Comparative biotransformation of aflatoxin B1 in swine,domestic fowls,and humans

Aflatoxin B₁ (AFB₁) is primarily biotransformed in the liver by cytochrome P450 (CYP) enzymes, which can yield either the genotoxic metabolite AFB₁-8,9 epoxide that causes liver carcinogenicity or less toxic compounds. The biotransformation of AFB₁ is better understood in humans, including gene expression of CYPs involved in the detoxification process. Studies on farm animals have demonstrated genes homologous to human CYPs that play similar roles in AFB₁ biotransformation. This review compares the activities of the most important CYPs related to the biotransformation of AFB₁ in humans, swine and domestic fowls (chickens, quail, turkeys and ducks), as well the main detoxification mechanisms in these species.     

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 of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B₁-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB₁ to the exo-AFBO and to detoxification products aflatoxin M₁ (AFM₁) and aflatoxin Q₁ (AFQ₁), respectively, complicates the kinetic analysis of AFB₁ in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB₁ in TLMs. In control TLMs, AFB₁ was converted to exo-AFBO in addition to AFM₁ and AFQ₁ confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB₁ concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM₁, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ₁ following the kinetic Hill equation. At 0.1 μM AFB₁, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM₁) compared to 3A37 (0.15: 1, exo-AFBO: AFQ₁), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB₁ bioactivation and metabolism at environmentally-relevant AFB₁ concentrations in turkey liver.     

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B₁ (AFB₁) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB₁ treatment. Competitive enzyme immunoassays indicated that after AFB₁ treatment, the AFB₁-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB₁-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB₁ in the cultural supernatants of cells exposed to AFB₁ showed that the AFM₁ level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB₁. Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB₁. Automated docking experiments showed that AFB₁ binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB₁ interaction is 44.9 μM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB₁, resulting in reduced AFB₁-DNA adducts formation and cell death.     

Trp266 determines the binding specificity of a porcine aflatoxin B1 aldehyde reductase for aflatoxin B1-dialdehyde

Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π–π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.     

Toxic Effects of Aflatoxin B1 in Chinese Sea Bass (Lateolabrax maculatus)

This study was performed to assess the effects of dietary aflatoxin B₁ (AFB₁) on the growth, antioxidant and immune response, digestive enzyme activities, and intestinal morphology of Lateolabrax maculatus during a 56-day feeding trial. Four diets were formulated including 0, 0.1, 0.5, and 1.0 mg/kg of AFB₁. Each diet was randomly assigned to 3 fish tanks with 40 fish per tank. Results indicated that the fish’s final body weight, weight gain rate, specific growth rate, feed intake, condition factor, viscerosomatic index, hepatosomatic index, and intestinesomatic index decreased (p < 0.01) as dietary AFB₁ increased. AFB₁ levels in diets increased (p < 0.05) serum total antioxidant capacity (TAOC), superoxide (SOD), catalase, malondialdehyde (MDA), alkaline phosphatase (AKP), and lysozyme (LZM), and increased (p < 0.05) the TAOC, SOD, MDA, AKP, LZM, and immunoglobulin M in the livers of the fish. Dietary AFB₁ decreased (p < 0.05) intestinal trypsin activity and induced intestinal injury. In summary, dietary AFB₁ up to 1.0 mg/kg was toxic to L. maculatus as judged by reduced growth, enhanced antioxidant and immune response, decreased intestinal trypsin activity, and impaired intestinal morphology.     

Molecular Mechanisms of Lipoic Acid Protection against Aflatoxin B1-Induced Liver Oxidative Damage and Inflammatory Responses in Broilers

Alpha-lipoic acid (α-LA) was evaluated in this study for its molecular mechanisms against liver oxidative damage and inflammatory responses induced by aflatoxin B₁ (AFB₁). Birds were randomly allocated into four groups with different diets for three weeks: a basal diet, a 300 mg/kg α-LA supplementation in a basal diet, a diet containing 74 μg/kg AFB₁, and 300 mg/kg α-LA supplementation in a diet containing 74 μg/kg AFB₁. In the AFB₁ group, the expression of GSH-P_X mRNA was down-regulated (p < 0.05), and the levels of lipid peroxide and nitric oxide were increased (p < 0.05) in the chicken livers compared to those of the control group. Additionally, the mRNA level of the pro-inflammatory factor interleukin-6 was up-regulated significantly (p < 0.05), the protein expressions of both the nuclear factor kappa B (NF-κB) p65 and the inducible nitric oxide synthase were enhanced significantly (p < 0.05) in the AFB₁ group. All of these negative effects were inhibited by α-LA. These results indicate that α-LA may be effective in preventing hepatic oxidative stress, down-regulating the expression of hepatic pro-inflammatory cytokines, as well as inhibiting NF-κB expression.     

Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts

Aflatoxins seriously threaten the health of humans and animals due to their potential carcinogenic properties. Enzymatic degradation approach is an effective and environmentally friendly alternative that involves changing the structure of aflatoxins. In this study, Trametes versicolor aflatoxin B₁-degrading enzyme gene (TV-AFB₁D) was integrated into the genome of Pichia pastoris GS115 by homologous recombination approach. The recombinant TV-AFB₁D was expressed in engineering P. pastoris with a size of approximately 77 kDa under the induction of methanol. The maximum activity of TV-AFB₁D reached 17.5 U/mL after the induction of 0.8% ethanol (v/v) for 84 h at 28 °C. The AFB₁ proportion of 75.9% was degraded using AFB₁ standard sample after catalysis for 12 h. In addition, the AFB₁ proportion was 48.5% using AFB₁-contaminated peanuts after the catalysis for 18 h at 34 °C. The recombinant TV-AFB₁D would have good practical application value in AFB₁ degradation in food crops. This study provides an alternative degrading enzyme for the degradation of AFB₁ in aflatoxin-contaminated grain and feed via enzymatic degradation approach.     

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB₁), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB₁ removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB₁ removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB₁ analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB₁ removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24–48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB₁ removal activity by whole cells was mainly due to AFB₁ binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB₁ binding process onto cell wall components.     

Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1

It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading foodborne carcinogens would reduce human exposure to these deleterious compounds. In the present study, the ability of eight strains of Lactobacillus casei to bind aflatoxin B₁ in aqueous solution was investigated. Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B₁ binding was assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage, human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain with the highest AFB₁ binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 μg/mL). In general, the human isolates bound the most aflatoxin B₁ and the cheese isolates the least. Stability of the bacterial–aflatoxin complex was assessed by repeated washings. Binding was to a limited degree (0.6–9.2% release) reversible; the L. casei 7R1–aflatoxin B₁ complex exhibited the greatest stability. L. casei L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the bacterial cells to bile significant increased aflatoxin B₁ binding and the differences between the strains was reduced.     

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans.     

Potential of Kale and Lettuce Residues as Natural Adsorbents of the Carcinogen Aflatoxin B1 in a Dynamic Gastrointestinal Tract-Simulated Model

Adsorption of the carcinogen aflatoxin B₁ (AFB₁) onto agro-waste-based materials is a promising alternative over conventional inorganic binders. In the current study, two unmodified adsorbents were eco-friendly prepared from kale and lettuce agro-wastes. A dynamic gastrointestinal tract-simulated model was utilized to evaluate the removal efficiency of the sorptive materials (0.5%, w/w) when added to an AFB₁-contaminated diet (100 µg AFB₁/kg). Different characterization methodologies were employed to understand the interaction mechanisms between the AFB₁ molecule and the biosorbents. Based on adsorption results, the biosorbent prepared from kale was the best; its maximum adsorption capacity was 93.6%, which was significantly higher than that of the lettuce biosorbent (83.7%). Characterization results indicate that different mechanisms may act simultaneously during adsorption. Non-electrostatic (hydrophobic interactions, dipole-dipole interactions, and hydrogen bonding) and electrostatic interactions (ionic attractions) together with the formation of AFB₁-chlorophyll complexes appear to be the major influencing factors driving AFB₁ biosorption.     

Multi-Toxic Endpoints of the Foodborne Mycotoxins in Nematode Caenorhabditis elegans

Aflatoxins B₁ (AFB₁), deoxynivalenol (DON), fumonisin B₁ (FB₁), T-2 toxin (T-2), and zearalenone (ZEA) are the major foodborne mycotoxins of public health concerns. In the present study, the multiple toxic endpoints of these naturally-occurring mycotoxins were evaluated in Caenorhabditis elegans model for their lethality, toxic effects on growth and reproduction, as well as influence on lifespan. We found that the lethality endpoint was more sensitive for T-2 toxicity with the EC₅₀ at 1.38 mg/L, the growth endpoint was relatively sensitive for AFB₁ toxic effects, and the reproduction endpoint was more sensitive for toxicities of AFB₁, FB₁, and ZEA. Moreover, the lifespan endpoint was sensitive to toxic effects of all five tested mycotoxins. Data obtained from this study may serve as an important contribution to knowledge on assessment of mycotoxin toxic effects, especially for assessing developmental and reproductive toxic effects, using the C. elegans model.     

Prevention of Aflatoxin B1-Induced DNA Breaks by β-D-Glucan

Aflatoxins are a group of naturally-occurring carcinogens that are known to contaminate different human and animal foodstuffs. Aflatoxin B₁ (AFB₁) is the most genotoxic hepatocarcinogenic compound of all of the aflatoxins. In this report, we explore the capacity of β-d-glucan (Glu) to reduce the DNA damage induced by AFB₁ in mouse hepatocytes. For this purpose, we applied the comet assay to groups of animals that were first administered Glu in three doses (100, 400 and 700 mg/kg bw, respectively) and, 20 min later, 1.0 mg/kg of AFB₁. Liver cells were obtained at 4, 10 and 16 h after the chemical administration and examined. The results showed no protection of the damage induced by AFB₁ with the low dose of the polysaccharide, but they did reveal antigenotoxic activity exerted by the two high doses. In addition, we induced a co-crystallization between both compounds, determined their fusion points and analyzed the molecules by UV spectroscopy. The data suggested the formation of a supramolecular complex between AFB₁ and β-d-glucan.     

