Guanine alkylation by the potent carcinogen aflatoxin B1: quantum chemical calculations

We report a series of ab initio and density functional theory simulations of guanine alkylation by aflatoxin B 1 exo-8,9-epoxide. This reaction represents an initial step of carcinogenesis associated with aflatoxin B 1, a potent genotoxic fungal metabolite. Effects of hydration were considered in the framework of the Langevin dipoles solvation model and the solvent reaction field method of Tomasi and co-workers. In silico-calculated activation free energies are in good agreement with the experimental value of 15.1 kcal/mol. This agreement presents strong evidence in favor of the validity of the proposed S(N)2 reaction mechanism and points to the applicability of quantum chemical methods in studies of reactions associated with carcinogenesis. In addition, we predict that the preference of aflatoxin B 1 exo-8,9-epoxide over the endo stereoisomer for the reaction with guanine exists in the aqueous solution and is only further amplified in the DNA duplex. Finally, through comparison with an analogous reaction between 3a,6a-dihydrofuro[2,3- b]furan exo-4,5-epoxide and guanine, we show that the large planar body of aflatoxin B 1 does not enhance its reactivity and related carcinogenicity. This explains why the planar region of related mycotoxins sterigmatocystin and aflatoxin G 1 could have been evolutionarily optimized in a different way.     

DNA damage checkpoint response to aflatoxin B1

Although most countries regulate the aflatoxin levels in food by legislations, high amounts of aflatoxin B1 (AFB1)-DNA adducts can still be detected in normal and tumorous tissue obtained from cancer patients. AFB1 cannot directly interact with DNA unless it is biotransformed to AFB1-8, 9-epoxide via cytochrome p450 enzymes. This metabolite spontaneously and irreversibly attaches to guanine residues to generate highly mutagenic DNA adducts. AFB1-induced mutation of ATM kinase results in the deterioration of the cell cycle checkpoint activation at the G2/M checkpoint site. Genomic instability and increased cancer risk due to A–T mutation is the result of diminished repair of DNA double strand breaks. The major point mutation caused by AFB1 is G-to-T transversion that is related with the high frequency of p53 mutation. Majority of AFB1 associated hepatocellular cancer cases carry TP53 mutant DNA, which is an indicator of AFB1 exposure, as well as hepatocellular cancer risk.     

Carcinogenic risk assessment: ethylene dibromide.

Ethylene dibromide has been shown to result in an increased incidence of gastric tumors in rats following intubation at 40 mg/kg/day. The U.S. Environmental Protection Agency (EPA) has used a one-hit carcinogenic model with parameters derived from this bioassay in rats to estimate the risk of cancer in humans arising from inhalation exposure to ethylene dibromide. The EPA estimated an almost 100% lifetime incidence of cancer to be expected in workers exposed to 0.4 ppm of ethylene dibromide at citrus fumigation centers. This communication reports a test of the validity of that prediction based on a comparison of the incidence of cancer predicted by the method used by the EPA with that observed in a group of 156 workers employed in the production of ethylene dibromide. The one-hit model, as used by the EPA, predicted a total of 85 tumors above the normal background incidence in this group of employees. A total of eight tumors has been observed. Therefore, use of this model to predict carcinogenic response in humans appears to result in highly exaggerated risk estimates.     

Repair of rat liver DNA in vivo damaged by ethylene dibromide.

Tube feeding of [¹⁴C]Jethylene dibromide (EDB) to non-fasted rats resulted in the incorporation of the radioactivity into liver DNA, RNA and protein. Using alkaline and neutral sucrose gradients, it was observed that administration of the pesticide to non-fasted rat caused slower sedimentation of liver DNA in alkaline and not in neutral sucrose gradients. Slower sedimentation of liver DNA in alkaline sucrose gradients was apparent within 2 hr after the administration of a dose of 22 mg/100 g or 4 hr after a dose of 7.5 mg/100 g of body weight. Using a dose of 7.5 mg/100 g, the EDB-induced liver DNA damage was repaired significantly by 17.5 hr and almost completely by 96 hr. Administration of diethyldithiocarbamate, a free radical scavenger, did not inhibit liver DNA damage caused by EDB.     

Two cases of ethylene dibromide poisoning

Ethylene dibromide (EDB) is commonly available as a liquid pesticide for use as fumigant and preservative for storage of cereals and grains in India. Accidental or suicidal ingestion is often associated with often fatal delayed sudden hepatic or renal failure. We report 2 cases of EDB poisoning in humans.     

Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels

This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 μg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 μg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health.     

Lophirones B and C prevent aflatoxin B1-induced oxidative stress and DNA fragmentation in rat hepatocytes

Context Despite the reported anticarcinogenic activity of lophirones B and C, no scientific information exists for its activity in rat hepatocytes.

Objective Effect of lophirones B and C on aflatoxin B₁ (AFB₁)-induced oxidative stress, and DNA fragmentation in rat hepatocytes was investigated.

Materials and methods Wistar rat hepatocytes were incubated with lophirones B and C (1?mg/mL) or sylimarin (1?mg/mL) in the presence or absence of AFB₁. For an in vivo study, rats were orally administered with lophirones B and C, and/or AFB₁ (20?μg/d) for 9 weeks.

Results Lophirones B and C lowered AFB₁-mediated increase in nitric oxide, superoxide anion radicals, caspase-3 and fragmented DNA. Lophirones B and C attenuated AFB₁-mediated decrease in superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and reduced glutathione. Also, lophirones B and C attenuated AFB₁-mediated increase in conjugated dienes, lipid hydroperoxides and malondialdehyde in rat hepatocytes. Furthermore, AFB₁-mediated alterations in alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin, total bilirubin and globulin in rat serum were significantly annulled in lophirones B and C-treated rats.

Conclusion This study revealed that lophirones B and C prevented AFB₁-induced oxidative damage in rat hepatocytes.     

Influence of aflatoxin B 1 and aflatoxin B 2 on rat liver lysosomal acid deoxyribonuclease

The in vitro effects of aflatoxins B₁ and B₂ were studied on the permeability of isolated rat liver lysosomes. Only aflatoxin B₁ altered the lysosomal membrane with the consequent release of lysosomal enzymes. The results of the in vivo experiment with aflatoxin B₁ show that the specific activity of acid DNase in liver lysosomes was markedly decreased in the rats dosed aflatoxin B₁ while the specific activity of the cytoplasmic acid DNase, or nonsedimentable acid DNase, was dramatically increased. The results are discussed in relation to a hypothesis concerning the role of lysosomes in carcinogenesis.     

Effects of chronic exposure to aflatoxin B1 and aflatoxin M1 on the in vivo covalent binding of aflatoxin B1 to hepatic macromolecules

Induction of resistance to aflatoxin B1 (AFB1) binding to cellular macromolecules in the rat by chronic exposure to AFB1 and aflatoxin M1 (AFM1) was investigated. The binding of [14C]AFB1 to liver macromolecules was measured in F-344 rats fed 0.5 ppb or 50 ppb AFM1 or 50 ppb AFB1 for 41 wk. The animals then received an intragastric dose of [14C]AFB1 at 5 micrograms/kg and were sacrificed 6 h later. Hepatic DNA, RNA, and protein were isolated by chloroform-phenol extraction and hydroxylapatite chromatography. In animals preexposed to 50 ppb AFB1, labeled AFB1 binding to DNA, RNA, and protein was decreased by 72%, 74%, and 61%, respectively. Preexposure to AFM1 resulted in a small reduction in binding to nucleic acids. Glutathione transferase activity was increased by 133% in animals fed 50 ppb AFB1, by 48% in those preexposed to 50 ppb AFM1, and remained at control values in rats fed 0.5 ppb AFM1. These results suggest that the induction of detoxification enzymes following chronic exposure to aflatoxin might contribute to the reduction in covalent binding of AFB1 to macromolecules.     

Role of diethyldithiocarbamate in ethylene dibromide metabolism and covalent binding

Diethyldithiocarbamate (DDC) pretreatment of rats prevented the ethylene dibromide (EDB) depression of liver glutathione (GSH) 2 hr after toxication. The levels of cytochrome P-450 seem to by unaffected by EDB. The steady-state kinetics of the reaction of GSH with [U-¹⁴C]EDB in vitro, in the presence of glutathione-S-transferase, was determined by GSH utilization and the formation of a labeled nonvolatile product. The inhibition of the enzyme by DDC is noncompetitive producing a change in V_max without a change in K_m. DDC also inhibited the covalent binding of [¹⁴C]EDB to microsomal proteins in the microsomal system supplemented with NADPH. EDB bound to microsomal protein in the absence of NADPH. The role of DDC in the metabolism and covalent binding of EDB to macromolecules is discussed.     

