Enhancement of butylated hydroxytoluene-induced mouse lung damage by butylated hydroxyanisole

The phenolic antioxidant butylated hydroxytoluene (BHT) is known to produce a dose-dependent increase in mouse lung weight which is characterized by the necrosis of pulmonary type I and endothelial cells. We studied the ability of butylated hydroxyanisole (BHA) to modify BHT-induced changes in lung weight in male CD-1 mice. BHA alone had no effect on lung weight up to a dose of 500 mg/kg (sc). However, when injected 30 minutes prior to sub-threshold doses of BHT (0-250 mg/kg, ip), BHA significantly enhanced lung weight in a dose-dependent manner. The ability of BHA to enhance BHT-induced changes in lung weight was dependent on both the time and the route of administration of BHA relative to BHT. Deuteration of BHT abolished the in vivo toxicity from the combination of BHA and BHT. These results suggest that the toxicity resulting from the combination of BHA and BHT is due to the formation of BHT-quinone methide and that the role of BHA might be either to deplete some protective mechanism in the target pulmonary cells or to enhance the biotransformation of BHT into BHT-quinone methide.     

Comparative metabolism of BHA, BHT and other phenolic antioxidants and its toxicological relevance

Following oral administration, butylated hydroxyanisole (BHA) is absorbed and rapidly excreted by the rat, rabbit and man, with little evidence of long-term tissue storage. The major metabolic pathways for BHA are conjugation (phase 2) reactions, oxidative metabolism (O-demethylation) being relatively unimportant. In the dog, the extent of absorption and urinary excretion is less, and oxidative metabolism is more important than in other species. In contrast, butylated hydroxytoluene (BHT) is cleared less rapidly from most species, enterohepatic circulation being partly responsible for the delay. Tissue accumulation is also greater for BHT than for BHA. Oxidative metabolism (phase 1 reactions) mediated by the microsomal monooxygenase system is the major route for BHT degradation; oxidation of the ring methyl group predominates in the rat, rabbit and monkey, and oxidation of the tert-butyl groups in man. Gallates and 2-tert-butylhydroquinone are mainly metabolized by non-oxidative pathways (methylation or conjugation with sulphate and glucuronic acid). The different biological properties of these compounds may be related to the differences in their absorption and metabolic disposition. Thus, whereas BHT, which is metabolized by oxidation reactions, is an inducer of the microsomal mono-oxygenase system, the other phenolic antioxidants, including BHA, are only weak inducers.     

Studies on the mechanism of enhancement of butylated hydroxytoluene-induced mouse lung toxicity by butylated hydroxyanisole

The studies described in this report were designed to probe possible mechanisms whereby butylated hydroxyanisole (BHA) is able to enhance butylated hydroxytoluene (BHT)-induced mouse lung toxicity. In experiments with mouse lung slices, BHA enhanced the covalent binding of BHT to protein, indicating that the interaction between BHA and BHT takes place in the lung. Subcutaneous administration of either BHA (250 mg/kg) or diethyl maleate (DEM, 1 ml/kg) to male CD-1 mice produced a similar enhancement of BHT-induced lung toxicity. In contrast to DEM, the administration of BHA (250 or 1500 mg/kg) did not decrease mouse lung glutathione levels, suggesting that the effect of BHA is not due to the depletion of glutathione levels. We previously observed that in the presence of model peroxidases a unique interaction occurs between BHA and BHT, resulting in the increased metabolic activation of BHT. Upon the addition of hydrogen peroxide or various hydroperoxides to mouse lung microsomes, BHA significantly increased the covalent binding of BHT to protein. BHA also stimulated the rate of formation of hydrogen peroxide by 4.7-fold in mouse lung microsomes. Likewise, hydrogen peroxide resulting from the NADPH cytochrome P-450 (c) reductase-catalyzed redox cycling of tert-butylhydroquinone, a microsomal metabolite of BHA, supported the peroxidase-dependent BHA-enhanced formation of BHT-quinone methide. These results suggest that BHA could facilitate the activation of BHT in the lung as a result of both the increased formation of hydrogen peroxide and the subsequent peroxidase-dependent formation of BHT-quinone methide from the direct interaction of BHA with BHT.     