NMR and Radical Scavenging Activities of Patuletin from Urtica urens. Against Aflatoxin B1

Abstract

In a previous study, we reported six flavonoids isolated from the methanol extract of Urtica urens L. Patuletin, the major component, was found to have powerful anti-inflammatory and antimicrobial activity. We have now investigated whether patuletin also has antioxidant activity and free radical scavenging effects in rats treated with aflatoxin B₁ (AFB₁). Male Sprague-Dawley rats (60) were assigned to 6 experimental groups including a control group and groups treated for 10 days with patuletin at low dose (7.5 mg/kg b.w) or high dose (10 mg/kg b.w) with or without AFB₁ (2 mg/kg b.w). Blood samples were collected at the end of the experimental period for biochemical analysis. The results showed significant changes typical to aflatoxicosis in rats treated only with AFB₁. Patuletin at the two tested doses caused an increase in glutathione peroxidase (GPx) and superoxide dismutase (SOD); high doses also caused an increase in liver and kidney enzymes, except total bilirubin (TB), which was in the normal value of the control animals. On the other hand, these enzymes were comparable to the controls in rats treated with low doses. Cotreatment of AFB₁ and patuletin resulted in a significant improvement in all parameters tested. We conclude that patuletin possesses an antioxidant activity and free radical scavenging effects in AFB₁-treated rats that can be observed at a dose as low as 7.5 mg/kg b.w.     

Determination of Urinary Biomarkers for Assessment of Short-Term Human Exposure to Aflatoxins in S?o Paulo,Brazil

In the present study, a longitudinal assessment was carried out to evaluate the short-term human exposure to aflatoxins in Pirassununga region, São Paulo, Brazil, by determination of urinary aflatoxins by a liquid chromatography coupled to mass sprectrometry (UPLC-MS/MS) method. Sixteen volunteers with ages ranging from 14 to 55 years old were instructed to collect the early morning first urine four times every three months, from June 2011 to March 2012, totaling 64 samples. Aflatoxin M₁ (AFM₁) was found in 39 samples (61%) at levels ranging from 0.19 to 12.7 pg·mg⁻¹ creatinine (mean: 1.2 ± 2.0 pg·mg⁻¹ creatinine). Residues of aflatoxins B₁, B₂, G₁, G₂ and aflatoxicol were not identified in any urine sample. No significant difference was found among the AFM₁ mean levels in urine samples collected in the four sampling periods. The levels of AFM₁ found in urine samples indicate a low short-term exposure of the population studied to aflatoxins through the diet, although further investigations are needed to assess other long-term biomarkers of exposure to AFB₁.     

Enzymatic Degradation of Multiple Major Mycotoxins by Dye-Decolorizing Peroxidase from Bacillus subtilis

The co-occurrence of multiple mycotoxins, including aflatoxin B₁ (AFB₁), zearalenone (ZEN) and deoxynivalenol (DON), widely exists in cereal-based animal feed and food. At present, most reported mycotoxins degrading enzymes target only a certain type of mycotoxins. Therefore, it is of great significance for mining enzymes involved in the simultaneous degradation of different types of mycotoxins. In this study, a dye-decolorizing peroxidase-encoding gene BsDyP from Bacillus subtilis SCK6 was cloned and expressed in Escherichia coli BL21/pG-Tf2. The purified recombinant BsDyP was capable of oxidizing various substrates, including lignin phenolic model compounds 2,6-dimethylphenol and guaiacol, the substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), anthraquinone dye reactive blue 19 and azo dye reactive black 5, as well as Mn²⁺. In addition, BsDyP could efficiently degrade different types of mycotoxins, including AFB₁, ZEN and DON, in presence of Mn²⁺. More important, the toxicities of their corresponding enzymatic degradation products AFB₁-diol, 15-OH-ZEN and C₁₅H₁₈O₈ were significantly lower than AFB₁, ZEN and DON. In summary, these results proved that BsDyP was a promising candidate for the simultaneous degradation of multiple mycotoxins in animal feed and food.     

Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells

Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB₁ (0, 5 and 20 μg/ml) and FB₁ (0, 35 and 70 μg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB₁ and all concentrations of FB₁ caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 μgAFB₁/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB₁ even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB₁ or FB₁ treated PBMC. The exposure to FB₁ for 7 days increased MDA (p < 0.05) only in cells treated with 70 μg/ml. Exposure of PBMC to AFB₁ reduced SOD mRNA while FB₁ decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB₁ and FB₁ may induce cytotoxicity through an impairment of the oxidative status of PBMC.     

Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1

Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B₁ (AFB₁) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 μg/L AFB₁ was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB₁ were strongly affected by the metal ions in which Cu²⁺ stimulated the degradation and Zn²⁺ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20–25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB₁, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.