Assessment of the protective effect of yeast cell wall ß-glucan encapsulating humic acid nanoparticles as an aflatoxin B1 adsorbent in vivo.

This study aimed to assess the protective effect of encapsulating humic acid-iron complexed nanoparticles (HA-Fe NPs) inside glucanmannan lipid particles (GMLPs) extracted from yeast cell wall against aflatoxin B (AFB1 ) toxicity in vivo. Four groups of male Sprague-Dawley rats were treated orally for 2 weeks included the control group, AFB1 treated group (80 µg/kg b.w); GMLP/HA-Fe NPs treated group (0.5 mg/kg b.w), and the group treated with AFB1 plus GMLP/HA-Fe NPs. GMLPs are empty 3-4 micron permeable microspheres that provide an efficient system for the synthesis and encapsulation of AFB1 -absorbing nanoparticles (NPs). Humic acid nanoparticles (HA-NPs) were incorporated inside the GMLP cavity by complexation with ferric chloride. In vivo study revealed that AFB1 significantly elevated serum alanine aminotransferase, aspartate aminotransferase, creatinine, uric acid, urea, cholesterol, triglycerides, LDL, malondialdehyde, and nitric oxide. It significantly decreased total protein, high-density lipoprotein, hepatic and renal CAT and glutathione peroxidase content and induced histological changes in the liver and kidney (p ≤ 0.05). The coadministration of the synthesized formulation GMLP/HA-Fe NPs with AFB1 has a protective effect against AFB1 -induced hepato-nephrotoxicity, oxidative stress and histological alterations in the liver and kidney.

Subcellular spatio-temporal intravital kinetics of aflatoxin B1 and ochratoxin A in liver and kidney.

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Lactic acid bacteria with promising AFB1 binding properties as an alternative strategy to mitigate contamination on brewers' grains.

Adsorption of molecules to the cell walls of microorganisms plays an important role in helping to prevent animal exposure to the toxic and carcinogenic effects of aflatoxins (AFs). The aim of this study was to evaluate the ability of LAB strains, isolated from brewers' grains, to adsorb aflatoxin B1 (AFB1). All LAB were able to reduce the bioavailability of AFB1 from phosphate buffered-saline (PBS). In addition, the strains retained their effectiveness even after heat treatment. The AFB1-LAB complex stability was first evaluated through sequential washing steps. These assays demonstrated that a low percentage of AFB1 was released after consecutive washes. After subjecting the complex to different pH and bile salt treatments, the percentage of bound AF decreased, as compared to the control, but remained at high levels. Finally, to simulate the formation of the AFB1-LAB complex at conditions similar to those of the gastrointestinal tract, LAB and AFB1 were homogenized in PBS adjusted at acidic conditions or under different bile salt concentrations. In general, LAB strains showed the highest AFB1 adsorption at the lowest pH (2) and bile salt concentration (0.05%). In conclusion, the studied strains represent promising biocontrol agents for preventing and/or ameliorating the AFB1 contamination of feed.

Lactobacillus rhamnosus GG modulates gastrointestinal absorption, excretion patterns, and toxicity in Holstein calves fed a single dose of aflatoxin B1.

