Mutagenicity of the products obtained from heated milk systems

Methylene chloride extracts of the browning reaction products prepared from model systems consisting of major milk components (casein and/or lactose, and non-fat dried milk) were tested for mutagenicity in the Ames Salmonella/microsome assay. Samples obtained by heating aqueous solutions of these components under either neutral or basic (pH 10) conditions exhibited no significant mutagenic activity when tested with or without S-9 mix. The addition of common food additives, such as sodium nitrite, butylated hydroxyanisole and butylated hydroxytoluene, to the aqueous solutions did not enhance the mutagenic activity of the browning samples. On the other hand, the tar samples prepared by heating the same milk components in the dry state exhibited strong mutagenicity, primarily to Salmonella typhimurium strain TA98 and only with S-9 mix. A casein/lactose mixture and non-fat dried milk were also heated with baking soda in the dry state. The presence of the baking soda enhanced the mutagencity of the browning products; the tar from the non-fat dried milk heated with baking soda was the most potently mutagenic of all the samples towards strain TA98 and also produced a positive response in strain TA100 in the presence of S-9 mix.     

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.     

Short-term pathological and proliferative effects of butylated hydroxyanisole and other phenolic anti-oxidants in the forestomach of Fischer 344 rats

Food grade butylated hydroxyanisole (BHA) when incorporated in the diet and fed to male Fischer 344 rats for 9 or 27 days induced proliferative squamous epithelial changes in the lesser curvature of the forestomach proximate to the glandular stomach. These changes were assessed histopathologically and by [methyl-³H]thymidine radioautography. It was shown that BHA mixed dry into powdered diet, incorporated into the diet in corn oil, or in a pelleted diet, induced similar effects. When levels of 2%, 1%, 0.5%, 0.25%, 0.1% and 0% BHA were incorporated in rat diet for 9 days the proliferative effect appeared to show a no effect level at 0.25% based on the [methyl-³H]thymidine-labelling index. Other food use antioxidants, namely butylated hydroxytoluene or tertiary butylhydroquinone, induced a lesser response that BHA at the maximum dose employed in the study. Propyl gallate was without effect. Propyyl-4-hydroxybenzoate, a food use phenol, on the other hand, induced a less pronounced response than BHA but was more effective than the other antioxidants. Because increased cellular proliferation often provides an optimal milieu for tumor formation, it is suggested that these observation may ne relevant to rat forestomach tumors induced by BHA.     

Effect of dietary antioxidants on benzo[a]pyrene metabolism in rat liver microsomes

Feeding of rats with 1% ethoxyquin (EQ) and butylated hydroxytoluene (BHT) but not butylated hydroxyanisole (BHA) increases the formation rate of benzo[a]pyrene (BP)-4,5-dihydrodiol from BP in hepatic microsomes. The production of other BP-dihydrodiols and of BP phenols is decreased after treatment with EQ, BHT and BHA. EQ and BHT are more effective than BHA in inducing epoxide hydrolase (EH) activity towards styrene oxide as the substrate.     

Influence of gallic acid esters on drug-metabolizing enzymes of rat liver

The effect of three antioxidants, propyl, octyl and dodecyl gallate, on hepatic drug metabolism in male rats was studied in vivo and in vitro. When fed at a dietary concentration of 1% for 14 days, only dodecyl gallate increased relative liver weight. Cytochrome P-450 content was not influenced, but a slight increase in cytochrome b₅ content was observed after the feeding of propyl gallate. Monooxygenase activity (benzo[a]pyrene-hydroxylase and ethoxycoumarin-deethylase activities) was not affected by propyl or octyl gallate. but a significant decrease in benzo[a]pyrene-hydroxylase activity was apparent in rats fed dodecyl gallate. Study of benzo[a]pyrene-metabolite formation in liver microsome preparations from control and propyl gallate-treated rats showed an overall decrease in metabolite production following gallate treatment, the decrease being statistically significant for the formation of the 9,10-dihydrodiol. Epoxide-hydratase activity was enhanced by a factor of 1·5 in rats fed propyl gallate; glutathione-transferase activity was unaffected. In vitro, the gallates proved to be potent inhibitors of ethoxycoumarin deethylation in liver microsomes from untreated and phenobarbital-treated rats; however, when cytochrome P-448 had been induced by pretreatment with 3-methylcholanthrene, ethoxycoumarin deethylase was less sensitive to the inhibitory action of the gallates.     