Disposition of single oral doses of butylated hydroxytoluene in man and rat

The kinetics and metabolism of butylated hydroxytoluene (BHT) in man and rats have been compared. Single oral doses of 200, 63 or 20 mg BHT/kg body weight were administered to rats and a single oral dose of 0.5 mg/kg body weight was ingested by human volunteers (non-smoking males). In rats, kinetic parameters (area under the plasma concentration-time curve, plasma BHT peak levels) showed a dose-dependent increase. Plasma BHT levels after oral administration were about four times higher than those that have been reported for another synthetic food antioxidant, butylated hydroxyanisole (BHA; Verhagen et al., Fd Chem. Toxic. 27, 151–158). This may be a reflection of a smaller volume of distribution for BHT, since there were no differences in plasma elimination half-life or plasma clearance between BHT and BHA. In man, the mean plasma concentration-time profile after oral BHT intake was well below the BHT profiles observed for rats and closely followed plasma BHA kinetics in man. In rats, the simultaneous administration of BHT (200 mg/kg body weight) and BHA (200 mg/kg) significantly decreased the absorption of BHT from the gastro-intestinal tract in the first few hours after treatment; the plasma kinetics of BHA were not influenced by the simultaneous administration of BHT. In human female volunteers no alterations in plasma BHT or BHA profiles were seen after the simultaneous ingestion of BHT (0.25 mg/kg body weight) and BHA (0.25 mg/kg). Rats excrete about 10% of an oral dose of 200 mg BHT/kg as unchanged BHT in the faeces, whereas in man no BHT could be detected in the faeces. Urinary excretion of (un)conjugated 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-COOH) accounts for only a small percentage of the administered dose in both rats and humans. It is concluded that the plasma BHT concentrations reached after the administration of a single medium to high dose of BHT to rats or a single low dose to man are very different.     

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.     

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.     

Inhibition of mutagenesis in Chinese hamster v-79 cells by antioxidants

The mutagenicity of benzo(a)pyrene (BP) on Chinese hamster V-79 cells cocultivated with X-irradiated hamster embryo cells was inhibited by phenolic antioxidants, such as tert-butyl-4-hydroxyanisole (BHA) and butyl-hydroxytoluene (BHT), but not by disulfiram and its related compounds such as tetraethylthiuram disulfide, diethyldithiocarbamic acid and dimethyl-dithiocarbamic acid. The mutagenicity of BP on V-79 cells was reduced 44% by BHA and 25% by BHT. BHA inhibited BP-induced mutagenesis, but not N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF)-induced mutagenesis. BHA is suggested to inhibit the mutagenic action of BP by altering its metabolism.     

Manipulation of mouse organ glutathione contents. II: Time and dose-dependent induction of the glutathione conjugation system by phenolic antioxidants

After 14 days of oral butylated hydroxyanisole (BHA) administration (1000 mg/kg/day) the tissue glutathione levels of male NMRI mice were increased by 74-141% in liver, lung, duodenum and intestine and after similar butylated hydroxytoluene (BHT) treatment by 18-85% in the liver, lung, spleen and the gastrointestinal tract. Doses of 100 mg/kg/day significantly elevated the glutathione content in the lung (BHA, BHT), duodenum (BHA) and intestine (BHA), while 10 mg/kg/day affected only lung glutathione content (BHA). BHA treatment (1000 mg/kg/day) induced GST activities significantly (138-1335%) in all organs investigated except the spleen, i.e. liver, lung, kidney and the entire gastrointestinal tract, while a similar dose of BHT increased GST activities in the liver, duodenum, intestine and colon by 26-339%. Daily doses of 100 mg/kg/day significantly induced GST activities only in the liver (BHA, BHT), lung (BHA) and kidney (BHA). Lower doses of BHA or BHT did not significantly affect GST activities in the organs investigated (except 10 mg BHA/kg/day in the lung). Comparison of the time course of induction of the glutathione conjugation system in various organs after different doses of antioxidants indicated no change between 5 and 14 days of treatment with all doses used (1-1000 mg/kg). Only the lung glutathione level showed a tendency to increase with low dose BHA by extending the time of treatment. The time course of the liver glutathione content between single doses of 100 mg/kg BHA or BHT revealed an initial decline followed by an increase above control values 2 days (BHA) or 5 days (BHT) after the first application. The glutathione levels of the lung and the duodenum increased without a preceding decline. Only the second dose of BHT caused a temporary decrease to control values of the elevated glutathione level in the duodenum. All animals (at any dose of BHA or BHT) showed control values of serum transaminase activities. These results suggest: The induction threshold of the glutathione conjugation system in various mouse organs is greater than or equal to 100 mg/kg for BHA and BHT. Chronic administration of these compounds did not change these results (except the lung glutathione level after low dose BHA). Elevated hepatic glutathione levels might be the result of an activated synthesis caused by a preceding loss of glutathione. Chronic BHA or BHT treatment did not cause hepatotoxic effects, as evaluated by serum transaminases, in male mice.     

