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.</p

Mode-of-action based risk assessment of exposure to mycotoxins in nixtamalized maize products from Mexico City

Mycotoxins are natural contaminants, unavoidable in food. In Mexico, the large consumption of maize, may place the population at risk, yet limited risk assessments of mycotoxins in Mexico are available (Chapter 1). This thesis assessed the risk of the main mycotoxins present in nixtamalized maize products consumed in Mexico City. To this end, a characterization of aflatoxin B1 (AFB1), fumonisin B1 (FB1), fumonisin B2 (FB2), and deoxynivalenol (DON) in samples collected from Mexico City was performed with an accompanying risk assessment. To enhance the use of mode-of-action based non-animal testing strategies in risk assessment of these mycotoxins, a physiologically based kinetic (PBK) model of AFB1 was developed. This enabled the use of a PBK model-based reverse dosimetry approach to predict the acute toxicity and genotoxicity of AFB1 in the liver using an in vitro&ndash;in silico testing strategy. The risk associated to the intake of AFB1 from nixtamalized maize was evaluated after analyzing its occurrence in nixtamalized maize samples from Mexico City (Chapter 2). A 4,5% of the samples contained AFB1 at levels above the limit of detection (1 ng/g) indicating a low frequency of AFB1 in the nixtamalized maize samples. The results obtained and additional AFB1 occurrence values from literature were combined with mean and P95 consumption values from literature. For a 70 kg body weight (bw) person, lower and upper bound exposure assessments resulted in estimated daily intakes (EDI) of 0.7&ndash;8.5 ng/kg bw/day, based on a mean maize consumption. Based on a P95 maize consumption, EDI values were 3.3&ndash;11.7 ng/kg bw/day. Corresponding Margin of Exposure (MOE) values amounted to 257-20 for the mean and 50-15 for the P95 consumers. The estimated increased cancer risks were 9-320 and 43-439 cases/106 individuals/lifetime of 75 years for the mean and P95 consumers, respectively. Altogether, continued risk management of AFB1 in Mexico City is needed. Given that mycotoxins can occur simultaneously in food, the levels of the mycotoxins most frequently encountered in maize, fumonisins (FB1+FB2) and DON, were also determined with an accompanying risk assessment. Chapter 3 presents the occurrence of FB1+FB2 and DON in the nixtamalized maize samples collected in Mexico City. It further analyses their distribution and the resulting EDI for Mexican consumers by a probabilistic approach using a two-dimensional Monte-Carlo simulation. The results revealed that for fumonisins 47% of the Mexican men and 30% of the Mexican women might exceed the provisional tolerable daily intake (PMTDI) of 2 &micro;g/kg bw/day for fumonisins and for DON the high consumers (9% of men and 5% of women) would be exceeding the PMTDI of 1 &micro;g/kg bw/day. The results raise a flag for risk managers in Mexico, to consider regulations and interventions to lower mycotoxin levels in nixtamalized maize for human consumption. To enhance the use of non-animal testing strategies in risk assessment, a generic PBK model of AFB1 was developed to predict in vivo toxicity by using a reverse dosimetry approach. Since PBK modelling-based reverse dosimetry enables quantitative in vitro in vivo extrapolation (QIVIVE), Chapter 4 presents an in vitro-in silico testing strategy to predict the doses causing acute liver toxicity of AFB1 in rats and humans. To this end PBK models for AFB1 in rats and humans were developed and evaluated. The models were further used to translate in vitro concentration&ndash;response curves for cytotoxicity in primary rat and human hepatocytes to in vivo dose&ndash;response curves using reverse dosimetry. From these data, the dose levels at which toxicity would be expected were obtained and compared to toxic dose levels from available rat studies and human case studies on AFB1 toxicity. The results showed that the in vitro&ndash;in silico testing strategy could predict dose levels observed to cause acute toxicity of AFB1 in rats and human in vivo. From this study, it was concluded that QIVIVE using PBK modelling-based reverse dosimetry can adequately predict AFB1 doses that cause acute liver toxicity in rats and human. The importance of genotoxicity in the mode of action of AFB1 made interesting to use this PBK model to predict its in vivo genotoxicity in liver. In Chapter 5, reported concentration-response curves for in vitro genotoxicity in hepatocytes were searched from literature. Suitable in vitro data, consisting of in vitro micronucleus (MN) data obtained in rat hepatocytes in vitro, were translated to an in vivo dose-response curve. Comparison of the predicted in vivo dose-response curve for MN induction with available in vivo data for MN induction in the liver of rats, revealed adequate dose predictions. The assessment of AFB1 target organ genotoxicity via PBK modelling-facilitated reverse dosimetry approach to perform QIVIVE, offers a promising alternative to obtain insight in the potential genotoxicity in rats taking in vivo kinetics into account. Despite the focus on one commodity, this study provides a promising starting point for future risk assessments in Mexico (Chapter 6). The risk assessment also revealed that the exposure to AFB1, fumonisins and DON is mainly driven by consumption rather than occurrence levels, and nixtamalization of maize may not be fully effective in reducing mycotoxins levels to a level of low concern. A PBK model approach for AFB1, and translation of in vitro data using PBK modelling-based reverse dosimetry to in vivo dose response curves was developed to estimate the acute liver toxicity without in vivo studies. Altogether, it was noticed that chronic exposure to AFB1 by Mexicans raise a concern. Also, it was observed that genotoxicity data available for AFB1 in vitro and in vivo in liver cells or liver tissue appear to be limited, and that improvement of liver cell models for studying chronic exposure in in vitro studies will also improve the PBK modelling-based reverse dosimetry approach. In conclusion, the results support the conclusion that further risk management actions to reduce potential health-risks of mycotoxins AFB1, fumonisins and DON in nixtamalized maize consumed by Mexican population are needed. QIVIVE results showed that PBK modelling-based reverse dosimetry is a promising tool to predict and study the toxicity of AFB1, but potentially also of other food borne mycotoxins