Effect of synthetic antioxidants on hydrogen peroxide formation, oxyferro cytochrome P-450 concentration and oxygen consumption in liver microsomes

Synthetic antioxidants lead in vitro to increased H2O2 formation in rat liver and lung microsomes and in guinea pig and hamster liver microsomes. Butylated hydroxyanisole and ethoxyquin are more potent than propyl-, octyl-, and dodecyl gallate; butylated hydroxytoluene is only weakly active. Extra production of H2O2 is maximal at antioxidant concentrations between 50 and 500 microM and is dependent on the concentration of NADPH. It is paralleled by increased microsomal oxygen consumption and decreased concentration of oxycytochrome P-450 and is enhanced by pretreatment of the animals with phenobarbital. Both the endogenous and the antioxidant-stimulated H2O2 production are inhibited by metyrapone. In vivo administration of ethoxyquin and butylated hydroxyanisole in the diet leads to decreased oxycytochrome P-450 concentrations but not to increased H2O2 formation in liver microsomes. No extra production of H2O2 was observed in a glucose oxidase or xanthine oxidase system; rather, inhibition occurred in the latter system. Our data suggest that antioxidants enhance the oxidase function of cytochrome P-450. This effect is discussed in view of the known toxicity of these food additives.     

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.     

Effect of micronutrients,antioxidants and related compounds on the mutagenicity of 3,2′-dimethyl-4-aminobiphenyl,a colon and breast carcinogen

The possible antimutagenic effects of butylated hydroxytoluene (BHT), disulfiram, indole-3-carbinol, indole-3-acetonitrile, sodium selenite and α-tocopherol on 3,2′-dimethyl-4-aminobiphenyl-induced mutagenicity were studied using the Ames Salmonella/mammalian microsome assay system with strains TA98 and TA100. All seven compounds were nonmutagenic in both bacterial tester strains. The addition of 50–250 μg of sodium selenite, 5–50 mg of α-tocopherol of 50–250 μg of BHT per plate inhibited DMAB-induced mutagenicity in TA98 and/or TA100. Ethoxyquin, disulfiram and indole-3-carbinol increased DMAB-induced mutagenicity in TA100, whereas these compounds had little or no effect in TA98. Indole-3-acetonitrile had very little effect in either strain.     

Inhibition by plant phenols of benzo[a]pyrene-induced nuclear aberrations in mammalian intestinal cells: a rapid in vivo assessment method

The polycyclic aromatic hydrocarbon, benzo[a]pyrene, induced dose-related nuclear damage (micronuclei, pyknotic nuclei and karyorrhectic bodies) in colonic epithelial cells of C57BL/6J mice within 24 hr when administered intrarectally in single doses of 0-200 mg/kg body weight. This damage was reduced when mice ingested the plant phenols, caffeic, ferulic and ellagic acids, and quercetin at levels of 4% or BHA at 2% (w/w) in the diet for 1 wk prior to the benzo[a]pyrene challenge (100 mg/kg body weight). Benzo[a]pyrene-induced nuclear damage was not significantly inhibited by 4% curcumin under similar conditions. The inhibition of nuclear damage is consistent with reported antimutagenic effects for these agents in vitro and in longer term animal studies. The procedure described here may provide a rapid in vivo method for assessing the potential of natural products to inhibit the carcinogenic process.     

Effects of dietary fat level and aflatoxin B1 treatment on rat hepatic lipid composition

