Administration Of Butylated Hydroxyanisole Research Articles

Retention of the synthetic antioxidant butylated hydroxyanisole in Atlantic salmon (Salmo salar) fillets

Butylated hydroxyanisole (BHA) is a phenolic antioxidant that is used as a food additive, particularly in fats and oils, and it is also authorized as a feed additive in the European Union for all animal species with a maximum concentration of 150 mg kg⁻¹ feed (alone or in combination with ethoxyquin and/or butylated hydroxytoluene). Information on the BHA levels in food of animal origin is scarce, as is literature on the transfer of BHA from feed to animal products. Consequently, the current feeding trial was conducted under realistic rearing conditions to determine the retention of BHA in Atlantic salmon (Salmo salar) fillets. Four different concentrations of BHA in feed were tested (0, 48.5, 92.5 and 225 mg BHA kg⁻¹ feed), and fish were sampled after 4, 8 and 12 weeks dietary exposure, and after a 2-week starvation period, which is representative of commercial salmon farming in Norway. The levels of BHA in salmon fillets were fairly dose dependent during the feeding period. A steady state in fillet residues was reached after 4 weeks of administration of BHA. After the 2-week withdrawal period, BHA was not detectable (<7 μg kg⁻¹) in either fish fillets or livers.

Induction of oxidative DNA damages and enhancement of cell proliferation in human lymphocytes in vitro by butylated hydroxyanisole.

The food additive butylated hydroxyanisole (BHA) has been shown to induce gastrointestinal hyperplasia in rodents by an unknown mechanism. The relevance of this observation for human risk assessment is not clear. We therefore analysed the effect of BHA and its primary metabolites tert-butylhydroquinone (TBHQ) and tert-butylquinone (TBQ) on 8-oxo-deoxyguanosine formation and labelling induces in human lymphocytes in vitro. Analysis of culture medium and cell lysate fractions after administration of BHA or metabolites of BHA revealed that BHA and TBHQ undergo biotransformation in whole blood cultures. Moreover, TBQ can be reduced to TBHQ. While in cultures treated with BHA 50-60% of the dose administered was recovered, a much lower dose recovery was found in cultures treated with either TBHQ or TBQ. This indicates a considerable binding of these compounds to macromolecules. BHA and TBHQ, as well as TBQ, induced a dose-dependent increase in cell proliferation of phytohaemagglutinin-stimulated lymphocytes, 50 microM being the optimal dose. Since BHA is metabolized to TBHQ, it is not clear which compound is responsible for the proliferation enhancing effects observed in culture. Inhibition of IBHQ metabolism to its semiquinone radical by acetylsalicylic acid (ASA) reduced the increase in labelling indices induced by TBHQ. This indicates that this metabolic pathway is involved in the enhancement of cell proliferation induced by the hydroquinone. HPLC-ECD analysis of oxidative DNA damage in lymphocytes exposed to 10, 50 and 100 microM BHA, TBHQ or TBQ respectively showed that BHA was not capable of inducing oxidative DNA damage to a significant degree. TBQ and, in particular, TBHQ at a dose of 50 microM (the optimal dose for induction of cell proliferation), however, increased lymphocyte 7-hydroxy-8-oxo-2'-deoxyguanosine formation by 320 and 680% respectively. Inhibition of prostaglandin H synthase by ASA in cultures treated with TBHQ decreased the oxidation ratio significantly, confirming the significance of this enzyme system in the mechanism of toxicity of BHA.

Selective induction of rat urinary bladder tumors by simultaneous administration of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and butylated hydroxyanisole or butylated hydroxytoluene is associated with increased DMAB-DNA adduct formation

Modification of 3,2'-dimethyl-4-aminobiphenyl (DMAB) multi-organ carcinogenesis by simultaneous treatment with butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) was studied using young and old male F344 rats. Animals, 4 or 54 weeks old, were given DMAB (s.c. injection of 50 mg/kg body wt once a week for 10 weeks) along with BHA (2.0% in diet for 11 weeks) or BHT (1.0% in diet for 11 weeks). The experiments were terminated 55 weeks after the commencement. Combined administration of BHA or BHT with the carcinogen resulted in development of urinary bladder tumors in greater than 90% of both young and old rats thus treated, whereas no tumors were induced in animals given DMAB alone. In contrast, the appearance of preneoplastic lesions in the liver and pancreas was reduced by BHA or BHT treatment. Tumor development (less than 30% incidence) was also evident in the small and large intestines, prostate, preputial glands, skin/subcutis and ear duct, with no modification by BHA or BHT. No ageing effects were evident. The formations of DMAB-DNA adducts, evaluated by the enzyme-linked immunosorbent assay and immunohistochemical staining, correlated well with tumorigenesis in the urinary bladder, liver and pancreas. The selective enhancement of urinary bladder tumor induction by BHA and BHT appeared to be due to both increased DMAB-DNA adduct formation caused by metabolic alteration of DMAB in the liver and increased DNA synthesis in the urothelial cells.