The aim of the present study was to evaluate the effects of Lactobacillus rhamnosus GG (LGG; ATCC 53013) on growth performance and hepatotoxicity in calves fed a single dose of aflatoxin B1 (AFB1) and to investigate the absorption, distribution, and elimination of AFB1 and the hydroxylated metabolite aflatoxin M1 (AFM1) in rumen fluid, blood, and excretions. Twenty-four male Holstein calves were blocked for body weight and age and were randomly assigned to 1 of 3 treatment groups: (1) untreated control, (2) treated with 4.80 mg of AFB1 (AFB1 only), or (3) treated with 1 × 1010 cfu of LGG suspension and 4.80 mg of AFB1 (AFB1 plus LGG). The calves received LGG suspension in 50 mL of phosphate-buffered saline daily via oral administration for 14 d before and on the day they received a single oral dose of AFB1. Body weight was recorded at the beginning of the study (before LGG administration), at the day of AFB1 administration, and at the end of the trial. Rumen fluid, blood, urine, and feces samples were collected continuously for 96 h after AFB1 administration. Average daily gain (ADG) and plasma biochemical parameters were analyzed, and concentrations of AFB1 and AFM1 in the samples were determined for monitoring excretion pattern and toxicokinetics. The results showed that ADG was lower in AFB1-treated animals; LGG administration partially mitigated the decrease in ADG (0.85 ± 0.08 vs. 0.76 ± 0.18 kg of gain/d). The AFB1 treatment increased plasma aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase levels. Administration of LGG alleviated the AFB1-induced increase in plasma enzymes activity. The excretion patterns of AFB1 and AFM1 were surprisingly regular; toxins were rapidly detected in all samples after a single oral dose of AFB1, and the peak of toxins concentrations was sequentially reached in rumen fluid, plasma, urine, and feces (except AFM1 in rumen fluid), followed by an exponential decrease. The excretion curves showed that AFB1 and AFM1 concentrations were the highest in feces and urine, respectively. Administration of LGG decreased the concentrations of free AFB1 and AFM1 in rumen fluid and reduced the release of toxins into plasma and urine. Toxicokinetic parameters (except for the time of maximum concentration and the terminal half-life) were reduced by LGG administration. In conclusion, the absorption, distribution, and excretion of AFB1 and AFM1 were rapid in calves fed a single dose of AFB1. Urine was the main route for the excretion of AFM1, and the clearance pattern from the peak of concentration was well fitted by exponential decreasing function. Administration of LGG reduced the absorption of AFB1 in the gastrointestinal tract by increasing the excretion via the feces, thus alleviating the hepatotoxic effect of AFB1.

The aflatoxin-detoxifizyme specific expression in mouse parotid gland.

The aflatoxin-detoxifizyme (ADTZ) gene derived from Armillariella tabescens was cloned into parotid gland-specific expression vector (pPSPBGPneo) to construct the parotid gland-specific vector expressing ADTZ (pPSPBGPneo-ADTZ). Transgenic mice were generated by microinjection and identified by using PCR and Southern blotting analysis. PCR and Southern blotting analysis showed that total six transgenic mice carried the ADTZ gene were generated. RT-PCR analysis indicated that the expression of ADTZ mRNA could be detected only in parotid glands of the transgenic mice. The ADTZ activity in the saliva was found to be 3.72 ± 1.64 U/mL. After feeding a diet containing aflatoxin B1 (AFB1) for 14 days, the effect of ADTZ on serum biochemical indexes and AFB1 residues in serum and liver of mice were evaluated. The results showed that total protein and globulin contents in the test treatment (transgenic mice) produced ADTZ were significantly higher than that of the positive control, while alanine aminotransferase and aspartate aminotransferase activity in serum of the test treatment (transgenic mice) were remarkably lower compared to that of the positive control (P < 0.05). Moreover, AFB1 residues in serum and liver of the test treatment (transgenic mice) were significantly lower compared with that of the positive control (P < 0.05). These results in the study confirmed that ADTZ produced in transgenic mice could reduce, even eliminate the negative effects of AFB1 on mice.

Cytochrome P450 2A13 mediates the neoplastic transformation of human bronchial epithelial cells at a low concentration of aflatoxin B1.

Cytochrome P450 2A13 (CYP2A13), mainly expressed in human respiratory tract, is highly efficient in the metabolic activation of aflatoxin (AF) B1 (AFB1) and is assumed to play a role in human lung tumorigenesis in airborne AFB1 exposure. To validate the assumption, we exposed human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13), CYP1A2 (B-1A2) and CYP2A6 (B-2A6) to 0.1-10 nM AFB1 for 30-50 passages. B-2A13 cells showed increased sensitivity to 0.1 nM AFB1-induced neoplastic transformation and the formation of tumors in nude mice were observed at passage 30 (P30) while it occurred at P50 B-1A2 cells. B-2A6, similar to vector control, showed no neoplastic transformation in this condition. Additionally, AFB1-DNA adducts and 8-OHdG significantly increased in transformed P40 B-2A13, in parallel with the upregulation of p-ATR, p-BRCA1, Mre11, Rad50 and Rad51. However, the apoptosis of P40 cells was near normal, while the expression of Bax, C-Caspase 3 and C-PARP increased passage-dependently. Inhibition of ATR (ATR siRNA or NU6027) reversely increased the apoptosis of P40 B-2A13 cells in parallel with the upregulation of Bax, C-Caspase 3 and C-PARP, suggesting that ATR plays an important role in maintaining cell survival via antiapoptosis. Additionally, activation of ATR was necessary to neoplastic transformation since blockage of ATR in P40 cells inhibited DNA damage repair response and anchorage-independent growth. Our data demonstrated that CYP2A13 played a critical role in AFB1-induced neoplastic transformation. ATR-mediated the dysfunction of apoptosis and DNA damage repair might be involved. These results help establish a linkage between airborne AFB1 and human respiratory carcinoma.