Sprague-Dawley rats were given either ten daily doses of aflatoxin B₁ (AFB₁) or the solvent tricaprylin intragastrically over a 2-wk period and were fed diets containing either 1.6 or 20% corn oil throughout the study. Hepatic lipid composition was analysed in groups of five rats both 3 and 13 wk after the start of treatment, in order to determine short-term and longer-term alterations. Total lipid and cholesterols (total, free and esterified) increased on the high-fat diet at wk 3. At wk 13 only total and esterified cholesterol were increased by 20% corn oil. AFB₁ treatment resulted in large intra-group variations in total lipid and cholesterol at wk 3, but these were no longer apparent by wk 13. AFB₁ produced various alterations in the fatty acid composition of hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE), apparent at wk 3 but not at wk 13. The unsaturation index decreased but no changes were seen in the saturated fatty acids. Only in animals fed 20% corn oil did AFB₁ result in significant changes in 18:2, 20:3 and 22:6 fatty acids, while 20:4 and 22:5 tended to decrease and 18:1 to increase in response to AFB₁ treatment with both diets in both phospholipids. The high-corn oil diet was found to increase 18:2, 22:6, and total unsaturation in PC and PE, while the ratio of 20:4 to 18:2 tended to decrease in these phospholipids, γ-Glutamyltranspeptidase, an indicator of liver damage, was significantly increased in AFB₁-treated animals, with the greatest increase over controls in those fed the high-fat diet.     

The acute toxicity of butylated hydroxytoluene and its metabolites in mice

The acute toxicity of butylated hydroxytoluene (BHT) and four of its metabolites, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien- 1-one(BHT-OOH), 2,6-di-tert-butyl-4-hydroxy-4-methyl-2,5-cyclohexadien- 1-one(BHT-OH), 2,6-di-tert-butyl-p-benzoquinone (DBQ), and 2,6-di-tert- butyl-4-[(methylthio)methyl] phenol (BHT-SCH₃) was studied in young male mice following intraperitoneal administration. The i.p. LD₅₀ values of BHT, BHT-OOH, BHT-OH, DBQ, and BHT-SCH₃ were 3550,190, above 1600, 2270, and 1840 $\text{mg}\text{kg}$, respectively. These results suggest that BHT-OOH probably is the most toxic metabolite of BHT.     

Kinetics of 3-tert-butyl-4-hydroxyanisole (BHA) in man

High-resolution capillary gas chromatography-mass spectrometry with selective ion monitoring and using deuterated 3-tert-butyl-4-hydroxyanisole (BHA) as an internal standard was used to measure BHA in the plasma and urine of human volunteers after oral administration of 30 or 5 mg of the compound in olive oil. Pharmacokinetic studies showed similar plasma-concentration profiles in subjects treated with either level of BHA. About 20% of the administered dose was excreted as BHA glucuronide in the urine within the first 24 hr.     

Chemistry,antioxidant and antimicrobial investigations on essential oil and oleoresins of Zingiber officinale

The essential oil and oleoresins (ethanol, methanol, CCl₄ and isooctane) of Zingiber officinale were extracted respectively by hydrodistillation and Soxhlet methods and subjected to GC–MS analysis. Geranial (25.9%) was the major component in essential oil; eugenol (49.8%) in ethanol oleoresin, while in the other three oleoresins, zingerone was the major component (33.6%, 33.3% and 30.5% for, methanol, CCl₄ and isooctane oleoresins, respectively). The antioxidant activity of essential oil and oleoresins were evaluated against mustard oil by peroxide, anisidine, thiobarbituric acid (TBA), ferric thiocyanate (FTC) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging methods. They were found to be better antioxidants than butylated hydroxyanisole (BHA). The antimicrobial properties were also studied using various food-borne pathogenic fungal and bacterial species. The essential oil and CCl₄ oleoresin showed 100% zone inhibition against Fusarium moniliforme. For other tested fungi and bacteriae, the essential oil and all oleoresins showed good to moderate inhibitory effects. Though, both essential oil and oleoresins were found to be effective, essential oil was found to be better than the oleoresins.     

