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.     

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.     

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.     

The neurotoxicity of aflatoxin B1 in the rat

The effects of repeated intraperitoneal administration of aflatoxin B₁ on the peripheral and central nervous systems of rats were investigated. Biochemical markers of neurotoxicity were monitored in nervous tissues following aflatoxin B₁ dosage and after the cessation of aflatoxin B₁ administration. Aflatoxin B₁ increased the activities of β-glucoronidase and β-galactosidase in the central and peripheral nervous systems. Repeated exposure of rats to aflatoxin B₁ also activated Na⁺ K⁺-ATPase and inhibited Mg²⁺-ATPase. Nervous tissue levels of DNA and total protein increased while the concentrations of RNA and phospholipid were depressed by aflatoxin B₁. The alterations in these parameters were specific for each of the tissues examined during the recovery of the rats. The findings indicate that the repeated administration of aflatoxin B₁ to rats results in degeneration in the central and peripheral nervous systems that may be related to the overt toxicity observed following aflatoxin administration.     

Effects of aflatoxin B1 on the activity of drug-metabolizing enzymes in rat liver

Aflatoxin B₁ (AFB₁) is one of the most active hepatotoxic and hepatocarcinogenic compounds known for rats. In order to evaluate the mechanism of action of the toxin on the liver, the effects of aflatoxin B₁ on the enzymes involved in its transformation, such as the monooxygenase-cytochromes P-450-dependent and conjugating enzymes, were studied. At the same time, liver damage was determined by measuring the activities of plasma γ-glutamyltransferase (GGT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) as well as the concentration of bilirubin.Male Sprague-Dawley rats received a single ip dose of AFB₁ (1 or 3 mg/kg). The effects of the toxic compound on the activity of drug-metabolizing enzymes were followed 20 days after this single exposure. Mortality (18%) within 7 days following administration was produced only by the higher dose. The same dose also significantly decreased the total level of hepatic proteins and impaired the activity of aminopyrine N-demethylase. AFB₁ lowered the content of cytochromes P-450 by 32 and 69% at the 1 and 3 mg/kg dose levels, respectively. Epoxide hydrase activity was increased by 121 and 170% at 1 and 3 mg/kg, respectively, whereas UDP-glucuronyltransferase activity was increased (44%) at 1 mg/kg, but also decreased at the same extent for the higher dose. The activity of GSH S-epoxide transferase was decreased by a maximum of 53% by 3 mg/kg AFB₁. Results obtained by the 3rd day following the administration of 3 mg/kg AFB₁ showed that blood levels of all the factors studied in this experiment were increased above control values, while at lower dose of AFB₁ (1 mg/kg), only the activities of AST and ALT were significantly increased. The activities of these enzymes were 27 to 42 times greater in rats treated with 3 mg/kg AFB₁ than in rats given 1 mg/kg AFB₁. Most of the biological features studied tended to return to control values between the 9th and 20th day after AFB₁ treatment. This study makes it possible to compare changes in tissue levels of drug-metabolizing enzymes at two doses of AFB₁. It can also be used to demonstrate any time lag or differing behavior of the serum enzymes, notably the sensibility of transaminases.     

Effect of repeated dietary exposure of aflatoxin B1 on brain biogenic amines and metabolites in the rat

Male Sprague-Dawley rats were treated po twice weekly for 3 weeks with a low (32.8 micrograms/kg) and high dose (327.9 micrograms/kg) of aflatoxin B1 (AFB1) in corn oil. A control group received corn oil only. At the end of the experiment the rats were killed, and the concentrations of the brain catecholamines, norepinephrine (NE) and dopamine (DA), catecholamine metabolites, 3-methoxy-4-hydroxymandelic acid (VMA), homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC), and the indoleamine serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were determined by high-pressure liquid chromatography in five brain regions. The major effects were found in striatal dopamine and serotonin concentrations, with decreases of 37 and 29%, respectively. A corresponding decline was observed in the dopamine metabolites, homovanillic acid (44%) and dihydroxyphenylacetic acid (30%). Concentrations of these neurotransmitters and metabolites were only marginally altered in cerebral cortex, cerebellum, hypothalamus, and medulla oblongata. It appears that a major effect of AFB1 is on dopaminergic pathways, possible by selectively perturbing the conversion of tyrosine to biogenic catecholamine neurotransmitters.     

