Effects Of Butylated Hydroxyanisole Research Articles

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

Migration analysis, antioxidant, and mechanical characterization of polypropylene-based active food packaging films loaded with BHA, BHT, and TBHQ.

Polypropylene (PP) based active composite films were prepared by adding butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylated hydroquinone (TBHQ) antioxidants using the extrusion molding process. All concentrations of BHT, 2% to 3% BHA, and 3% TBHQ significantly increased the tensile strength (TS) of the composite films compared with control films. Increasing antioxidant concentration decreased TS values for BHT films, whereas an opposite trend was observed for BHA and TBHQ films. BHA at <2%, BHT at >2%, and TBHQ at all added concentrations significantly reduced elongation at break (Eb ) of the composite films compared to control films. Water vapor permeability (WVP) of 1% BHT film was not significantly different from control. However, other antioxidants especially at increased concentrations significantly increased WVP values. TBHQ films with 300% to 662% increase had the highest WVP and BHT films with 5% to 81% increase had the lowest WVP among composite films. All three antioxidants had a negative effect on the transparency of the films; however the effect of BHA at higher concentrations was greater. The antioxidants did not change the color attributes of the films. Films containing all antioxidants showed 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, which increased with increase in their concentration, especially for those containing 3wt.% BHT and TBHQ. Overall, incorporating BHA and BHT into a PP matrix improved mechanical, barrier, antioxidant properties, and film appearance and consequently were proposed for the development of antioxidant active PP films. TBHQ film is not recommended for food packaging because of its weak mechanical properties (lower Eb and TS values, higher WVP, and greater migration).

Effects of butylated hydroxyanisole on the steroidogenesis of rat immature Leydig cells

Butylated hydroxyanisole (BHA) is a synthetic antioxidant used for food preservation. Whether BHA affects testosterone biosynthesis is still unclear. The effects of BHA on the steroidogenesis in rat immature Leydig cells were investigated. Rat immature Leydig cells were isolated from 35-old-day rats and cultured with BHA (50 μM) for 3 h in combination with 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone or dihydrotestosterone, and the concentrations of 5α-androstanediol and testosterone in the media were measured. Leydig cells were cultured with BHA (0.05–50 μM) for 3 h. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1 and Akr1c14. The testis microsomes were prepared to detect the direct action of BHA on 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), 17α-hydroxylase (CYP17A1) and 17β-hydroxysteroid dehydrogenase 3 activities. In Leydig cells, BHA (50 μM) significantly inhibited LH- and 8Br-cAMP-mediated androgen production. BHA directly inhibited rat testis CYP17A1 and HSD3B1 activities. At 50 μM, it also reduced the expression levels of Hsd17b3 and Srd5a1 and their protein levels. In conclusion, BHA directly inhibits the activities of CYP17A1 and HSD3B1, and the expression levels of Hsd17b3 and Srd5a1, leading to the lower production of androgen in Leydig cells.

Comparative antioxidant capacities of quercetin and butylated hydroxyanisole in cholesterol-modified erythrocytes damaged by tert-butylhydroperoxide

Phenolic compounds are potent antioxidants that scavenge reactive oxygen species (ROS), protecting the cells against oxidative damage. Their antioxidant capacities are governed by their structural features and the nature and physical state of the cell membrane. Our study compares the protective effects of butylated hydroxyanisole (BHA) and quercetin against the cellular injury induced by oxidative stress, and the influence of membrane cholesterol contents in their antioxidant capacities, analyzing the structural changes and cellular stability of native and cholesterol-modified erythrocytes exposed to tert-butylhydroperoxide in presence of each antioxidant. The data provide clear evidence that BHA affords better protection than quercetin against ROS generation, lipid peroxidation and lipid and GSH losses in oxidized erythrocytes. However, cellular integrity and stability are better protected by quercetin owing to the hemolytic effect of BHA. Both antioxidants suppress the alterations in membrane fluidity with similar efficiency, reducing methemoglobin formation in all oxidized erythrocytes. Membrane cholesterol depletion decreases the protection against the oxidative damage provided by both antioxidants. This lower preservation may be due to low antioxidant contents, a lower antioxidant capacity, or even to an increased oxidative damage in this membrane type as a consequence of environment modifications after cholesterol depletion.

