene pretreatment in the rat enhanced the total in vitro metabolism of aflatoxin B1 by the hepatic postmitochondrial fraction (S-9) and increased the formation of aflatoxin M1, aflatoxin Q1 and a metabolite tentatively identified as the aflatoxin-glutathione conjugate, the latter being the major metabolite produced. Addition of diethyl maleate, a glutathione depletor, to the incubation mix, reduced formation of the conjugate. No significant difference between treated and control animals was observed in the S-9-mediated binding of aflatoxin B1 to calf thymus DNA. However, the mutagenicity of aflatoxin B1 in Salmonella typhimurium TA98 was significantly lower in the presence of S-9 from BHT-treated rats than with S-9 from controls.     

The biliary metabolites of butylated hydroxytoluene in the rat

The biliary metabolites from intravenous and intraperitoneal doses of small amounts of [¹⁴C]butylated hydroxytoluene have been separated and estimated. The metabolites recognized were the alcohol (II), aldehyde (III) and acid (IV) together with small amounts of the diphenyl ethane (VI). Three other metabolites appear to be present in relatively large amounts. The pattern of metabolites in bile and urine has been compared. It is suggested that the relatively low excretion of the acid (IV) in the urine compared to bile is due to a selective reabsorption of this compound after biliary excretion.     

Mutachromosomal effects of tert-butylhydroquinone in bone-marrow cells of mice

When given to mice either in a single ip injection of 200 mg/kg body weight or in 30 daily oral doses of 2 mg/kg, tert-butylhydroquinone had severe clastogenic effects on the bone-marrow cells. However mitostatic activity was observed only with the acute treatment.     

Modification of lung tumor development in A/J mice

Strain A mice were injected with urethan, 3-methylcholanthrene or dimethylnitrosamine and given repeated injections of butylated hydroxytoluene (BHT). This treatment significantly increased multiplicity of lung tumors induced by all 3 carcinogens. Two other antioxidants, butylated hydroxyanisole (BHA) or α-tocopherol (vitamin E) did not enhance tumor formation, nor did methylcyclopentadienyl manganese tricarbonyl (MMT), an agen capable of producing cell proliferation in lung. Lungs were more susceptible to the carcinogenic action of urethan 2 weeks following BHT-induced injury, but not during the phase of acute cell proliferation in lung. It is concluded that the effects of BHT on lung tumor development in mice are not related to its properties as an antioxidant or to its capability to produce extensive cell proliferation in lung.     

Antioxidant effect of butylated hydroxytoluene on ferric nitrilotriacetate induced renal hyper proliferation and injury in rats

This study was designed to investigate the effect of butylated hydroxy toluene (BHT), a phenolic antioxidant used in foods, cosmetics and pharmaceutical products, on Fe-NTA-induced nephrotoxicity in rats. Fe-NTA (alone) treatment enhances ornithine decarboxylase activity to 5.3-fold, and [³H] thymidine incorporation in DNA to 3.5-fold compared with the corresponding saline treated control. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 2.12–2.15-fold respectively at a higher dose of 2 mg BHT/day/animal, compared with the Fe-NTA treated group. Fe-NTA treatment also enhanced the renal microsomal lipid peroxidation to 2.0-fold and decreased the activities of glutathione and antioxidant enzymes to a range of 2.2–2.5-fold in kidney. These changes were reversed significantly in animals receiving a pretreatment of BHT. Present data suggests that BHT can prevent the toxic effects of Fe-NTA and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and nephrotoxicity in rats.