Aflatoxin B1 and ethanol co-exposure induces hepatic oxidative damage in mice.

The present study investigated the effects of aflatoxin B1 (AFB1) and ethanol co-exposure on biomarkers of hepatic damage in mice. Four groups of adult male mice were treated for 7 consecutive days. Control mice received corn oil alone at a dose of 2 mL/kg bw. One group was treated with ethanol at a dose of 500 µL/kg bw and another group administered 9 mg/kg bw of AFB1 dissolved in corn oil. The fourth group was co-administered with ethanol and AFB1. The body and liver weights of treated mice decreased significantly when compared with corresponding control. Alone, ethanol and AFB(1) treatment separately increased serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP). Alcohol dehydrogenase (ALD) activity was markedly elevated in ethanol-treated mice but was unaffected by AFB1 treatment. Co-exposure of AFB1 and ethanol escalated the activities of these serum enzymes. Administration of ethanol and AFB1 separately resulted in significant decrease in both non-enzymatic antioxidant glutathione (GSH) level and enzymatic antioxidant catalase (CAT) and glutathione-S-transferase (GST) activities, whereas lipid peroxidation was markedly elevated. Superoxide dismutase activity and vitamin C level remained unaffected in all treatment groups. Co-exposure of animals to ethanol and AFB1 showed additive effects on the activities of GST and CAT as well as on the GSH level. Histopathological study revealed that these compounds interact together to exacerbate their individual effects on the liver. In summary, the data presented showed that AFB1 and ethanol co-exposure induced severe oxidative damage to the liver of mice and as such humans consuming excessive amount of ethanol and diets contaminated with AFB1 simultaneously may be at greater risk of the hepatotoxic effects of these compounds.

Influence of benzoflavone on aflatoxin B1-induced cytotoxicity, mutation, and transformation of C3H/10T1/2 cells.

Aflatoxin B1 (AFLB1), a metabolite of the fungus Aspergillus flavus, is hepatotoxic and hepatocarcinogenic in several animal species and is thought to play an etiological role in human liver cancer. C3H/10T1/2 clone 8 mouse embryo fibroblasts are killed, mutated, and morphologically transformed byAFLB1. 7,8-Benzoflavone, a known inhibitor of aryl hydrocarbon hydroxylase, inhibits this enzymatic activity in C3H/10T1/2 cells. Furthermore, benzoflavone inhibits the binding of AFLB1, to the DNA of C3H/10T1/2 cells. Benzoflavone also inhibits AFLB1-induced cytotoxicity and mutation of C3H/10T1/2 cells, as well as inhibiting the activation of AFLB1 into mutagenic metabolites capable of reverting the Ames Salmonella tester strain TA98. Interestingly, benzoflavone had no effect on the oncogenic transformation of these cells by AFLB1. Therefore, benzoflavone inhibits the DNA binding, cytotoxic, and mutagenic effects of AFLB1 but does not reduce the morphological transformation of C3H/10T1/2 cells by this mycotoxin.

Mutation of Chinese Hamster V79 cells and transformation and mutation of mouse fibroblast C3H/10T1/2 clone 8 cells by aflatoxin B1 and four other furocoumarins isolated from two Nigerian medicinal plants.

Mutation by aflatoxin B1 (AFB1), imperatorin, marmesin, chalepin, and 8-methoxypsoralen (MOP), with and without black light (BL; long-wavelength ultraviolet light) activation, was determined at the hypoxanthine-guanine phosphoribosyltransferase locus (8-azaguanine resistance) in Chinese hamster V79 cells and at the ouabain locus in mouse C3H/1OT1/2 cells. Transformation by these furocoumarins under the same activation conditions was also investigated in C3H/1OT1/2 cells. In V79 cells, AFB1 induced a 4-fold maximum mutation frequency over controls under BL activation at a concentration of 5 micrograms/ml; marmesin induced a 2-fold increased mutation frequency at 1.5 micrograms/ml; MOP induced a 19-fold increase at 10 micrograms/ml; chalepin induced a 3-fold increase at 5 micrograms/ml; and imperatorin induced a 20-fold increase at 10 micrograms/ml. Essentially no mutation was observed at the ouabain-resistant (Ouar) locus in C3H/1OT1/2 cells with any of these compounds. In the transformation assays, type II and type III foci were observed at a 1-microgram/ml addition of AFB1 with or without BL activation; while with MOP and imperatorin, these types of foci were observed only with BL activation. Marmesin, although relatively more cytotoxic than the other furocoumarins studied, with a 50% lethal dose of less than 0.5 micrograms/ml, was not as mutagenic or potentially carcinogenic as were AFB1, imperatorin, or MOP with BL activation. These furocoumarins are considered to be involved in the etiology of the high incidence of skin cancer in Nigeria. Our experiments reinforce that concept and suggest that exposure to these furocoumarins may constitute a real carcinogenic hazard.

The reduced mutagenicity of aflatoxin B1 due to hydroxylation: observations on five Salmonella typhimurium tester strains.