Abstract 5366: NAD(P)H-Quinone oxidoreductase (NQO1): Potential role in the prevention of estrogen-induced breast cancer

Abstract The importance of estrogens in the etiology of breast cancer is widely recognized. However, the exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. The objective of the current study was to characterize the role of NQO1 in the prevention of estrogen-induced mammary tumorigenesis by antioxidants vitamin C or butylated hydroxyanisole (BHA). NQO1 converts 17β-estradiol (E2)-quinones back to E2-catechols, thus making E2-quinones unavailable for oxidant damage. Female ACI rats were treated with E2 (3 mg); vitamin C (1% in drinking water); E2 + vitamin C; BHA (0.7% in diet); and E2 + BHA for up to 8 months. The female ACI rat is an established animal model to study the mechanisms of E2-induced breast cancer. The protein isolated from breast tissue and breast tumor tissue of rats treated with E2 and antioxidants was analyzed for NQO1 expression. NQO1 was suppressed in E2-exposed mammary tissue after treatment of rats with E2 for 8 months and in mammary tumors. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Co-treatment of rats with E2 and vitamin C resulted in a 4-fold induction of NQO1 compared to control mammary tissue. Similarly, co-treatment of rats with E2 and BHA induced NQO1 4.4-fold compared to control mammary tissue. There was no change in NQO1 expression during early time points of E2 exposure (7 and 15 days). The up-regulation of NQO1 by vitamin C or BHA was evident after 4 months of treatment. There was no effect of BHA or vitamin C treatment on NQO1 expression on other non-target organs like liver, lung and heart except kidney. These studies suggest a protective role of NQO1 in E2-induced breast carcinogenesis. NQO1 may thus have a great potential in the development of therapeutic strategies for the prevention of estrogen-induced neoplasia. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5366.

Molecular and cellular influences of butylated hydroxyanisole on Chinese hamster V79 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine: antimutagenicity of butylated hydroxyanisole.

The antioxidant butylated hydroxyanisole (BHA) is a rodent carcinogen that also reduces the mutagenicity and carcinogenicity of other agents. In this study, we have evaluated possible mechanisms for the antimutagenicity of BHA by investigating its effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated Chinese hamster V79 cells. Mutant frequency was determined using the hprt/V79 assay, while plating efficiency was used to measure cytotoxicity, and apoptosis was measured by flow immunofluorocytometry. In addition, DNA strand breaks and the kinetics of strand-break rejoining were investigated by the alkaline elution of DNA and by single-cell gel electrophoresis (SCGE). Although the higher concentration of BHA (0.5 mM) increased the cytotoxicity of MNNG and the lower concentration of BHA (0.25 mM) did not change it, both concentrations were antimutagenic in MNNG-treated cells, with the greater effect occurring at the lower BHA concentration. Neither BHA nor MNNG nor BHA + MNNG increased the level of apoptotic nuclei, and BHA did not change the level of MNNG-induced DNA strand breaks, though it did inhibit their rejoining. Determination of O(6)-methylguanine-DNA-methyltransferase (MGMT) activity confirmed that V79 cells do not synthesize active MGMT protein; MGMT activity was also undetectable after MNNG and BHA + MNNG treatment. The ability of BHA to reduce the level of MNNG-induced mutations did not correlate with cytotoxicity, induction of apoptosis, the level of DNA strand break induction, or MGMT activity. A modified SCGE assay showed that BHA significantly reduced the level of formamidopyrimidine-DNA-glycosylase + endonucleaseIII-sensitive sites, which at least partially are caused by oxidative DNA lesions. The results suggest that the protective effect of BHA on MNNG-induced mutagenicity is best explained by the antioxidative activity of BHA, which may scavenge free radicals that participate in MNNG-induced mutagenicity.