Clinical features of aflatoxin B1-exposed patients with liver cancer and the molecular mechanism of aflatoxin B1 on liver cancer cells

Aflatoxin B1 (AFB1) induces hepatocellular carcinoma (HCC) through consumption of contaminated food in Southern China. Aldo-keto reductase-7A (AKR7A) functionally plays a potent role in the biodetoxification in the liver. In addition, hepatocellular lipid disorder has found to be closely linked to the development of HCC. This study was, therefore, designed to investigate the potent bioeffect of AKR7A on the lipid metabolism in AFB1-exposed hepatocellular carcinoma cells through assaying human cancerous samples and cell culture. In the baseline data, the HCC patients showed increased contents of AFB1 in sera and cancerous samples. In the clinical parameters, the HCC patients demonstrated changed lipid settings in sera. As revealed by immunostaining and immunoblotting, AFB1-elevated HCC sections showed marked down-regulation of AKR7A expression, accompanied with reduced ApoB expression and increased CD36, S6K1 expressions in the HCC. Studies in the human hepatocarcinoma line HepG2 also showed AFB1-exposure to increase ApoA1, LDL, TC, and TG contents; induce cell proliferation; and reduce hepatocellular AKR7A expression. Furthermore, AKR7A bioactivity was inactivated after treatment with perfluorooctane sulfonate (PFOS), an ApoB activator, in AFB1-dosed HepG2 cells. Collectively, our current findings suggest that hepatocellular AKR7A has a protective role against AFB1-induced cytotoxicity through the regulation of CD36, S6K1 and ApoB expression through the reduction of lipid utilization in malignant liver cells.     

Activation of alpha(1)-adrenergic receptors potentiates the nephrotoxicity of ethylene dibromide

Ethylene dibromide (EDB) has been used as a model compound for eliciting hepato- and nephrotoxicity. Conjugation with glutathione (GSH) has been shown to play a role in the bioactivation of EDB. The aim of this study was to determine whether activation of alpha(1)-adrenergic receptors, which causes a decrease in cellular GSH levels, could modulate the nephrotoxicity of EDB. For this purpose, male ICR mice were treated with EDB and/or the alpha-adrenergic agonist, phenylephrine (Pe), or the alpha-adrenergic antagonist, phentolamine (Phe). Animals treated with EDB (40 mg/kg, i.p.) had a 9.3-fold increase in urinary gamma-glutamyltranspeptidase (GGTP: EC 2.3.2.2) activity and a 38% decrease in renal non-protein bound sulfhydryl (NPSH) levels; however, animals co-treated with EDB and Pe (50 mg/kg, i.p.) exhibited a 27.8-fold increase in urinary GGTP activity and a 60% decrease in NPSH levels. The enhanced presence of urinary GGTP and decrease in cellular levels of NPSH was nearly blocked by treating animals concomitantly with EDB and Phe (10 mg/kg, i.p.) or EDB, Pe, and Phe. Histopathological examination revealed the enhanced degree of tissue damage and necrosis following treatment with EDB and Pe, and the protective effect of Phe at ameliorating EDB toxicity. These results indicate that factors that can influence alpha-adrenergic receptors may be critical in assessing dose-response data used in the risk assessment process.     

Binding of aflatoxin B1 to melanin

Whole-body autoradiography of pigmented C57BL mice injected with 3H-labelled aflatoxin B1 (AFB1) showed a high degree of labelling of melanin-containing tissues, such as the melanin of eyes and hair follicles. The corresponding tissues in albino NMRI mice contained only low amounts of the label. Liquid chromatography of extracts of pigmented mouse eyes showed the presence of only non-metabolized AFB1. Analysis of the binding of AFB1 to melanin from bovine eyes using the method of Scatchard revealed two classes of binding sites. Incubations in the presence of metallic cations did not affect the binding, indicating that ionic forces play no role in the affinity. Comparisons of the binding of AFB1 to the bovine-eye pigment in media containing n-propanol, ethanol or methanol indicated that hydrophobic interactions play a role in the affinity. Apposition of the aromatic rings in AFB1 and the indole nuclei of the melanin may also result in van der Waals' forces, and the combination of these two types of forces may underly the binding of AFB1 to melanin. The melanin binding implies that AFB1 is retained in pigmented tissues at higher concentrations than in any other tissue of the body. The biological implication of this accumulation is now known, but the possibility that there may be an increased risk for the induction of melanomas is discussed.     

Inhalation of ethylene dibromide during gestation by rats and mice.