Activity of some respiratory enzymes and cytochrome contents in rat hepatic mitochondria following aflatoxin B1 administration

Effects of aflatoxin B₁ (AFB₁) administration (7 mg/kg body weight i.p.) on rat hepatic mitochondrial respiratory components have been examined. Succinoxidase and cytochrome oxidase activities were decreased in liver mitochondria isolated from rats 12–24 h after AFB₁ treatment. Both enzyme activities returned to normal levels after 48 h. Glutamate dehydrogenase and β-hydroxybutyrate dehydrogenase activities did not show any alterations up to 24 h and thereafter increased at 48–72 h. Succinate dehydrogenase activity was impaired by 41% at 12 h and thereafter was found to be normal. The intramitochondrial cytochrome b content declined at 24–72 h, whereas cytochrome aa₃ content was decreased maximally at 72 h after AFB₁ administration. These observations on mitochondrial enzyme activities and cytochrome contents correlate well with our earlier observations made on hepatic mitochondrial respiratory rates after AFB₁ treatment. The impairment of respiratory functions possibly results from membrane damage and selective modification of gene expression in mitochondria imparted by AFB₁.     

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.     

Effect of phospholipases A2 and C on structure and phospholipids of the electroplax

Phospholipid hydrolysis after 30 min exposure to phospholipase A₂ or phospholipase C was determined in intact electroplax cells and in the separated conducting and non-conducting membranes. These enzymes, in concentrations of 0.2 and 2.0 mg per ml, caused approximately the same percentage hydrolysis of phosphatidylcholine phosphatidylethanolamine and phosphatidylserine (40–80%); in addition phospholipase C hydrolyzed sphingomyelin.Phospholipase A₂ (0.2 and 2.0 mg per ml) caused mitochondrial swelling, and a pinching off of the membrane inpocketings into clusters of small rounded vesicles external to the membrane. Lysophosphatidylcholine (2 mg per ml) caused some vesicular formation, although not nearly as numerous as with phospholipase A₂; and no mitochondrial alterations. Phospholipase C (2 mg per ml) caused some mitochondrial swelling, but no vesicle formation.Disruption by phospholipase C of hydrophilic or electrostatic interactions between phospholipids and proteins has less effect on the ultrastructural organization of the membrane than does disruption of hydrophobic interactions by phospholipase A₂. The results are discussed in relationship to the fluid mosaic model of membrane organization.     

The effects of aflatoxin B1 on some testicular and kidney enzyme activity in rat

The effects of chronic intra-peritoneal administration of aflatoxin B₁ on the activity of alkaline and acid phosphatases; glutamic oxaloacetate (GOT) and pyruvaate transaminases (GPT); 5′-nucleotidase and lactic dehydrogenase enzymes were monitored in the testis and kidney opf adult albino rats. Results showed that aflatoxin B₁ depressed the activity of alkaline phosphatase in both tissues, but increased that of acid phosphatase in only the testis. While GOT and 5′-nucleotidase were inhibited, GPT and lactic dehydrogenase activity was enhanced by this carcinogen. These responses were similar for the testis and kidney.The above findings coupled with the microscopical observation of the testis tissue seem to indicate that the essential lesion of this toxin on the testis may be a modification of the enzymes of germinal cells resulting from a gradual depletion of the latter. Furthermore, the results appear to show that by and large, aflatoxin B₁ exerts only slightly different effects on the testis and kidney at the enzyme level.     