Effects of butylated hydroxyanisole and butylated hydroxytoluene on DNA adduct formation and arylamine N-acetyltransferase activity in human bladder tumour cells.

In this study, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine the inhibition of arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in human bladder tumour cell line T-24. The activity of NAT was measured by high-performance liquid chromatography, assaying for the amounts of N-acetyl-2-aminofluorene and N-acetyl-p-aminobenzoic acid and remaining 2-aminofluorene and p-aminobenzoic acid. Human bladder tumour cell line T-24 cytosols and intact cells were used for examining NAT activity and carcinogen-DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene-DNA adduct formation in human bladder tumour cells were inhibited and decreased by BHA and BHT in a dose-dependent manner. The effects of BHA and BHT on the values of the apparent K(m) and V(max) also were determined in both systems examined. The results indicated that BHA and BHT decreased the apparent values of K(m) and V(max) from human bladder tumour cells in both cytosol and intact cells.

Effect of inducers of DT-diaphorase on the toxicity of 2-methyl- and 2-hydroxy-1,4-naphthoquinone to rats

It has previously been shown that rats pre-treated with butylated hydroxyanisole (BHA), a well-known inducer of the enzyme DT-diaphorase, are protected against the toxic effects of 2-methyl-1,4-naphthoquinone but are made more susceptible to the harmful action of 2-hydroxy-1,4-naphthoquinone. In the present experiments, the effects of BHA have been compared with those of other inducers of DT-diaphorase. Rats were dosed with BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), dimethyl fumarate (DMF) or disulfiram (DIS) and then challenged with a toxic dose of the naphthoquinones. All the inducers protected against the haemolytic anaemia induced by 2-methyl-1,4-naphthoquinone in rats, with BHA, BHT and EQ being somewhat more effective than DMF and DIS. A similar order of activity was recorded in the relative ability of these substances to increase hepatic activities of DT-diaphorase, consistent with a role for this enzyme in facilitating conjugation and excretion of this naphthoquinone. In contrast, all the compounds increased the haemolytic activity of 2-hydroxy-1,4-naphthoquinone. DMF and DIS were significantly more effective in this regard than BHA, BHT and EQ. DMF and DIS also caused a much greater increase in levels of DT-diaphorase in the intestine, suggesting that 2-hydroxy-1,4-naphthoquinone is activated by this enzyme in the gut. BHA, BHT and EQ had no effect on the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the renal lesions was decreased in rats pre-treated with DMF and DIS. The results of the present experiments show that modulation of tissue levels of DT-diaphorase may not only alter the severity of naphthoquinone toxicity in vivo, but may also change the relative toxicity of these substances to different target organs.

Increased Longevity, Reduced Fecundity, and Delayed Development in Fruitfly (Zaprionus paravittiger) Fed on Butylated Hydroxy Anisole

Oxygen free radicals are generated as a by-product during normal metabolism, and they cause damage to proteins, lipids, and DNA in organisms. The defense system of the body counteracts these highly reactive chemical moieties and neutralizes them. However, a small fraction of free radicals escapes, which causes lipid peroxidation and hence aging of the organism. It has been hypothesized that, if the free radicals are arrested/reduced, then aging can be delayed or life span could be enhanced. To test the above hypothesis, we fed butylated hydroxy anisole (BHA) to a drosophilid insect, Zaprionus paravittiger, and observed its effect on life span, fecundity, and developmental period. The insects were reared and maintained on standard corn meal agar (CMA) medium at 26 degrees +/- 2 degrees C. Various concentrations (1, 5, 10, 25, 50, and 100 mM) of BHA were mixed with CMA medium, and the cultures were reared and maintained on these mixtures to study the life span of insects. Survivor curves showed that lower concentrations (5, 10, 25 mM) of BHA increased the life span, while higher concentrations (50, 100 mM), which were rather toxic, decreased life span. The most suitable concentration was 10 mM, which increased median (LT50) (27% and 15% in male and female) and maximum (LT100) (18% and 27% in male and female) life spans of insects maximally. Females exhibited longer life spans compared with males. The cultures were fed on the optimal concentration (10 mM) of BHA to study its effect on developmental period and egg-laying rate. The total developmental period was delayed by 7.2%, and egg-laying rate was reduced by 19.7% on BHA feeding. The extension in developmental period and reduction in egg-laying capacity could be the contributory factors to the favorable effect of BHA on life span.

