Effect of butylated hydroxytoluene on the disposition of [14C]aflatoxin B1 in the lactating rat

The distribution and metabolism of an ip dose of [14C]aflatoxin B1 (AFB1) were studied in lactating Sprague-Dawley rats fed for the previous 13 days on a diet containing 0.5% butylated hydroxytoluene (BHT). Compared with ingestion of a BHT-free diet, treatment with BHT increased the biotransmission of AFB1 metabolites, predominantly aflatoxin M1 (AFM1), into the mammary gland and its content of milk, decreased AFB1 binding to liver nuclear DNA and enhanced the excretion of water-soluble metabolites of AFB1, all measured 6 hr after an oral dose of [14C]AFB1. These changes are related to the induction by BHT of hepatic enzymes involved in the transformation and detoxification of AFB1. The results suggest that exposure to BHT may protect the lactating animal from the carcinogenic effect of AFB1 but may increase the risk of exposure of the newborn infant to the carcinogenic metabolite AFM1.     

The excretion of [14C] butylated hydroxytoluene in the rat

The excretion of small doses of [¹⁴C]labelled butylated hydroxytoluene has been examined in the rat. Parenteral doses are excreted slowly in urine and faeces over 4 days. Biliary excretion is rapid. The biliary radioactivity is absorbed readily from the gut and a rapid enterohepatic cycle has been found to operate over at least 96 hr.     

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.     

Characterization of water-soluble glucuronide and sulphate conjugates of aflatoxin B1. 1. Urinary excretion in monkey, rat and mouse

Water-soluble aflatoxin conjugates prepared from urine samples from rats, mice and rhesus monkeys dosed with [14C]aflatoxin B1 (AFB1) ip or iv were hydrolysed by enzymes (beta-glucuronidase and sulphatase), acid or a combination of both treatments. Different amounts of AFB1 and its metabolites were found in hydrolysates from different sources, indicating the presence of glucuronide, sulphate and possibly mercapturate conjugates of aflatoxins. In addition to aflatoxins M1, P1, Q1 and B2a, AFB1 was frequently identified in the products released from the hydrolysates. These water-soluble aflatoxin conjugates were not mutagenic to Salmonella typhimurium TA98 in the presence of rat-liver S-9 mix. However, chloroform extracts of the hydrolysates from beta-glucuronidase and sulphatase treatment showed mutagenic activity in these bacteria in the presence of S-9 mix. Although very low levels of AFB1 radioactivity were detected in the hydrolysates, the potent mutagenic activity of AFB1 contributed to the high numbers of revertant colonies. AFP1 was detected in urine samples from monkeys that were pretreated with phenobarbital before an iv dose of AFB1. No mutagenic activity was detected in the enzymatic hydrolysate of the sample from these monkeys. The results thus indicate that AFB1 can form glucuronide and/or sulphate conjugate(s) directly and be excreted in the urine.     

Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis--inhibition of aflatoxin B1 metabolism

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B₁ (AFB₁) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB₁ metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB₁ to the reactive intermediate AFB₁-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB₁in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB₁ epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.     

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.     

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.     

Effect of disulfiram pretreatment on the tissue distribution, macromolecular binding, and excretion of [U-1,2-14C]dichloroethane in the rat

The effect of disulfiram (DSF) pretreatment on the distribution of [14C]ethylene dichloride (EDC) in selected organs and/or tissues of control and EDC-pretreated rats was studied. The presence of EDC metabolites and their binding to an acid-insoluble extract of the tissues, as well as purified protein and DNA, were evaluated. Dietary DSF was found to modulate the distribution, excretion, and macromolecular binding of EDC and/or its metabolites at 4 and 24 hr following ip administration. The urinary excretion of [14C]EDC metabolites was not affected by subchronic inhalation exposure to nonradiolabeled EDC. However, DSF pretreatment increased the fat deposition of EDC and decreased the urinary excretion of its metabolites. DSF also increased the binding of EDC metabolites to DNA and decreased the binding to protein in the liver, kidneys, spleen, and testes. However, prior exposure to EDC alone increased the binding of its metabolites to DNA in the kidneys only.     

Reduction in binding of [14C] aflatoxin B1 to rat liver macromolecules by phenobarbitone pretreatment.