Toxicological evaluation of compounds found in food using rat renal explants

A rat kidney expiant system was developed and its respiratory characteristics in the presence and absence of a series of compounds found in food were studied. Cubes taken from the whole kidneys of male Osborne-Mendel rats were incubated under sterile conditions for up to 18 hr. The respiratory activity of these expiants after incubation was measured by determining the amount of ¹⁴CO₇ evolved as a result of the metabolism of [¹⁴C]glucose. Changes in membrane permeability as a result of treatment were assessed by measuring the total protein content in the medium. Measurements of respiratory activity showed that the biochemical integrity of the control expiants was well maintained over the test period. Various compounds were tested at a range of concentrations from 0·1 to 1·0 mM. In the initial screen, several of the compounds tested at 1mM—butylated hydroxytoluene (BHT), butylated hydroxy-anisole (BHA), DDT, polychlorinated biphenyl (Aroclor 1254), patulin, zearalenone and cadmium chloride—markedly inhibited respiration; neither ascorbic acid nor caffeine was effective at this concentration. Two of the fat-soluble compounds, DDT and zearalenone, inhibited respiration at concentrations of 0·1 and 0·25 mM concentrations, respectively, while patulin, a water-soluble mycotoxin, did not give significant inhibition at concentrations below 0·5 mM. In some cases, e.g. patulin and BHA, the dose-response curves appeared to be bimodal, with stimulation of respiration occurring at lower concentrations. The two mycotoxins, patulin and zearalenone, and the antioxidant BHA produced losses of cellular protein at concentrations of 1·0, 0·50 and 1·0 mM, respectively, indicating membrane perturbation. Thus, this kidney expiant system responded consistently to a variety of known toxins and can be considered as another in vitro method of determining which compounds show the potential for in vivo toxicity.     

Effect of butylated hydroxytoluene,curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

1.?The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes.

2.?Treatment of human hepatocytes for 72 h with 2–200 µM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 µM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 µM BHT.

3.?In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels.

4.?The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6β-hydroxylase activity.

5.?In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.     

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

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

A carcinogenicity study with mutagenic organic concentrates of drinking-water in the Netherlands

The carcinogenicity in male and female Wistar SSP TOX rats of organic drinking-water concentrates that are positive in the Ames test was studied at three doses. The organic mutagenic concentrates were prepared weekly from drinking-water from one location in The Netherlands by adsorption onto XAD-4/8 resins and elution with dimethylsulphoxide. The organic concentrates in dimethylsulphoxide were mixed with non-mutagenic drinking-water before exposure of the rats. Dose levels were based on multiples of expected human exposure levels. For the calculation the average human daily intake of drinking-water was taken as 2 litres for a body weight of 70 kg. There was no significant increase in tumour induction when male Wistar SSP TOX rats were exposed for 106 wk to 4.5, 14 or 40 times the expected human exposure level and females to 7,22 or 68 times the human level. The development and types of tumours were similar in the treated and control groups. The numbers of animals with tumours and of animals that died as a result of tumours in the exposed groups did not differ significantly from those in the control groups. These results suggest that these organic mutagenic drinking-water concentrates did not contain very potent carcinogens in effective concentrations.     

Dietary antioxidant effects on the hepatic mixed-function oxidase system of rainbow trout (Salmo Gairdneri)

Rainbow trout (Salmo gairdneri) were fed a control diet with or without an antioxidant--3,5-di-tert-butyl-4-hydroxytoluene (BHT), 2(3)-tert-butyl-4-hydroxyanisole (BHA), mono-tert-butylhydroquinone (TBHQ) or ethoxyquin (EQ)--at a level of 5.56 mmol in 100 g oil/kg diet for 6 wk. The treated trout had reduced liver weight/body weight ratios. In comparison with trout fed control diet, microsomal protein content was lowered by 13% in TBQH-fed trout and elevated by 28% in EQ-fed trout, cytochrome P-450 content was 21% lower in BHA- and TBHQ-fed and 18% lower in EQ-fed trout and cytochrome b5 content was 46% lower in EQ-fed trout. Activities of benzo[a]pyrene hydroxylase, epoxide hydratase and ethoxycoumarin-O-deethylase were, respectively, 3.2-4.8, 1.2-1.7 and 1.3-5.5 times higher in antioxidant-fed trout. NADPH-cytochrome c reductase was elevated 1.2-1.3 times over the control value with dietary BHA, TBHQ and BHT, but was lowered with EQ. p-Nitroanisole-O-demethylase activity was completely suppressed in antioxidant-fed trout. The content of post-mitochondrial acid-soluble sulphydryl groups was 42% lower in BHA- and BHT-fed trout. Alterations in the enzyme activities of the mixed-function oxidase system, changes in the ethyl isocyanide binding ratio and decreases in cytochrome P-450 content suggest that dietary antioxidants could alter carcinogen activation and/or detoxification mechanisms in the hepatic microsomes of rainbow trout.     

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

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

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

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