The mutagenicity of aflatoxin M1 relative to that of aflatoxin B1, the parent compound, was studied in 5 Ames' tester strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537, TA 1538). Aflatoxins B1 and M1 are both highly mutagenic in microsome-mediated system in TA 100. The prediction of the relative carcinogenicity of aflatoxin M1 to aflatoxin B1 posed by the mutation of TA 100 is probably more authentic than by TA 98.

Mutagenicity of chamuvaritin: a benzyldihydrochalcone isolated from a medicinal plant.

The mutagenic effects of chamuvaritin, dihydrobenzylchalcone isolated from Uvaria chamae, were investigated using Salmonella typhimurium tester strains TA92, TA94--98, TA100--1535, TA1537 and TA1538. The phytochemical was mutagenic in tester strains TA98 and TA100 and required activation by the hepatic S-9 microsomal enzyme preparation.

The presence of aflatoxin and some polycyclic aromatic hydrocarbons in human foods.

An analysis of several common food items (fish, meat, crops and spices) as sold in the Nigerian markets has shown the presence of (a) benzo[a]-pyrene and benz[a]anthracene in fish and meat samples, and (b) aflatoxin in crops and spices. These results are discussed in relation to the relatively high incidence of cancer in tropical Africa.

Isolation and partial characterization of pigeon pea protease inhibitor: its effect on the genotoxic action of aflatoxin B1.

Protease inhibitor was isolated and purified from pigeon pea Cajanus capan. By using gel filtration analysis the inhibitor was found to have an Mr of 18,200. It inhibits trypsin competitively with a specific inhibitor constant Ki of 1.53 x 10(-7) M. The purified inhibitor produced a marked reduction in aflatoxin B1-induced beta-galactosidase activity in Escherichia coli PQ37. This reduction is independent of whether the protease inhibitor was added to the reaction medium prior to or after aflatoxin B1 activation. The observed reduction may therefore be a result of the inhibitor's activity on the RecA protease produced in response to aflatoxin B1-induced DNA damage in the bacteria.

Inhibition of oxygen uptake in liver slices of three mammalian species (rat, rabbit, guinea-pig) by aflatoxin B1 (AFB1).

Oxygen uptake in liver slices of rats, rabbits and guinea pigs were determined manometrically in the presence of different concentrations of aflatoxin B1 (AFB1). AFB1 inhibited oxygen uptake at all concentrations of AFB1 tested (3.2 microM, 16.0 microM, 48.1 microM, 64.1 microM, 80.0 microM, 112.2 microM). Inhibition was directly proportional to the concentration of AFB1 inducing the inhibition. The degree of inhibition of oxygen uptake in the 3 mammalian species seems to correlate with their respective susceptibilities to AFB1 toxicity. The highest inhibition was in guinea pig and the least in rat; that in the rabbit was intermediate between rat and guinea pig.

Comparative effects of three naturally occurring furanocoumarins on mitochondrial bioenergetics.

Oxygraphic measurements of the rates of mitochondrial respiration in the presence of varying amounts of chalepin, imperatorin and marmesin, three naturally occurring furanocoumarins, revealed that the oxidation of NAD(+)-linked substrates was inhibited by chalepin and imperatorin and less significantly by marmesin. The order of potency being rotenone much greater than chalepin imperatorin greater than marmesin. There was no effect whatsoever on succinate oxidation by the furanocoumarins tested (up to 60 microM). State 3 respiration was also inhibited by these furanocoumarins; by at least 80% by 10 microM chalepin and by 48 and 29% with 60 microM imperatorin and 60 microM marmesin, respectively. Consequently, ADP control of respiration was diminished by those concentrations of furanocoumarins that inhibited respiration. At 60 microM, respiratory control ratio was reduced by about 88, 49 and 28% with chalepin, imperatorin and marmesin, respectively. A measurement of the rate of proton and Ca2(+)-movements across the mitochondrial coupling membrane demonstrated that succinate-supported transport was not affected by these furanocoumarins. On the other hand, pyruvate/malate-supported proton ejection was significantly inhibited by chalepin, imperatorin and marmesin. The order of the degree of inhibition of proton flux is rotenone much greater than chalepin greater than imperatorin greater than marmesin. The pattern of the inhibition of pyruvate/malate-supported Ca2(+)-transport was identical to that seen during proton transport. A comparison of the effects of chalepin to that of rotenone suggests that chalepin might be about 10 times less potent than rotenone.

In vitro effects of aflatoxin B1 on the activities of three hydrolases.

The in vitro effects of aflatoxin B1 on the activities of trypsin, amylase and lipase in the pancreatic extract of male albino Wistar rats have been studied. Activities of the 3 hydrolases were inhibited at 1 microgram/ml, 200 microgram/ml and 400 microgram/ml concentrations of aflatoxin B1, with the exception that at the 1 microgram/ml concentration the activity of lipase was stimulated. Lineweaver-Burk reciprocal plots indicate non-competitive inhibition of the 3 hydrolases.

Prophage induction by 4 antimalaria drugs (daraprim, fansidar, nivaquine and camoquine) and in combination with aflatoxin B1.