Induction of Hepatic Tumors with Butylated Hydroxyanisole in the Self‐fertilizing Hermaphroditic Fish Rivulus ocellatus marmoratus

The three‐day‐old larvae of self‐fertilizing hermaphroditic fish Rivulus ocellatus marmoratus were fed a diet containing the antioxidant butylated hydroxyanisole (BHA) at levels of 0.01–0.8% for 12 days. Six months after BHA administration, hepatic tumors were found in all groups of BHA‐treated fish. The BHA‐induced tumor incidences were clearly dose‐dependent. These results show that dietary BHA is hepatocarcinogenic in Rivulus even at 0.01% dose.

Effect of butylated hydroxyanisole on the level of DNA adduction by aristolochic acid in the rat forestomach and liver.

Administration of butylated hydroxyanisole (BHA) orally at either 0.5 g or 1 g/kg daily for 14 days to rats did not produce any DNA adducts in the forestomach as measured by the 32P‐postlabeling method using (1) limiting concentrations of 32P‐ATP; (2) nuclease P1 enhancement; or (3) butanol extraction. Experiments were conducted to establish the effects of BHA administration on aristolochic acid (AA) DNA adduct formation in the forestomach and liver, when BHA was administered prior to, together with or after AA administration. Adduct levels per 109 nucleotides in the liver after oral dosing daily for 5 days with 1 mg/kg AA and BHA (1 g/kg) or corn oil (5 ml/kg) for 7 days were as follows: (a) BHA and AA given simultaneously; 235±71, (b) AA+corn oil; 63±39, (c) AA followed by BHA; 57±13, (d) AA followed by corn oil; 91±38, (e) BHA followed by AA; 90±12, (f) corn oil followed by AA; 83±24. For the forestomach the values were: (a) 236±86, (b) 77±25, (c) 367±97, (d) 296±47, (e) 217±81, (f) 70±64. These data suggest that BHA could have an enhancing effect on AA‐induced lesions in the forestomach if dosed together with, or prior to, AA as adduct levels are significantly higher than in controls.

Effect of subacute oral intake of the food antioxidant butylated hydroxyanisole on clinical parameters and phase-I and -II biotransformation capacity in man.

A study is presented in which eight healthy male non-smoking volunteers ingested a daily amount of 0.5 mg/kg butylated hydroxyanisole (BHA) for 10 consecutive days. Blood samples were taken on days -6 and 0 before and on days 4 and 7 after the first BHA administration for the assessment of standard clinical plasma parameters (L-aspartate aminotransferase, L-alanine-aminotransferase, L-gamma-glutamyltranspeptidase, creatine phosphokinase, lactate dehydrogenase, total protein, albumin, urea, creatinine, Na+, and Cl-). Antipyrine (500 mg p.o.) and paracetamol (500 mg p.o) were administered before and during BHA administration as test substances to measure phase-I and phase-II biotransformation capacity. Saliva samples and urine were subsequently collected for the assessment of kinetic parameters (e.g. saliva elimination half-life, saliva clearance, apparent volume of distribution) and urinary excretion of metabolites. Kinetic plasma parameters of BHA itself were determined in plasma samples obtained via a catheter in an arm vein after oral BHA intake on days 0 and 7. Levels of antipyrine, paracetamol, BHA and metabolites in plasma, saliva or urine were quantified by standard or newly developed reversed-phase high-performance liquid chromatography methods. Urinary excretion of Na+, K+, and Cl-, as well as osmolality of urine were measured on three days before and six days during BHA administration. Generally, no significant differences were detected in the parameters measured, indicating that oral administration of BHA to men for 10 days remains without effects on clinical biochemical parameters and phase-I and phase-II biotransformation capacity. In contrast, urinary excretion of metabolites of BHA was significantly increased on days 3 and 7 vs. the first day of BHA administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of butylated hydroxyanisole administration on pancreatic carcinogenesis in Syrian hamsters initiated with N-nitrosobis(2-oxopropyl)amine