An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride.

The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.

Modified hydra bioassay to evaluate the toxicity of multiple mycotoxins and predict the detoxification efficacy of a clay-based sorbent.

Food shortages and a lack of food supply regulation in developing countries often leads to chronic exposure of vulnerable populations to hazardous mixtures of mycotoxins, including aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)). A refined calcium montmorillonite clay [i.e. uniform particle size NovaSil (UPSN)] has been reported to tightly bind these toxins, thereby decreasing bioavailability in humans and animals. Hence, our objectives in the present study were to examine the ability of UPSN to bind mixtures of AFB(1) and FB(1) at gastrointestinally relevant pH in vitro, and to utilize a rapid in vivo bioassay to evaluate AFB(1) and FB(1) toxicity and UPSN efficacy. Isothermal sorption data indicated tight AFB(1) binding to UPSN surfaces at both pH 2.0 and 6.5, but substantially more FB(1) bound at pH 2.0 than 6.5. Site-specific competition occurred between the toxins when exposed to UPSN in combination. Importantly, treatment with UPSN resulted in significant protection to mycotoxin-exposed hydra maintained at pH 6.9-7.0. Hydra were exposed to FB(1), AFB(1) and FB(1) /AFB(1) combinations with and without UPSN. A toxic response over 92 h was rated based on morphology and mortality. Hydra assay results indicated a minimum effective concentration (MEC) of 20 µg ml(-1) for AFB(1), whereas the MEC for FB(1) was not reached. The MEC for co-exposure was 400 µg ml(-1) FB(1) + 10 µg ml(-1) AFB(1). This study demonstrates that UPSN sorbs both mycotoxins tightly at physiologically relevant pH levels, resulting in decreased bioavailability, and that a modified hydra bioassay can be used as an initial screen in vivo to predict efficacy of toxin-binding agents.

Exploring the Impact of Efavirenz on Aflatoxin B1 Metabolism: Insights from a Physiologically Based Pharmacokinetic Model and a Human Liver Microsome Study.

Physiologically based pharmacokinetic (PBPK) models were utilized to investigate potential interactions between aflatoxin B1 (AFB1) and efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor drug and inducer of several CYP enzymes, including CYP3A4. PBPK simulations were conducted in a North European Caucasian and Black South African population, considering different dosing scenarios. The simulations predicted the impact of EFV on AFB1 metabolism via CYP3A4 and CYP1A2. In vitro experiments using human liver microsomes (HLM) were performed to verify the PBPK predictions for both single- and multiple-dose exposures to EFV. Results showed no significant difference in the formation of AFB1 metabolites when combined with EFV (0.15 µM) compared to AFB1 alone. However, exposure to 5 µM of EFV, mimicking chronic exposure, resulted in increased CYP3A4 activity, affecting metabolite formation. While co-incubation with EFV reduced the formation of certain AFB1 metabolites, other outcomes varied and could not be fully attributed to CYP3A4 induction. Overall, this study provides evidence that EFV, and potentially other CYP1A2/CYP3A4 perpetrators, can impact AFB1 metabolism, leading to altered exposure to toxic metabolites. The results emphasize the importance of considering drug interactions when assessing the risks associated with mycotoxin exposure in individuals undergoing HIV therapy in a European and African context.