Aflatoxin B1 in nixtamalized maize in Mexico; occurrence and accompanying risk assessment

Maize is a staple food in Mexico that might contain Aflatoxin B1 (AFB1). Nonetheless, data on the exposure and risk assessment of AFB1 from maize for the Mexican population are limited. The aim of the present study was to analyse the occurrence of AFB1 in Mexican nixtamalized maize samples, and to assess the accompanying exposure and risk. Four out of 88 samples contained AFB1 at levels above the limit of detection (1 ng/g). AFB1 occurrence values obtained in this study and additional occurrence values from literature were combined with available literature data for mean and P95 consumption of maize based products. For a 70 kg body weight person, lower bound and upper bound exposure assessments resulted in estimated daily intakes (EDI) of 0.7–8.5 ng/kg bw/day, based on a mean maize consumption. Based on the P95 maize consumption these EDI values amounted to 3.3–11.7 ng/kg bw/day. The corresponding Margin of Exposure (MOE) values amounted to 257-20 for the mean and 50-15 for the P95 consumers. The estimated increased cancer risks were 9-320 and 43-439 cases/106 individuals/lifetime of 75 years for the mean and P95 consumers, respectively. Altogether, the assessment reveals the need for continued risk management of AFB1 in Mexico.</p

Occurrence and probabilistic risk assessment of fumonisin B1, fumonisin B2 and deoxynivalenol in nixtamalized maize in Mexico City

Fumonisins (FB1+FB2) and deoxynivalenol (DON) are mycotoxins produced by Fusarium species that might be present in maize and maize products. Knowledge on their occurrence in nixtamalized maize from Mexico together with an accompanying risk assessment are scarce, while nixtamalized maize is an important food in Mexico. This study presents the occurrence of FB1 + FB2 and DON in nixtamalized maize samples collected in Mexico City and analyses their distribution and resulting estimated daily intake for Mexican consumers by a probabilistic approach using a two‐dimensional Monte‐Carlo simulation. The results obtained reveal that for FB1 + FB2, 47% of the Mexican men and 30% of the Mexican women might exceed the provisional tolerable daily intake (PMTDI) of 2 μg/kg bw/day for fumonisins and for DON, 9% of men and 5% of women would be exceeding the PMTDI of 1 μg/kg bw/day, corresponding to the high consumers. The results raise a flag for risk managers in Mexico, to consider regulations and interventions that lower mycotoxin levels in nixtamalized maize for human consumption.</p