Effects of butylated hydroxyanisole, butylated hydroxytoluene and tertiary butylhydroquinone on growth and luteoskyrin production byPenicillium islandicum

Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) alone in cultural media were tested for the inhibition of growth and luteoskyrin production by two toxigenic strains ofPenicillium islandicum UST-11 andP. islandicum HLT-6. In potato dextrose agar, the concentrations of BHA and TBHQ from 0.2 mg/disc, BHT from 5.0 mg/disc did affect the growth of both tested strains, but the initial concentrations of these antioxidants to reduced luteoskyrin production by UST-11 strain were BHA 0.5 mg/disc, BHT 1.0 mg/disc and TBHQ 0.4 mg/disc, while for HLT-6, BHA 0.4 mg/disc, BHT and TBHQ were 0.2 mg/disc, respectively. In grainy and powdery rice media, the effects of BHA, BHT and TBHQ on luteoskyrin production byP. islandicum UST-11 and HLT-6 were clearly demonstrated. The efficiency of the inhibitory effect was not only closely related to the concentration of antioxidants, but also completely inhibited the luteoskyrin production at a concentration of 200 mg/kg or higher. Also, the antioxidants at a concentration higher than 20 mg/kg reduced significantly the growth and luteoskyrin production by both strains ofP. islandicum.

Evaluation of Antioxidant Butylated Hydroxyanisole and Fungicide Prochloraz for Control of Post-Harvest Anthracnose of Avocado Fruit During Storage.

The incidence of post-harvest anthracnose of avocado caused by Colletotrichum gloeosporioides was reduced significantly after dip or spray treatment with the antioxidant butylated hydroxyanisole (BHA) or BHA + prochloraz. In small, medium, and semicommercial experiments in California on cv. Hass and in Israel on cv. Fuerte there was a significant reduction of decay by single treatments of 1,200 μg a.i. of BHA per ml or of 1,200 μg a.i. of BHA + 250 μg a.i. of prochloraz per ml. Prochloraz at 1,000 μg a.i./ml alone did not always reduce the incidence of decay while BHA or BHA + prochloraz reduced decay consistently. The effect of BHA + prochloraz lasted longer than that of BHA alone. It is suggested that the antioxidant might reduce post-harvest decay in avocado by modulating the natural fruit resistance.

Antileishmanial activity and modification of hepatic xenobiotic metabolizing enzymes in golden hamster by 2(3)-tert-butyl-4-hydroxyanisole following infection with Leishmania donovani

Butylated hydroxyanisole (BHA) inhibited the growth of Leishmania donovani promastigotes (strain AG83) in vitro. The inhibition was dose dependent: the concentration of BHA required for 50% inhibition of the rate of growth was 1 μg/mL. BHA also prevented growth of L. donovani in vivo in golden hamsters infected with L. donovani. In addition, the effect of BHA on several enzymes involved in the metabolism of xenobiotics both in uninfected animals and animals infected with L. donovani is reported.