The hepatoprotective effect of sea buckthorn (Hippophae rhamnoides) berries on induced aflatoxin B1 poisoning in chickens 1.

The leaves and berries of sea buckthorn (SB; Hippophae rhamnoides; family Elaeagnaceae) are medically claimed as having phytoantioxidant, antiinflammatory, and anticancerous properties in humans. This study evaluated the hepatoprotective activity of oil from SB berries against toxicity induced by aflatoxin B1 (AFB1) in broiler chickens. The toxicity of AFB1 led to lower total serum proteins and specifically reduced albumin (P < 0.001). Serum aspartate aminotransferase increased from 191.14 ± 11.56 to 218.80 ± 13.68 (P < 0.001). When chickens were simultaneously dosed with AFB1 and an extract of SB berries, subsequent histology of the liver showed a significant reduction of necrosis and fatty formation compared with chickens treated with AFB1 alone. Immunohistochemical results indicated that COX2, Bcl-2, and p53 were highly expressed in the liver of AFB1-treated chickens and their expression was significantly reduced by SB oil supplementation. The levels of AFB1 residues in chickens livers were significantly reduced by SB oil from 460.92 ± 6.2 ng/mL in the AFB1 group to 15.59 ± 6.1 ng/mL in the AFB1 and SB oil group. These findings suggest that SB oil has a potent hepatoprotective activity, reducing the concentration of aflatoxins in liver and diminishing their adverse effects.

Excretion pattern of aflatoxin M1 in milk of goats fed a single dose of aflatoxin B1.

The feedstuffs used in dairy animals must be able to give consumers confidence about the wholesomeness of milk with regard to aflatoxin contamination. The aim of this study was to determine the excretion patterns of aflatoxin M(1) (AFM1) in the milk of dairy goats fed a single dose of pure aflatoxin B(1) (AFB1), which can occasionally occur if feeds are infected by hot-spot growth of molds that produce aflatoxins. Five dairy goats in midlactation were administered 0.8 mg of AFB1 orally. Individual milk samples were collected for 84 h after AFB1 dosage. Aflatoxin M(1) was found in milk in the highest concentration. In all goats, AFM1 was not detected in milk before AFB1 administration, but was detected in the first milking following AFB1 administration. The excretion pattern of AFM1 concentration in milk was very similar in all goats even if the values of the concentration differed between animals. The peak values for AFM1 concentration in milk was observed in milk collected during the milking at 3 and 6h. After the peak, the AFM1 in milk disappeared with a trend that fitted well a monoexponential decreasing function, and the toxin was not detected after 84 h. Only about 0.17% of the amount of AFB1 administered was detected as AFM1 in milk, and about 50% of this was excreted in the first liter of milk yielded after AFB1 intake. Correct procedures to prevent growth of molds, and consequent AFB1 contamination, on the feedstuffs for lactating goats represent the key to providing consumers a guarantee that milk is not contaminated by AFM1.

Effect of adding a mycotoxin-sequestering agent on milk aflatoxin M1 concentration and the performance and immune response of dairy cattle fed an aflatoxin B1-contaminated diet.

This project aimed to examine the effects of adding 2 doses of a montmorillonite-based mycotoxin adsorbent on milk aflatoxin M(1) (AFM(1)) concentrations and the performance and innate immune response of dairy cows fed an aflatoxin B(1) (AFB(1))-contaminated diet. Eight lactating cows were used in a duplicated 4×4 Latin square design with 12-d periods. Treatments included the following: (1) control diet (C), (2) aflatoxin diet (T) containing C and 75 µg of AFB(1)/kg, 3) low-clay (LC) diet containing T and Calibrin A (Amlan International, Chicago, IL) added at 0.2% of the diet dry matter (DM), and 4) high-clay diet (HC) containing T and Calibrin A added at 1% of the diet DM. Milk production and DM intake were recorded daily and milk was sampled twice daily on d 5, 9, 10, 11, and 12 in each period. Blood samples were collected on d 5 and 9 of each period. Dietary treatments did not affect DM intake, milk yield, or feed efficiency. Even though cows were limit fed, feeding T instead of C reduced milk fat yield (0.67 vs. 0.74 kg/d) and milk protein concentration (3.28 vs. 3.36%). Concentrations of AFM(1) in milk of cows fed the T and LC diets were similar (0.57 and 0.64 µg/kg) and greater than those of cows fed the HC diet (0.46 µg/kg). Haptoglobin concentration was greater (22.0 vs. 14.4) and ß(2)-integrin expression (220 vs. 131) tended to be greater in cows fed diet T instead of C, but values for cows fed LC, HC, and C did not differ. In comparison to C, feeding T increased the innate immune response and decreased milk fat yield and milk protein concentration, but feeding LC and HC did not affect these measures. Only the HC diet reduced milk AFM(1) concentration.