Various dietary regimens have been shown to alter the acute and chronic toxicity of aflatoxin B₁ (AFB₁) in rats^1–4. Phenobarliitone (PB), an inducer of liver mixed function oxidases⁵ reduces the liver carcinogenioity of AFB₁ contaminated peanut meal⁶, decreases the LD₅₀ after an intraperitoneal injection of AFB₁ from 1 to 5 mg/kg (Gamer, Miller and Miller, unpublished) and reduces the inhibitory action of AFB₁ on rat liver ribonucleic acid synthesis⁷. The protective effect of PB contrasts with $\text{in vitro}$ findings that AFB₁ metabolism is increased by this enzyme-inducing agent, both to detoxification products such as aflatoxin B_2a⁸ and aflatoxin M₁⁹ as well as to AFB₁ 2, 3-oxide, the probable ultimate carcinogenic form of this compound¹⁰. To investigate this apparent discrepancy further the macro-molecular binding of [¹⁴C] AFB₁ after a single intraperitoneal injection has been studied in control and PB pretreated animals.     

Decreased utilization of [2-14C]orotic acid for the synthesis of cytidine nucleotides in rat liver after administration of alpha-hexachlorocyclohexane.

Administration of alpha-1,2,3,4,5,6-hexachlorocyclohexane (alpha-HCH) to rats decreased the utilization of [2-14C]orotic acid for the synthesis of liver cytidine nucleotides. The specific radioactivities of uridine components of the acid-soluble pool and rRNA increased during the first hours of treatment with the drug. Later on the specific radioactivities of uridine nucleotides remained unchanged, while those of cytidine components decreased gradually. Administration of hydrocortisone increased the incorporation of labelled orotic acid into rRNA cytidylic acid.     

Effects of butylated hydroxytoluene pretreatment on the metabolism and genotoxicity of aflatoxin B1 in primary cultures of adult rat hepatocytes: selective reduction of nucleic acid binding

To elucidate biochemical mechanisms underlying the anticarcinogenic activity of butylated hydroxytoluene (BHT), studies were undertaken to characterize the influence of BHT pretreatment on the metabolism and genotoxicity of aflatoxin B1 (AFB1) in primary cultures of rat hepatocytes. During a 10-day pretreatment period, adult male rats were fed either a control diet or a diet supplemented with 0.5% BHT. Hepatocytes were subsequently isolated from each animal and cultured in chemically defined medium. Cultures prepared from rats which had been fed BHT metabolized AFB1 more rapidly than did controls. BHT pretreatment also enhanced oxidation of AFB1 to aflatoxin M1 (AFM1), and accelerated the rate of AFM1 conjugation. Covalent binding to DNA and RNA in BHT-pretreated cultures was reduced by 91 and 82%, respectively, while protein binding decreased by only 29%. AFB1 did not stimulate detectable DNA repair synthesis in BHT-pretreated cultures, although stimulation of DNA repair was clearly evident in control cultures. In a separate experiment, consistently higher baseline concentrations of reduced glutathione were observed in BHT-pretreated cells, indicating that BHT pretreatment may enhance formation of detoxified glutathione conjugates of AFB1. These findings suggest that the anticarcinogenic activity of BHT is due in part to preferential enhancement of hepatic detoxification mechanisms, with the result that intracellular concentrations of reactive metabolites are reduced and fewer covalently bound adducts are formed.     

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.     

Identification and mutagenicity of aflatoxicol-M1 produced by metabolism of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout (Salmo gairdneri) fed beta-naphthoflavone

beta-Naphthoflavone (beta NF) fed to rainbow trout (Salmo gairdneri) at 50 or 500 ppm in the diet, modified the in vitro metabolism of aflatoxin B1 (AFB1) by the postmitochondrial fraction (PMF) of the liver. Production of aflatoxicol (AFL) was significantly less in the 500 ppm beta NF-fed group (33.9 ng/mg protein) than in the control group (45.7 ng/mg protein), aflatoxin M1 production was dependent on the dose of beta NF, being greatest in the 500 ppm beta NF-fed group (48.9 ng/mg protein), intermediate in the 50 ppm beta NF-fed group (3.7 ng/mg protein), and was not detected in controls. A new trout metabolite, 4-hydroxyaflatoxicol (aflatoxicol M1, AFLM1) was also detected in small amounts from in vitro metabolism by liver PMF from beta NF-fed trout. Sufficient quantities of AFLM1 for confirmation of identity by ultraviolet spectra, mass spectra and nuclear magnetic resonance spectra were prepared by biotransformation of AFL using liver microsomes and isolation by HPLC. In a modified Ames mutagen assay with Salmonella typhimurium TA98, ALFM1 was 4.1% as mutagenic as AFB1 in a previous determination. The carcinogenicity of AFLM1 to rainbow trout is expected to be considerably less than that of AFB1.     