Pregnant rats and mice inhaled ethylene dibromide (EDB) at concentrations of 20, 38, and 80 ppm for 23 hr a day. The exposures started on Day 6 of gestation and lasted for a total of 10 days. Observations were made on maternal welfare and fetal development. The results of this study indicated that: (1) EDB was more toxic to pregnant mice than pregnant rats; (2) adverse effects on maternal welfare, as measured by weight gain, feed consumption, and survival, were observed in both mice and rats; and (3) morphological changes were observed in fetuses from dams exposed to EDB. However, the latter occurred at concentrations that also affected maternal welfare. Consequently, EDB was judged to have little primary effect on development in this study.     

Toxic exposure to ethylene dibromide and mercuric chloride: Effects on laboratory-reared octopuses

The effects of acute and chronic exposure to either ethylene dibromide (EDB) or mercuric chloride (MC) were studied in laboratory-reared Octopus joubini, O. maya and O. bimaculoides. The advantages of using octopuses were that the responses were immediate, highly visible and sensitive. All species demonstrated signs of toxicity to acute and chronic exposure to EDB and to MC. A dosage-sensitive relationship for the loss and subsequent recovery of locomotor response and of chromatophore expansion was found for each species after acute exposure. For each species the LC₅₀ for chronic exposure occurred within 12 hr at 100 mg/l for EDB and within 3 hr at 1.000 mg/l for MC. This study demonstrated the potential usefulness of laboratory-reared octopuses in evaluating the toxicity of marine environmental pollutants.     

Aflatoxin B1 and aflatoxin M1 induced cytotoxicity and DNA damage in differentiated and undifferentiated Caco-2 cells

Aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) are natural mycotoxins that frequently present in food and feed and pose risks to human health. There are few data in the literature regarding the impairment of them in the intestine. Therefore, the present study investigated their cytotoxic effect on Caco-2 cells, especially the differentiated ones that resemble mature small intestinal enterocytes. Both undifferentiated (UC) and differentiated (DC) cells were treated with AFB1 and AFM1 at various concentrations for up to 72 h. Cell viability, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and DNA damage were determined. Data showed that AFB1 and AFM1 significantly inhibited UC and DC cell growth, increased LDH and caused genetic damage in a time- and dose-dependent manner (p < 0.05). In comparison, AFB1 was found to be more toxic than AFM1 on both UC and DC. All these cytotoxic outcomes might be associated with intracellular ROS generation, leading to membrane damage and DNA strand break. Additionally, DC was found to be more sensitive to aflatoxins, which might be due to the alteration of enzymes during cell differentiation. The present study provided the first in vitro evidence of DNA damage of DC induced by AFB1 and AFM1.     

Temperature-modulated aflatoxin B1 hepatic disposition and formation and persistence of DNA adducts in rainbow trout.

Previous work showed that the incidence of chemically induced tumors in fish increased with environmental temperature. The present study assessed genotoxicity as a mechanism to explain such results. Rainbow trout (2 g) were acclimated to 10 or 18 degrees C for 1 month and then immersed in 0.1 ppm [3H]aflatoxin B1 (AFB1) solutions for 30 min at their respective acclimation temperatures, or at 14 degrees C. The total radioactivity in liver immediately after immersions was 50% higher for 18- than 10 degrees C-acclimated and exposed fish. Conversely, adduction of [3H]AFB1 to hepatic DNA of 10 degrees C-acclimated fish was higher than that of 18 degrees C-acclimated fish after exposure. A similar DNA adduction result was observed after [3H]AFB1 immersion concentrations were varied to achieve similar hepatic [3H]AFB1 equivalents. After acute shifts in temperature (10 to 14 degrees C or 18 to 14 degrees C), no differences were found in hepatic [3H]AFB1 equivalents. However, adduction of [3H]AFB1 to hepatic DNA was higher for 10- than for 18 degrees C-acclimated fish 1 day after exposures at 14 degrees C. This was probably explained by effects of temperature acclimation on the status of the membrane-bound cytochrome P-450 system, affecting its competition with a detoxifying cytosolic enzyme. After [3H]AFB1 immersions at respective acclimation temperatures, or acute temperature shifts to 14 degrees C, DNA adducts were less persistent in fish maintained for 21 days at 18 degrees C than at 10 degrees C. Our data demonstrated that temperature-modulated AFB1 genotoxicity occurred via three mechanisms: hepatic disposition and formation and persistence of DNA adducts.