Red ginseng extract protects against aflatoxin B1 and fumonisins-induced hepatic pre-cancerous lesions in rats

The current study was conducted to evaluate the chemoprevention effects of ginseng extract (GE) against pre-cancerous lesions in female Sprague–Dawley rats treated with aflatoxin B₁ (AFB₁) and fumonisin (FB). Six experimental groups treated for 12 weeks and included: the control group; the GE alone-treated group (150 mg/kg b.w); the group treated orally with AFB₁ (17 μg/kg b.w) during the first 2 weeks and fed FB-contaminated diet (250 mg/kg diet) during the 6th to 8th weeks; the group treated with GE during the mycotoxin protocol and continued till week 10; the group treated with GE 2 weeks before AFB₁ administration and continued till the end of FB treatment and the group treated with GE for 4 weeks after the toxin protocol stopped. The sequential mycotoxins treatment induced significant changes in serum biochemical parameters accompanied by severe histological and histochemical changes of the liver tissue. Treatment with GE during, before or after the treatment with the mycotoxins improved all biochemical parameters and histological picture of the liver. Moreover, treatment with GE after the administration of the mycotoxins was found to be more effective. It could be concluded that GE has a protective effects as pre-cancerous lesions and therapeutic effects as well.     

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.     

Effect of aflatoxin B1 on brain serotonin and catecholamines in chickens

A single oral dose of aflatoxin B₁ at 3 mg/kg body weight caused a significant increase in brain serotonin (5-HT) in 5-week-old chickens. Norepinephrine (NE) brain concentration significantly decreased, whereas the dopamine (DA) concentration remained unchanged. These results suggest that in modifying the concentrations of biogenic amines in the brain, aflatoxin B₁ may affect brain function.     

Changes in hepatic polyamine levels during acute and chronic administration of aflatoxin B1 to rats

A subacute dose of aflatoxin B₁ (3 mg/kg body weight) increases liver putrescine levels within 1 hr after administration, with high levels persisting over 24 hr. Higher doses of the carcinogen elicited larger increases in liver polyamine levels. A marked elevation of putrescine, spermidine and spermine were noted in aflatoxin B₁-induced preneoplastic liver. Pretreatment of rats with phenobarbital prior to aflatoxin B₁ administration resulted in no synergistic or additive effects.     

The comparative metabolism and toxic potency of aflatoxin B1 and aflatoxin M1 in primary cultures of adult-rat hepatocytes

Both aflatoxin B₁ (AFB₁) and a hydroxylated metabolite, aflatoxin M₁ (AFM₁), were potent cytotoxins and genotoxins to primary cultures of rat hepatocytes. However, AFB₁ stimulated the release of lactate dehydrogenase into the culture medium and the loss of viable cells from the monolayer at lower doses than did AFM₁. The lowest toxic doses of AFB₁ and AFM₁ were 0·05–0·1 and 0·6 μg/ culture, respectively. Genotoxicity, determined by an assay for stimulation of DNA repair, was apparent at lower doses than was cytotoxicity. AFB₁ was again more potent than AFM₁, stimulating DNA repair at 0·025 μg/culture. compared to the lowest genotoxic dose of AFM₁ of 0·05 μg/culture. At higher doses (1·2–2·4 μg/culture) the responses due to both aflatoxins in the cytotoxicity and DNA-repair assays were approximately equal. The metabolism of a low dose (c. 0·17 μg/culture) of [¹⁴C]AFB₁ and [³H]AFM₁ by cultured hepatocytes differed significantly. After 1 hr, 50% of the [¹⁴C]AFB₁ remained unchanged in the culture medium, whereas about 18 hr were required for the same amount of [³H]AFM₁ metabolism to occur. [¹⁴C]AFB₁ was metabolized to AFM₁, to polar metabolites recovered in the aqueous phase after chloroform extraction, and to metabolites covalently bound to hepatocyte macromolecules. [³H]AFM₁ was also metabolized to polar metabolites and to forms bound to macromolecules. The degree of covalent binding of the aflatoxins correlated with their cytotoxicity and genotoxicity at lower doses. After a 24-hr incubation, 12·5% of the dose of [¹⁴C]AFB₁ was covalently bound to macromolecules compared to 1·5% of [³H]AFM₁. Although AFM₁ was less potent than AFB₁ in cytotoxicity, DNA-repair and covalent-binding assays using primary cultures of hepatocytes, AFM₁ was still active at relatively low doses and therefore is probably a potent hepatotoxin in vivo.     