Effects of Butylated Hydroxyanisole on Ornithine Decarboxylase Activity and Its Gene Expression Induced by Phorbol Ester Tumor Promoter

Butylated hydroxyanisole (BHA) is a phenolic antioxidant that has been found to suppress the activity of skin tumor promoters. In this study, we investigated the effect of BHA on the activity of ornithine decarboxylase (ODC, an indicator of tumor promotion) and its gene expression induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse skin. TPA-induced ODC activity was markedly inhibited by the topical application of 55 mumol of BHA (the inhibition rate at 6 h was about 80%). In Northern and dot-blot analysis, the TPA-induced increase in ODC mRNA was shown to be markedly reduced by the same dose of BHA (the inhibition rate at 4 h was about 60%). These results suggest the involvement of a decrease in ODC gene expression in the mechanism of the inhibition of ODC activity by BHA.

Antioxidants Delay the Onset of Thyroiditis in Obese Strain Chickens*

Dietary iodine has been shown to be important in the induction of thyroiditis in susceptible chicken strains although the underlying mechanism remains unknown. Iodine may exert its effects through the formation of reactive oxidative radicals which would cause thyroidal injury and initiate infiltration. We have tested this hypothesis by examining the ability of butylated hydroxyanisole (BHA), ethoxyquin, and other antioxidants to prevent thyroiditis in Obese strain (OS) chickens, a strain that develops severe disease by 4 weeks of age. BHA, when administered from hatching until death at 5 weeks of age, reduced thyroidal infiltration and serum levels of antibodies binding thyroglobulin, T3, T4. Similar effects were observed with the antioxidant ethoxyquin. Weaker antioxidants such as vitamins C and E and beta-carotene had only slight or negligible effects on these parameters. BHA reduced thyroiditis in OS chicks killed at 3 and 5 weeks of age, but not at 8 weeks. When BHA treatment was initiated after the development of severe disease, it did not reduce thyroglobulin antibody levels. To determine the mechanism by which BHA reduces thyroiditis, studies were performed to assess the effect of BHA on thyroid function and on the immune responses to exogenous antigens. BHA had no effect on thyroid function in normal strain chickens since thyroidal radioiodine uptake and organification and serum T3 and T4 levels were unaffected. BHA did not alter immune responses to exogenous antigens such as sheep red blood cells or Brucella abortus in OS chickens. In summary, potent antioxidant drugs delayed the onset of thyroiditis when treatment was initiated before the onset of disease, suggesting that reactive oxygen intermediates are involved in the early stages of pathogenesis. However, the site of action remains unknown since they had no detectable effects on thyroid function or general immune responses.

Dose-dependent effects of short-term dietary administration of the food additive butylated hydroxyanisole on cell kinetic parameters in gastro-intestinal tract

Groups of ten 5-week old male Wistar rats were fed a diet containing 0, 0.25, 0.50, 0.75, 1.0 or 2.0% butylated hydroxyanisole (BHA) ad libitum for 2 weeks; another group of rats served as a pair-fed control (PFC) group for the 2% BHA-fed animals. Subsequently, rats were injected i.p. with the thymidine-analogue bromodeoxyuridine (BrdU) which was incorporated into the DNA of cells during DNA synthesis. Cell kinetic parameters in gastro-intestinal tract tissues were determined by means of bivariate BrdU/DNA analysis applying flow-cytometry to randomized tissue samples or by applying immunohistology to randomized tissue sections. In the forestomach, glandular stomach, small intestine and colon/rectum, mean tissue labelling index (LI) was significantly increased in rats on a diet supplemented with 2% BHA, in comparison with rats fed the basal diet (0% BHA) ad libitum or restricted to the mean daily intake of 2% BHA-fed rats (PFC). In the oesophagus of rats fed 2% BHA, the LI was significantly higher in comparison with their PFC group, but not with the group of rats fed 0% BHA ad libitum. In rat forestomach, an apparent no observed effect level for ad libitum fed rats was found at 0.5% BHA (LI) and at 0.75% BHA (potential doubling time). Thus, the oesophagus, glandular stomach, small intestine and large bowel, in addition to the forestomach, are possible target tissues in rats for the proliferation enhancing effects of BHA. At the time of termination of the experiment, plasma BHA concentrations were dose dependently increased and were in the range that is easily attained in man after ingestion of a dose equal to the acceptable daily intake for BHA (0.5 mg/kg).