Effects of saturated and unsaturated dietary fat on aflatoxin B1 metabolism

Male Fisher 344 rats were fed diets containing either 20% corn oil, 20% coconut oil or 18% coconut oil plus 2% corn oil for 3 wk. A single dose of [3H]aflatoxin B1 [(3H]AFB1) was administered ip and the biliary excretion of aflatoxin metabolites and the binding of 3H to nucleic acids were studied. In other experiments the in vitro metabolism of AFB1 by liver postmitochondrial supernatants prepared from rats fed the different sources of dietary fat was determined. The major nonextractable aqueous metabolite of AFB1 was the aflatoxin B1-glutathione conjugate (AFB1-GSH). Variation in the source of dietary fat did not affect production of the conjugate, nor was in vivo binding of AFB1 to nucleic acids affected. Aflatoxin P1 ( AFP1 )--mostly conjugated--and aflatoxin M1 (AFM1) were also identified in the bile, and the quantities of these metabolites produced were unaffected by the dietary treatments. The metabolites recovered in the in vitro study included aflatoxins Q1, P1 and M1. The corn oil diet produced a higher microsomal cytochrome P-450 level than the coconut oil diet. Associated with the higher level of cytochrome P-450 was increased in vitro conversion of AFB1 to AFQ1 and AFM1, but not to AFP1 . The in vitro production of aqueous metabolites and the covalent binding of metabolites to protein was unaffected by the dietary treatments. The results of the in vivo and in vitro studies suggest that the formation of the putative carcinogenic metabolite, AFB1-epoxide, which undergoes detoxification through glutathione conjugation, is not affected by the type of dietary fat. These observations further suggest that dietary fat, in particular unsaturated fat, affects AFB1 carcinogenesis through a mechanism other than by alteration of the metabolic activation of AFB1.     

The distribution and excretion of [3H, 14C] lofepramine in the rat.

1. Lofepramine hydrochloride (N-methyl-N-[4-chlorobenzoylmethyl-3-(10, 11-dihydro-5H-dibenz(b,f)azepin-5-yl)]-propylamine hydrochloride) labelled with 3H in the dihydrobenzazepine moiety and 14C in the chlorobenzene moiety was synthesized and its distribution, urinary and biliary excretion were studied in female rats after oral administration. 2. For both isotopes, high ratios were established between tissues and blood. 3. Extensive N-dealkylation of lofepramine occurred in the rat. One of the metabolites, desmethylimipramine, was found in high concentrations in most tissues, especially the lungs and brain. 4. Desmethylimipramine was further metabolized to 2-hydroxydesmethyl-imipramine and 2-hydroxy-iminodibenzyl, and the corresponding glucuronides. These metabolites were mainly excreted via the bile. 5. p-Chlorobenzoic acid was found in the liver and lungs but not in the urine where most of the excreted 14C-activity was found. The identity of the major urinary metabolite is discussed.     

Butylated hydroxytoluene chemoprevention of aflatoxicosis – Effects on aflatoxin B1 bioavailability, hepatic DNA adduct formation, and biliary excretion

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB₁ toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB₁ bioactivation. To test whether dietary BHT alters hepatic AFB₁–DNA adduct formation, excretion, and bioavailability of AFB₁ in vivo, turkeys were given diets with BHT (4000 ppm) for 10 days, given a single oral dose of [³H]-AFB₁ (0.05 μg/g; 0.02 μCi/g), then sampled at intervals up to 24 h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB₁–DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24 h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M₁ (AFM₁) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB₁, aflatoxin Q₁ or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB₁ bioavailability in addition to inhibition of bioactivation.     

Metabolism, pharmacokinetics, tissue distribution, and excretion of [14C]CP-424391 in rats.