Saccharomyces cerevisiae Cell Wall-Based Adsorbent Reduces Aflatoxin B1 Absorption in Rats.

Mycotoxins are naturally occurring toxins that can affect livestock health and performance upon consumption of contaminated feedstuffs. To mitigate the negative effects of mycotoxins, sequestering agents, adsorbents, or binders can be included to feed to interact with toxins, aiding their passage through the gastrointestinal tract (GI) and reducing their bioavailability. The parietal cell wall components of Saccharomyces cerevisiae have been found to interact in vitro with mycotoxins, such as, but not limited to, aflatoxin B1 (AFB1), and to improve animal performance when added to contaminated diets in vivo. The present study aimed to examine the pharmacokinetics of the absorption of radiolabeled AFB1 in rats in the presence of a yeast cell wall-based adsorbent (YCW) compared with that in the presence of the clay-based binder hydrated sodium calcium aluminosilicate (HSCAS). The results of the initial pharmacokinetic analysis showed that the absorption process across the GI tract was relatively slow, occurring over a matter of hours rather than minutes. The inclusion of mycotoxin binders increased the recovery of radiolabeled AFB1 in the small intestine, cecum, and colon at 5 and 10 h, revealing that they prevented AFB1 absorption compared with a control diet. Additionally, the accumulation of radiolabeled AFB1 was more significant in the blood plasma, kidney, and liver of animals fed the control diet, again showing the ability of the binders to reduce the assimilation of AFB1 into the body. The results showed the potential of YCW in reducing the absorption of AFB1 in vivo, and in protecting against the damaging effects of AFB1 contamination.

Toxicokinetics of aflatoxin in pregnant mice.

Our objective was to study the toxicokinetics of aflatoxin (AF) in pregnant mice. Aflatoxin B1 (AFB1) was administered intraperitoneally (IP) to groups of pregnant mice in single doses of 20 mg/kg on gestation day (GD) 13 and orally at the same gestational age. Controls received (IP and oral) a proportionate volume of solvent only. Maternal blood was collected at 15, 30, 45, 60, 90, 120, and 150 minutes posttreatment. Their AFB1 contents were determined. Aflatoxin B1 concentrations following maternal exposure to AFB1 were highly correlated with time after exposure. The serum concentrations were predictable and the highest serum levels were seen immediately at 15 minutes in mice given AFs IP and at 30 minutes in those given it orally. The absorption was 5.0 microg/min and elimination was 3.0 microg/min. The toxicokinetics of AFB1 have been delineated. Aflatoxins are easily and rapidly absorbed both from the gastrointestinal tract (GI) tract and through the peritoneum.

Occurrence and bioacessibility of mycotoxins in fish feed.

In this study Aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEN) occurrence in fish feed, regarding its chemical composition, were investigated. Besides, AFB1 bioaccessibility to fish was evaluated by in vitro digestion. Mycotoxins were extracted by QuEChERS and quantified by HPLC-FLD. Results showed that 93.3% of the samples were contaminated at maximum levels of 16.5, 31.6, and 322 µg/kg in the cases of AFB1, OTA, and ZEN, respectively. A positive correlation between OTA, ZEN contamination, and lipid content was observed. Risk estimation of feed consumption by fish at the highest levels of AFB1, OTA, and ZEN shows that the younger the fish, the higher the risk of exposure to mycotoxins. The AFB1 bioaccessibility assay showed that 85% of this mycotoxin may be absorbed by fish. Therefore, establishing maximum levels in the fishing sector is fundamental to contribute to feed quality and nutritional safety of fish species.