Inhibition of binding of 2-acetylaminofluorene to DNA by butylated hydroxytoluene and butylated hydroxyanisole in vitro

Numerous studies have shown that the food antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), under specific exposure conditions, can inhibit hepatocarcinogenesis induced by various carcinogens. The purpose of the present work was to study the biochemical mechanisms responsible for the anticarcinogenic activity of BHA and BHT using in vitro systems. The effects of BHA and BHT on the binding of 2-acetylaminofluorene (2-AAF) to DNA was determined in a microsomal system and in primary cultures of rat hepatocytes. It was found that both antioxidants reduce the binding of 2-AAF and that of N-OH-2-acetylaminofluorene (N-OH-2-AAF) to calf thymus DNA in the presence of liver microsomes. The inhibition was however more pronounced with the parent compound. Lower levels of DNA binding were also detected in hepatocytes incubated with 2-AAF along with BHA or BHT. These results suggest that phenolic antioxidants can exert anticarcinogenic activity through modulation of carcinogen interaction with DNA which may reflect on alteration in carcinogen metabolic activation.

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.

Sequential changes of the forestomach of F344 rats, Syrian golden hamsters, and B6C3F1 mice treated with butylated hydroxyanisole.

Butylated hydroxyanisole (BHA) was given to F344 rats, Syrian golden hamsters and B6C3F1 mice at 2 doses for up to 104 weeks. The two doses were 2.0% and 1.0% for rats and hamsters, and 1.0% and 0.5% for mice. Animals were sacrificed sequentially at 8-week intervals from week 8 to week 104, and the carcinogenic effects of BHA on the forestomach were examined histopathologically. Papillomas and carcinomas were found in rats, hamsters and mice. In rats, papillomas first appeared in week 8 in the group given the higher level of BHA and in week 56 in that given the lower level. The first carcinoma was observed in week 48 in rats given the high level, while no carcinoma was observed in rats given the lower level. In hamsters, papillomas appeared in week 8 in both BHA-treated groups, and in both groups, the incidence of papillomas was much higher than in BHA-treated rats. Squamous cell carcinomas were observed in 4 hamsters (10.0%) among those that survived more than 64 weeks on treatment with the higher level of BHA and in 4 (7.3%) among those treated with the lower level. In mice, papillomas were induced by BHA in both BHA-treated groups after more than 88 weeks. Although the incidence was not statistically significant, carcinoma was also seen in mice, suggesting that BHA may also be carcinogenic to mouse forestomach.

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), kindney 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.

Effects of BHA, d-alpha-tocopherol and retinol acetate on TCDD-mediated changes in lipid peroxidation, glutathione peroxidase activity and survival.

Daily treatment of female rats with butylated hydroxyanisole (BHA) protected against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. This protective effect was associated with reduced microsomal lipid peroxidation, increased glutathione peroxidase (GSH-PX) activity and decreased aryl hydrocarbon hydroxylase (AHH) activity. Retinol acetate (vitamin A) inhibited lipid peroxidation, elevated GSH-PX activity, and enhanced AHH activity. Thirty per cent of vitamin A-treated animals were alive 25 d after a lethal dose of TCDD. d-alpha-Tocopherol (vitamin E) inhibited markedly microsomal lipid peroxidation, enhanced AHH activity, and had no effect on GSH-PX activity. Only 10% of the vitamin E-treated animals were alive 25 d after a lethal dose of TCDD. The mechanism of TCDD toxicity may involve in part inhibition of GSH-PX activity with resultant lipid peroxidation by hydrogen peroxide.