Effects of butylated hydroxyanisole and butylated hydroxytoluene on DNA adduct formation and arylamines N-acetyltransferase activity in PC-3 cells (human prostate tumor) in vitro.

The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the N-acetyltransferase (NAT) activity and DNA adduct formation in PC-3 cells (human prostate tumor) was studied. PC-3 cells were placed into tissue culture flasks and grown in an incubator as cytosols and intact cells. The BHA or BHT were added to the cytosols and intact cells. The NAT activity in cytosol and intact PC-3 cells were measured by HPLC assaying exhibited for the amounts of N-acetyl-2-aminofluorene and N-acetyl-p-aminobenzoic acid, 2-aminofluorene and p-aminobenzoic acid. The NAT activity in PC-3 cells and cytosols were inhibited by BHA or BHT in a dose-dependent manner; that is, the higher the concentrations of BHA or BHT the higher inhibition of NAT activity. The NAT values of K(m) and V(max) from PC-3 cells were also decreased by BHA or BHT in both cytosols and intact cells. The data also demonstrated concomitant exposure to BHA or BHT decreased AF-DNA adduct formation which was seen in the PC-3 cells. In addition, the formation of DNA adduct was decreased after BHA or BHT exposure. These findings suggested the usefulness of using human cultured PC-3 cells for assessing arylamine-induced DNA adduct formation. Furthermore, the findings illustrate how effectively BHA or BHT reduce the adduct formation.     

Choleresis and increased biliary efflux of glutathione induced by phenolic antioxidants in rats

The mechanism by which high doses of the synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) raise hepatic glutathione levels above physiological values was investigated in rats. A single dose of an antioxidant (200 mg/kg; p.o.) reduced the hepatic glutathione content by 17% (BHA) or 36% (BHT) after 5 h, but in contrast levels of 55% (BHA) or 34% (BHT) above controls (7.1 +/- 0.5 mumol GSH-equivalents/g liver wt) were measured 48 h after dosing. Both antioxidants increased basal bile flow (1.37 +/- 0.11 microliter/min per g liver wt) and biliary efflux of total glutathione, i.e. GSH and GSSG, (4.18 +/- 0.97 nmol GSH-eq./min per g) severalfold (up to 250%) over controls 24 h after in vivo antioxidant treatment. The sinusoidal efflux of reduced glutathione (14.9 +/- 2.2 nmol GSH-eq./min per g) was significantly reduced (BHA: 23%; BHT: 41%). The increased glutathione excretion into bile is likely to be independent of the induction of the choleresis. The secretion of bile salts was unaffected by BHA treatment and only temporarily reduced by BHT. Conclusion: phenolic antioxidants increase the hepatic turnover of glutathione by stimulating the biliary efflux of GSH. The resulting shift from a predominantly sinusoidal efflux of GSH in controls (hepato-renal circulation) to a predominantly biliary efflux of GSH in antioxidant-treated animals (entero-hepatic circulation) may lead to increased concentrations of cysteine, glycine and glutamic acid in the portal vein and consequently may stimulate the biosynthesis of GSH by enhanced substrate availability in the liver.     

In vivo protective role of antioxidants against genotoxicity of metronidazole and azanidazole

The mutagenicity of metronidazole and azanidazole has been extensively reported. Previous experiments demonstrated, by means of the intrasanguineous host-mediated assay, that they significantly induced mutagenicity in liver, kidney and lung of mice. The treatment of mice with butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) by two different routes of administration (i.p. injection and oral intubation) significantly reduced liver- and kidney-mediated mutagenicity of azanidazole and metronidazole. No significant differences were observed between the routes of treatment in terms of protective effect on genotoxicity of azanidazole in the considered organs, whereas i.p. administration was the most suppressive on the mutagenicity of metronidazole. Even if BHT was the most effective agent in preventing mutation induction in mice, a detectable toxicity, in terms of increased mutagenicity, was evaluated in the liver. Evidence of lung abnormalities was also seen. The results suggest that the possible adverse effects on biological systems limit the prophylactic use of BHA and BHT in preventing the action of chemical carcinogens in man.     