Predicting the Acute Liver Toxicity of Aflatoxin B1 in Rats and Humans by an In Vitro-In Silico Testing Strategy.

SCOPE: High-level exposure to aflatoxin B1 (AFB1) is known to cause acute liver damage and fatality in animals and humans. The intakes actually causing this acute toxicity have so far been estimated based on AFB1 levels in contaminated foods or biomarkers in serum. The aim of the present study is to predict the doses causing acute liver toxicity of AFB1 in rats and humans by an in vitro-in silico testing strategy. METHODS AND RESULTS: Physiologically based kinetic (PBK) models for AFB1 in rats and humans are developed. The models are used to translate in vitro concentration-response curves for cytotoxicity in primary rat and human hepatocytes to in vivo dose-response curves using reverse dosimetry. From these data, the dose levels at which toxicity would be expected are obtained and compared to toxic dose levels from available rat and human case studies on AFB1 toxicity. The results show that the in vitro-in silico testing strategy can predict dose levels causing acute toxicity of AFB1 in rats and human. CONCLUSIONS: Quantitative in vitro in vivo extrapolation (QIVIVE) using PBK modeling-based reverse dosimetry can predict AFB1 doses that cause acute liver toxicity in rats and human.

In vitro hepatic aflatoxicol production is related to a higher resistance to aflatoxin B1 in poultry.

A study was conducted to determine the cytosolic in vitro hepatic enzymatic kinetic parameters Vmax, KM, and intrinsic clearance (CLint) for aflatoxin B1 (AFB1) reductase [aflatoxicol (AFL) production] and AFL dehydrogenase (AFB1 production) in four commercial poultry species (chicken, quail, turkey and duck). Large differences were found in AFB1 reductase activity, being the chicken the most efficient producer of AFL (highest CLint value). Oxidation of AFL to AFB1 showed only slight differences among the different poultry species. On average all species produced AFB1 from AFL at a similar rate, except for the turkey which produced AFB1 from AFL at a significantly lower rate than chickens and quail, but not ducks. Although the turkey and duck showed differences in AFL oxidation Vmax and KM parameters, their CLint values did not differ significantly. The ratio AFB1 reductase/AFL dehydrogenase enzyme activity was inversely related to the known in vivo sensitivity to AFB1 being highest for the chicken, lowest for the duck and intermediate for turkeys and quail. Since there is no evidence that AFL is a toxic metabolite of AFB1, these results suggest that AFL production is a detoxication reaction in poultry. Conversion of AFB1 to AFL prevents the formation of the AFB1-8,9-exo-epoxide which, upon conversion to AFB1-dihydrodiol, is considered to be the metabolite responsible for the acute toxic effects of AFB1.

Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers.

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.

Structure, genetic mapping, and function of the cytochrome P450 3A37 gene in the turkey (Meleagris gallopavo).

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB1 is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB1-8,9-epoxide (AFBO). Using 3'-5'-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB(1). Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB(1), this study will contribute to the identifying allelic variants of this important gene in poultry.

Effects of probiotic bacteria on the bioaccessibility of aflatoxin B(1) and ochratoxin A using an in vitro digestion model under fed conditions.

In the present study, we aimed at determining the release of aflatoxin B(1) (AFB(1)) and ochratoxin A (OTA) from different food products in the gastro-intestinal tract in the absence and presence of probiotics, a possible adsorbent. The average bioaccessibility of AFB(1) and OTA without probiotics was about 90%, and 30%, respectively, depending on several factors, such as food product, contamination level, compound and type of contamination (spiked versus naturally contaminated). The six probiotic bacteria showed varying binding capacity to AFB(1) and OTA depending on the bacterial strain, toxin studied, type of food and contamination level. A reduction to a maximum of 37% and 73% as observed for the bioaccessibility of AFB(1) and OTA in the presence of probiotic bacteria, respectively. This is the first report on the effect of probiotic bacteria on reducing the fraction of mycotoxins available for absorption in the gastrointestinal tract from different food products.