The modifying potential of butylated hydroxyanisole (BHA) administration on pancreatic carcinogenesis was evaluated in 70 female Syrian golden hamsters. Groups of animals received saline, 70 mg/kg body weight of N-nitrosobis(2-oxopropyl)amine (BOP) or 70 mg/kg plus 20 mg/kg body weight of BOP followed by basal diet or diet containing 2% BHA from week 3. Although the body weights of hamsters receiving the 2% BHA supplement decreased, caloric restriction was not observed. All hamsters were killed at week 18 and histopathologically examined for lesion development. The incidences of pancreatic carcinomas in hamsters receiving 70 mg/kg plus 20 mg/kg body weight of carcinogen followed by 2% BHA was 7.1%, significantly lower than the 64.3% evident in hamsters given the same doses of BOP followed by basal diet. The total numbers of pancreatic lesions including carcinomas, atypical ductal hyperplasias and ductal hyperplasias and ductular proliferations in the liver were also significantly decreased in animals receiving BOP followed by 2% BHA. The results thus indicate that both pancreatic and cholangiocellular carcinogenesis initiated by BOP in Syrian hamsters can be inhibited by 2% BHA treatment for a relatively short experimental period.

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.

Early lesions induced by DHPN in Syrian golden hamsters: influence of concomitant Opisthorchis infestation, dehydroepiandrosterone or butylated hydroxyanisole administration.

The effects of concomitant Opisthorchis infestation and dehydroepiandrosterone (DHEA) or butylated hydroxyanisole (BHA) administration on dihydroxy-di-n-propylnitrosamine (DHPN) induction of preneoplastic lesions were investigated in Syrian golden hamsters. Whereas parasite infection was primarily associated with first-order ductular proliferation in the liver and a secondary appearance of cholangiofibrotic lesions, DHEA treatment brought about increased carcinogen toxicity and enhanced generation of glutathione-S-transferase P (GST-P)-positive hepatocellular foci, liver cysts and focal proliferative changes in the pancreas. BHA also exerted an enhancing influence on pancreatic but not liver carcinogenesis. The results suggest that whereas alteration of DHPN metabolism by DHEA and BHA treatment effected changes at the initiation level, opisthorchiasis principally exerted an enhancing influence subsequent to carcinogen withdrawal.

Effect of subchronic dietary administration of butylated hydroxyanisole on canine stomach and hepatic tissue

Beagle dogs, 29 males and 30 females, were assigned to feeding groups of 0, 1.0 and 1.3% butylated hydroxyanisole (BHA) for 180 days. Animals were observed, daily for physical signs of pharmacological or toxicological effect. Except for the production of a reddish-brown urine, no physical signs attributable to BHA administration were observed. Both food consumption and body-weight gain were reduced in the BHA-treated animals. Fifty-one animals completed the study. At termination, tissues were examined for gross BHA-related effects, and specimens were taken for enzyme analysis, light microscopy, electron microscopy and morphometric analysis. The liver showed a significant weight increase over the control in both sexes at both BHA dose levels. Ultrastructural examination of the liver of BHA-treated animals revealed proliferation of smooth endoplasmic reticulum and hepatocytic cytoplasmic myelinoid bodies. Enzyme analysis of hepatic tissue showed a significant increase in mixed-function oxidases, UDP glucuronyl transferase, glutathione S-transferase and epoxide hydratase in the BHA-treated dogs compared with the controls. Light microscopy revealed no proliferative/hyperplastic lesions of the stomach/gastric epithelium. Electron microscopic examination of the lower oesophagus and stomach specimens from representative animals from each treatment group failed to reveal any treatment-related effect as compared with controls.

2 Final Report on the Safety Assessment of Butylated Hydroxyanisole

Butylated hydroxyanisole (BHA) is used in cosmetic formulations as a chemical preservative and as an antioxidant. Both animal and human studies have shown that BHA is absorbed from the gastrointestinal tract and metabolized. Tissue storage may occur with BHA because of its lipid solubility. However, the amount stored is limited by rapid metabolism and excretion. Reported acute oral LD50 values for BHA in rats varied from 2.0 to &gt; 5.0 g/kg. Formulations containing BHA elicited, at most, minimal or moderate skin and eye irritation in rabbits. An extensive number of subchronic and chronic oral studies have been conducted and are reviewed. BHA given orally or parenterally to mice and rats was shown to inhibit the carcinogenic effects of a broad range of chemical carcinogens. BHA has been shown to inhibit mutagenesis and was not a mutagenic agent in standard in vitro tests. No evidence of carcinogenicity was observed when BHA was administered to mice by subcutaneous injection, by intraperitoneal injection, or by topical application. No carcinogenesis was demonstrated following dietary administration of BHA to either rats or dogs. An increased incidence of forestomach papillomas and squamous cell carcinomas has been observed in rats fed BHA. Studies with pregnant rabbits, mice, rats, and hamsters receiving BHA during gestation by a variety of oral dosage regimens revealed no significant embryotoxic or teratogenic effects. Clinical data for BHA in cosmetic formulations indicated that they were generally nonsensitizing, nonphotosensitizing, and only minimally or mildly irritating. It is concluded that BHA is safe as a cosmetic ingredient in the present practices of use.