高效液相色谱-荧光检测法测定沉香中黄曲霉毒素B1、B2、G1、G2

目的建立高效液相色谱–光化学衍生–荧光检测法测定沉香药材中黄曲霉毒素B1、B2、G1、G2。方法采用高效液相色谱法,通过免疫亲和柱提取和净化,荧光检测器检测。Agilent Zorbax Ecilpse Plus C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇–水(45∶55);体积流量:0.8 m L/min;柱温:30℃;进样盘温度:4℃;荧光激发波长为360 nm,发射波长为450 nm。结果黄曲霉毒素B1、B2、G1、G2分别在9.3~74.4、3.0~24.0、9.3~74.4、3.5~28.0 pg线性关系良好,r均大于0.998 0;检测限分别为1.86、0.60、1.86、0.70 pg,定量限分别为7.44、2.40、7.44、2.80 pg。平均回收率分别为78%、92%、82%、99%,RSD值分别为4.4%、3.0%、4.3%、2.8%。结论所建立的方法结果准确、重复性、稳定性均良好,可用于沉香药材中黄曲霉毒素的质量控制。     

Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice

Aflatoxin B₁ (AFB₁)-mediated hepatic damage is involved in production of AFB₁-8,9-epoxide-bound DNA adducts and this is also affected by a pro-oxidant potential of the toxin. In this study we investigated the effects of quercetin on AFB₁-treated HepG2 cells. We also examined the biochemical mechanisms associated with the effects of quercetin on AFB₁-mediated liver damage in mice. Our results revealed that quercetin and isorhamnetin inhibit production of reactive oxygen species and cytotoxicity, and block the decrease of reduced glutathione (GSH) levels in AFB₁-treated HepG2 cells. Isorhamnetin have inhibitory ability on lipid peroxidation stronger than quercetin in the cells. Oral supplementation with quercetin decreased serum lactate dehydrogenase levels, increased hepatic GSH levels and superoxide dismutase activity, and reduced lipid peroxidation in both the liver and kidney in AFB₁-treated mice. However, quercetin did not show a significant reduction on serum levels of alkaline phosphate, alanine aminotransferase and aspartate aminotransferase that were increased in AFB₁-treated mice. HPLC analysis revealed that quercetin in plasma is mainly present as glucoronides and/or sulfates of quercetin. Collectively, it is suggested that quercetin does not directly protect against AFB₁-mediated liver damage in vivo, but exerts a partial role in promoting antioxidative defense systems and inhibiting lipid peroxidation.     

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.     

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.     

Essential oil composition and antioxidant activity of Thymus longicaulis C. Presl subsp. longicaulis var. longicaulis

This study is designed to examine the chemical composition and in vitro antioxidant activity of the hydrodistillated essential oil and various extracts obtained from Thymus longicaulis subsp. longicaulis var. longicaulis. GC and GC–MS analysis of the essential oil were resulted in determination 22 different compounds, representing 99.61% of total oil. γ-terpinene, thymol and p-cymene were determined as the major compounds of the oil (27.80, 27.65 and 19.38%, respectively). Antioxidant activities of the samples were determined by four different test systems namely β-carotene/linoleic acid, DPPH, reducing power and chelating effect. Essential oil showed the highest antioxidant activity in β-carotene/linoleic acid system among the experiments examined. In the case of other test systems, in general, methanol and water extracts exhibited the strongest activity profiles. Especially, reducing power of water extract was found superior than those of synthetic antioxidants. As well as the antioxidant activities of the extracts, they were evaluated in terms of their total phenolic and flavonoid contents. Hexane and water extracts were found to be rich-in phenolics. However, flavonoids were determined in the highest level in methanol extract.