CP-424391, 2-amino-N-[3aR-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1R-benzyloxymethyl-2-oxoethyl]-isobutyramide, is an orally active growth hormone secretagogue currently being developed. In this study, we investigated the metabolic fate and disposition of radiolabeled CP-424391 in rats. Following 15 mg/kg single oral administration to Sprague-Dawley rats, 91% of the radiolabeled dose was recovered. Feces was the major route of excretion: 77% of the dose recovered in feces of the female rat and 84% in the male. Excretion in the urine was 15% in the female rat compared with 7% in the male. Both fecal and urinary metabolic profiles were consistent in both genders. The metabolic pathways of CP-424391 were oxidation at the benzyl group of the O-benzylserine moiety, N-demethylation of pyrazolidine, and/or O-debenzylation. In circulation, CP-424391 was absorbed within the first hour to an average apparent C(max) of 1.44 microg/ml. CP-424391 accounts for about 40% of radioactivity area under the plasma concentration-time curve and C(max) in circulation. The plasma terminal elimination half-life of CP-424391 was 2.4 h and for total radioactivity was 2.8 h. The radioactivity was widely distributed in all tissues except for the central nervous system. [(14)C]CP-424391 radioactivity was eliminated from most tissues by 9 h with the exception of liver, skin, and uvea. By 168 h, [(14)C]CP-424391 radioactivity remained localized only in the uvea.     

Characterization of water-soluble glucuronide and sulphate conjugates of aflatoxin B1. 2. Studies in primary cultures of rat hepatocytes

Aflatoxin B1 and some of its metabolites were released from water-soluble aflatoxin conjugates isolated from rat primary hepatocyte cultures and hydrolysed by enzymes (beta-glucuronidase and sulphatase), by acid or by a combination of both treatments. The presence of AFB1 in the hydrolysates was detected on TLC plates, or indicated indirectly by the Ames mutagenicity assay. The aflatoxin conjugates were not mutagenic to Salmonella typhimurium strain TA98 in the presence of rat-liver S-9 mix. However, following enzymatic hydrolysis, the chloroform extract of the hydrolysate was highly mutagenic to the bacteria, indicating the presence of mutagenic AFB1. The conjugates AFB1-glucuronide and AFB1-sulphate are therefore produced from AFB1 in primary cultures of rat hepatocytes.     

Comparative tissue distribution and excretion of [1-14C]acrylamide in beagle dogs and miniature pigs

Male beagle dogs and miniature pigs were given acrylamide in the diet for 3-4 wk at a dosage of 1 mg/kg/day. They were then given [1-14C]acrylamide as a single oral dose of 1 mg/kg. The animals were killed 6 hr or 1, 2, 4 or 14 days after administration of the radioactive compound and tissues were analysed for radioactivity. The radiolabelled material was distributed to a major extent in muscle tissue in both species (31-35% of the dose at 6 hr and 5-7% at 14 days). Although the nervous system is the primary target for acrylamide monomer toxicity, less than 1% of the administered 14C was found in the brain in both species. No neurotoxic signs were evident during the exposure period at the dosage used. Analysis of discrete areas of the brain for radioactivity revealed that the levels of penetration of [1-14C]acrylamide in brain paralleled the vascularization pattern of the tissues. Approximately 60% of the administered radiolabel was excreted in the urine in both species and smaller amounts were excreted in the faeces. However, recovery in the faeces was higher in pigs (c. 25%) than in dogs (c. 7%) and this and the considerably higher levels demonstrated in the gastro-intestinal tract of the pigs indicated that the absorption of acrylamide was more rapid and more extensive in dogs than in pigs.     

Butylate hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin B1 in primary hepatocyte cultures

Primary cultures of adult rat hepatocytes were used to characterize the effect of butylated hydroxytoluene (BHT) pretreatment on the metabolic disposition and cytotoxicity of a single dose of aflatoxin B₁ (AFB₁). Four male Sprague-Dawley rats were fed a control diet, and five were fed a diet containing 0.5% BHT. After 10 days, hepatocytes were prepared and cultured in chemically defined, hormone-supplemented medium. After 20–22 hr in culture, 120–150 ng of [¹⁴C]aflatoxin B₁ was added to dishes containing 2.5 × 10⁶ cells. By 10 hr, control cells had converted 59% of the AFB₁ to aqueous metabolites, while 15.5% was bound covalently. During the same interval, cells from BHT-fed rats produced 69% aqueous metabolites, and only 6.6% was bound covalently. The rate of AFB₁ disappearance in the two groups was not statistically different. AFB₁ produced marked cytotoxicity in hepatocyte cultures from control rats but had no apparent toxic effect on hepatocyte cultures from BHT-pretreated rats, as indicated by light microscopic examination and release of lactate dehydrogenase into the medium. These results suggest that reduction of cytotoxicity by BHT was associated with increased output of nontoxic, water-soluble metabolites and decreased binding of metabolites to macromolecules. These results also indicate that BHT may protect against the acute toxicity and carcinogenicity of aflatoxin B₁in vivo.