Manipulation of mouse organ glutathione contents I: Enhancement by oral administration of butylated hydroxyanisole and butylated hydroxytoluene

Administration of either butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) (1000 mg/kg/day for 5 days) to male mice increased the content of reduced glutathione by 50-100% in liver, lung, duodenum and intestine. In colon, glandular stomach, spleen and kidney no effect on glutathione level was observed. BHA and BHT led also to 100-1000% induction of glutathione transferases in liver, lung (only BHA), kidney and digestive tract (except the colon); the relative increase in transferase activity was greater with 1-chloro-2,4-dinitrobenzene (DCNB) as a substrate than with CDNB in all organs investigated. The effects of BHA, administered in olive oil by gavage, on different parts of the gastrointestinal tract revealed maximum increase of the glutathione content and transferase activities in the duodenum, smaller increase of these parameters in the upper intestine and no significant effects in the lower intestine and the colon. Starving mice for 1 day decreased the glutathione content of the liver by 50% to 21.3 +/- 4.5 nmol/mg protein in controls and to 39.4 +/- 3.3 in BHA-treated animals. Intravenous injection of 0.5 mmol GSH/kg restored the fed state (C: 37.4 +/- 2.8 nmol GSH/mg protein; BHA: 84.9 +/- 7.7) within 2 h. This indicates a much faster de novo synthesis of liver glutathione in BHA-pretreated animals. The mechanistic aspects of phenolic antioxidant effects on GSH metabolism are discussed.     

Lack of lung damage in mice following administration of tertiary butylhydroquinone

Tertiary butylhydroquinone (TBHQ), a phenolic antioxidant used in foods, was tested for its potential to produce lung damage in mice similar to that seen following administration of butylated hydroxytoluene (BHT). Male mice (CRL:CD-1) were given single intraperitoneal injections of 62.5, 125, 250 or 500 mg/kg TBHQ or 300, 625 or 1230 mg/kg BHT in corn oil. Survivors were killed five days after treatment and the lungs were removed, weighed, and processed for histologic examination. Lung weights of the TBHQ treated animals were comparable to the controls, while BHT produced a statistically significant increase in lung weight. Histologically, BHT produced hyperplasia of pulmonary pneumocytes as previously reported. No treatment-related lung lesions were seen in the TBHQ mice. The results of this study indicate that TBHQ does not produce pulmonary lesions in mice.     

Studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogenesis

Studies were conducted on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats and hamsters. To obtain information concerning the mechanism of action of BHA on the forestomach, the following areas were examined: the effects of 12 phenolic compounds structurally related to BHA on the hamster forestomach, the effects of combinations of BHA and other antioxidants on the rat forestomach, and the metabolism of BHA in the forestomach. Also examined were the effects of several antioxidants on two-stage carcinogenesis in rats. Squamous-cell carcinomas were induced in the forestomach of rats and hamsters fed BHA. In a limited study, 1 of 13 hamsters developed a squamous-cell carcinoma. The tumorigenic action of crude BHA on the forestomach was largely due to the action of 3-tert-BHA. p-tert-Butylphenol and 2-tert-butyl-4-methylphenol induced pronounced hyperplasia and papillomas in the hamster forestomach. BHA and other antioxidants, particularly propyl gallate and ethoxyquin, showed additive effects in inducing forestomach hyperplasia and cytotoxicity. Neither BHA nor its metabolites were found in the forestomach epithelium, although small amounts of metabolites were detected in the stomach contents. Thus, a direct action on the stomach epithelium may be exerted by BHA itself or by metabolites formed on interaction of BHA with gastric juice. BHA enhanced forestomach carcinogenesis initiated in rats by N-methyl-N'-nitro-N-nitrosoguanidine or N-methylnitrosourea (MNU) and enhanced urinary bladder carcinogenesis initiated by MNU or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In contrast, it inhibited carcinogenesis initiated in the liver by either diethylnitrosamine or N-ethyl-N-hydroxyethylnitrosamine (EHEN) and mammary carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA). BHT promoted urinary bladder carcinogenesis initiated by BBN or MNU and thyroid carcinogenesis initiated by MNU, but inhibited ear-duct carcinogenesis initiated by DMBA. Ethoxyquin promoted EHEN-initiated kidney carcinogenesis, but inhibited both DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. Sodium ascorbate promoted forestomach and urinary bladder carcinogenesis, and sodium erythorbate also enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear-duct carcinogenesis. No antioxidants tested had any effect on glandular stomach carcinogenesis. Thus antioxidants have independent modifying (promoting or inhibitory) effects in different organs.     

Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid)

Caffeic acid (3,4-dihydroxycinnamic acid) is among the major hydroxycinnamic acids present in wine; sinapic acid, which is a potent antioxidant. It has also been identified as one of the active antioxidant. In the present study, the antioxidant properties of the caffeic acid were evaluated by using different in vitro antioxidant assays such as 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH) scavenging, total antioxidant activity by ferric thiocyanate method, total reductive capability using the potassium ferricyanide reduction method, superoxide anion radical scavenging and metal chelating activities. alpha-Tocopherol, trolox, a water-soluble analogue of tocopherol, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) were used as the reference antioxidant compounds. At the concentrations of 10 and 30 microg/mL, caffeic acid showed 68.2 and 75.8% inhibition on lipid peroxidation of linoleic acid emulsion, respectively. On the other hand, 20 microg/mL of standard antioxidant such as BHA, BHT, alpha-tocopherol and trolox indicated an inhibition of 74.4, 71.2, 54.7 and 20.1% on peroxidation of linoleic acid emulsion, respectively. In addition, caffeic acid is an effective ABTS(+) scavenging, DPPH scavenging, superoxide anion radical scavenging, total reducing power and metal chelating on ferrous ions activities.     

Intake of butylated hydroxyanisole and butylated hydroxytoluene and stomach cancer risk: results from analyses in the Netherlands Cohort Study.

Both carcinogenic and anticarcinogenic properties have been reported for the synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). The association between dietary intake of BHA and BHT and stomach cancer risk was investigated in the Netherlands Cohort Study (NLCS) that started in 1986 among 120,852 men and women aged 55 to 69 years. A semi-quantitative food frequency questionnaire was used to assess food consumption. Information on BHA or BHT content of cooking fats, oils, mayonnaise and other creamy salad dressings and dried soups was obtained by chemical analysis, a Dutch database of food additives (ALBA) and the Dutch Compendium of Foods and Diet Products. After 6.3 years of follow-up, complete data on BHA and BHT intake of 192 incident stomach cancer cases and 2035 subcohort members were available for case-cohort analysis. Mean intake of BHA or BHT among subcohort members was 105 and 351 microg/day, respectively. For consumption of mayonnaise and other creamy salad dressings with BHA or BHT no association with stomach cancer risk was observed. A statistically non-significant decrease in stomach cancer risk was observed with increasing BHA and BHT intake [rate ratio (RR) highest/lowest intake of BHA = 0.57 (95% confidence interval (CI): 0.25-1.30] and BHT = 0.74 (95% CI: 0.38-1.43). In this study, no significant association with stomach cancer risk was found for usual intake of low levels of BHA and BHT.     

Lack of Lung Damage in Mice Following Administration of Tertiary Butylhydroquinone

ABSTRACT

Tertiary butylhydroquinone (TBHQ), a phenolic antioxidant used in foods, was tested for its potential to produce lung damage in mice similar to that seen following administration of butylated hydroxytoluene (BHT). Male mice (CRL:CD-1) were given single intraperitoneal injections of 62.5, 125, 250 or 500 mg/kg TBHQ or 300, 625 or 1230 mg/kg BHT in corn oil. Survivors were killed five days after treatment and the lungs were removed, weighed, and processed for histologic examination. Lung weights of the TBHQ treated animals were comparable to the controls, while BHT produced a statistically significant increase in lung weight. Histologically, BHT produced hyperplasia of pulmonary pneumocytes as previously reported. No treatment-related lung lesions were seen in the TBHQ mice. The results of this study indicate that TBHQ does not produce pulmonary lesions in mice.     

Estimate of the maximal daily dietary intake of butylated hydroxyanisole and butylated hydroxytoluene in The Netherlands

The daily dietary intake of the phenolic antioxidants butylated hydroxyanisole (BHA) and/or butylated hydroxytoluene (BHT) was estimated using data obtained from a nationwide dietary record survey carried out in The Netherlands in 1987/1988. The estimates were based on the fat content of selected food categories and their respective maximum permitted levels of BHA and/or BHT. The results indicate that it is unlikely that the current acceptable daily intake for BHA (0-0.05 mg/kg body weight) is surpassed, even in individuals with an extremely high caloric intake, except in extreme cases in 1-6-year-olds. However, it cannot be excluded that the acceptable daily intake for BHT (FAO/WHO: 0-0.125 mg/kg; EEC: 0-0.05 mg/kg) is exceeded in all age and sex groups, but particularly in children aged 1-6 years.     

Intake of phenolic antioxidants from foods in Canada

The potential dietary intake of butylated hydroxyanisole (BHA) in Canada was estimated using dietary recall data on food consumption and maximum permitted use levels for this antioxidant. Based on these estimates, it is concluded that the dietary intake of BHA and other permitted phenolic antioxidants (BHT and propyl gallate) is unlikely to exceed 1 mg/kg body weight/day and on average is less than 0.4 mg/kg body weight/day. Canadian dietary intake estimates compare favourably with those of the United States.     

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.