The abilities of 4 antimalaria drugs (Daraprim, Fansidar, Nivaquine and Camoquine) on their own and in combination with aflatoxin B1 to induce prophage in tester strains E. coli D21 and D22 were studied. The 4 drugs were found to induce prophage in the tester strains; Daraprim and Fansidar without the need for activation by liver mixed function oxidases (S9 microsomal fraction), while Nivaquine and Camoquine did require activation by this system. Aflatoxin B1 was used as a positive control in all the tests. The combined effects of each of the drugs with aflatoxin B1 were lower than that of the individual drugs acting alone. From these results, it may be concluded that the drugs may, on their own, pose a carcinogenic hazard to the population in the Nigerian environment exposed to them. In addition, the depression of prophage induction observed when the drugs were combined with aflatoxin B1 may be indicative of a common target site of action in the tester strains.

Effect of antiserum against aflatoxin B1-bovine serum albumin complex on aflatoxin B1-induced lysogenesis.

Escherichia coli K12 bacteria lysogenic for the lambda phage were used to study the effect of antiserum against aflatoxin B1-induced lysogenesis. The antiserum was obtained from rabbits immunized with water in oil emulsion of aflatoxin B1-bovine serum albumin complex (AFB1-BSA). A marked reduction in the degree of lysogenesis was observed when the antiserum was added to the reaction medium prior to microsomal enzyme activation of aflatoxin B1. There was no detectable effect when the antiserum was added after aflatoxin B1 activation. The result presented suggests that the antibodies in the AFB1-BSA antiserum can interact with aflatoxin B1 prior to its activation. This implies that an immune-protective effect can only be exerted if the antibodies intervene before activation.

Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens.

AIM: To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and / or culture) in order to determine which of the three PCR methods (ureA, glmM and 26-kDa, SSA gene) was most appropriate in the diagnosis of Helicobacter pylori (H pylori ) infection and also to evaluate the detection of a putative virulence marker of H pylori, the cagA gene, by PCR in biopsy specimens. METHODS: One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms. The PCR methods used to detect H pylori DNA directly from biopsies were the glmM, 26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS: Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods, while 68% of these were positive for the cagA gene. Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened. The remaining 41% were either positive for ureA gene only, glmM only, 26-kDa only, or ureA + glmM, ureA + 26-kDa, glmM + 26-kDa. Out of the 35% positive biopsies, 41% and 82% were positive by culture and CLO respectively, while all negative biopsies were also negative by culture and cagA. Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION: This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.

In vitro effects of a beer-foam stabilizer on the activities of three pancreatic hydrolases.

The chemical composition of a foam stabilizer and its in vitro effects on the activities of trypsin, amylase and lipase in the pancreatic extract of male albino Wistar rats have been studied. Activities of two of the three hydrolases (amylase and trypsin) were stimulated while that of lipase was inhibited. Lineweaver--Burk reciprocal plot indicates non-competitive inhibition of lipase.

Binding reaction of aflatoxin B1 with immunoglobulin G against aflatoxin B1-bovine serum albumin complex.

Polyclonal immunoglobulin G antibodies were raised against aflatoxin B1-bovine serum albumin complex and characterised. Antibodies against the complex were obtained after a single intradermal multiple site injection of water in oil emulsion of the complex into adult female albino rabbits. Equilibrium dialysis and Scatchard plot analysis of the interaction of the antibodies with aflatoxin B1 showed that the antibodies have high affinity for binding aflatoxin B1. The average number of binding sites on the antibody molecules for aflatoxin B1 is 1.74 +/- 0.20 with mean standard free energy change (delta F1(0) of -23.10 KJ/mol, while the average association constant is 2.35 +/- 0.73 x 10(-4)M-1. Male wistar strain albino rats after immunization with the complex showed lower mortality when challenged with a single dose of aflatoxin B1. The results suggest that such antibodies with high affinity for aflatoxin B1 could be used in the immunointerception of the toxin.

Influence of four Nigerian food additives on the mutagenicity of aflatoxin B1.

The mutagenicity of aqueous extracts of four Nigerian food additives namely Xylopia aethiopica (Xa), Monodora species (Ms); fermented Litrillus lanatus-ogiri (Og) and fermented Parikia africans (African locust bean)-Iru (Ir) alone and in combination with different concentrations of aflatoxin B1 (0.05 microgram-0.25 micrograms) in the presence and absence of fecalase was studied using the Ames' salmonella mutagenicity assay system. Preliminary screening tests show the tester strain TA98 to be the most sensitive of the four tester strains (TA97, TA98, TA100, TA102) screened. The most mutagenic of the doses of the extracts are 3mg each of Xa and Ms per plate and 5mg each of Og and Ir per plate. A combination of these doses with different concentrations of aflatoxin B1 resulted in an enhanced mutagenicity of aflatoxin B1. The increases could not be accounted for by additive mutagenicity of the extracts and aflatoxin B1. Fecalase further increased the effects resulting from these combinations with the exception of Xa which showed a decrease in mutagenic induction. The increase may be indicative of the presence of some mutagenic glycosides in the extracts.