Promoting activities of butylated hydroxyanisole, butylated hydroxytoluene and sodium L-ascorbate on forestomach and urinary bladder carcinogenesis initiated with methylnitrosourea in F344 male rats.

The promoting effects of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and sodium L-ascorbate on two-stage carcinogenesis initiated with methylnitrosourea (MNU) in F344 male rats were investigated. Animals were given injections of MNU (20 mg/kg ip) twice a week for 4 weeks, and then basal diet containing 2% BHA, 1% BHT or 5% sodium L-ascorbate for the next 32 weeks. Administration of BHA, BHT or sodium L-ascorbate in the diet significantly increased the incidences per group and numbers per rat of papilloma and papillary or nodular hyperplasia of the urinary bladder, and BHA and BHT also increased the number of cancers per rat. Furthermore BHA significantly increased the incidences of cancer and papilloma in the forestomach of rats initiated with MNU, whereas treatment with BHA alone was associated with papilloma but no carcinoma development in the rat forestomach. The incidence of adenoma, but not adenocarcinoma, of the thyroid was significantly increased by treatment with MNU plus BHT. These results show that BHA, BHT and sodium L-ascorbate have promoting activities on urinary bladder carcinogenesis in rats initiated with MNU, and that BHA also has a promoting effect on forestomach carcinogenesis after initiation with MNU.

Effects of administration to mice of butylated hydroxyanisole by oral intubation on benzo[a]pyrene-induced pulmonary adenoma formation and metabolism of benzo[a]pyrene.

Administration of butylated hydroxyanisole (BHA) by oral intubation 4 hours before challenge with benzo[a]pyrene (BP) inhibited the formation of pulmonary adenomas in A/HeJ mice. Incubation of BP with liver microsomes from mice that received BHA 2,4, or 8 hours before being killed resulted in less binding of BP metabolites to added DNA than occurred with control microsomes. High-pressure liquid chromatography studies of the BP metabolite pattern produced by the incubation of BP with liver microsomes from mice given BHA by oral intubation showed a decrease in formation of BP-4,5-oxide and 9-hydroxybenzo[a]pyrene. In contrast, the formation of 3-hydroxybenzo[a]-pyrene was increased. The was increased. The short interval between the administration of BHA by oral intubation and the observed biochemical changes indicated that BHA could exert a direct effect on the microsomal metabolism of BP. These changes in metabolism of BP occurred under conditions of BHA administration that produced a decreased neoplastic response to this carcinogen.

Liver response tests. IV. Application to short-term feeding studies with butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)

Preliminary studies involved oral administration to rats of 6 daily doses (500 mg/kg) of butylated hydroxytoluene (BHT; 3,5-di- tert-butyl-4-hydroxytoluene) or butylated hydroxyanisole (BHA; 3- tert-butyl-4-hydroxyanisole). Both BHA and BHT induced liver enlargement and increased daily output of urinary ascorbic acid. These changes paralleled each other closely in onset, degree and duration, being rapid but transient with BHA, and slower in onset yet more prolonged with BHT. Administration of BHA or BHT at a level of 0·1% in the diet of rats for 16 wk elicited similar changes, particularly in females. With BHT the effects were more pronounced and more prolonged, but despite continuation of treatment relative liver weight and urinary ascorbic acid had returned to control levels by 16 wk. In the case of BHA the effects were no longer manifest after 4 wk. There was no histopathological evidence of damage to the liver. Equally negative findings regarding liver response were obtained in the biochemical measurements and histochemical assessments of glucose 6-phosphatase and glucose 6-phosphate dehydrogenase activities. Both BHT and BHA brought about increases in adrenal weight. It is concluded that measurement of ascorbic acid excretion in the urine affords a promising means of detecting hyperfunctional liver enlargement and distinguishing it from toxic liver enlargement. Under the conditions of the present experiments neither BHT nor BHA exhibited evidence of hepatotoxicity.