In vitro-in silico study on the influence of dose, fraction bioactivated and endpoint used on the relative potency value of pyrrolizidine alkaloid N-oxides compared to parent pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) are phytotoxins found in food, feed and the environment. Yet, limited data exist from which the relative potency of a PA-N-oxide relative to its corresponding PA (REPPANO to PA) can be defined. This study aims to investigate the influence of dose, fraction bioactivated and endpoint on the REPPANO to PA of a series of pyrrolizidine N-oxides using in vitro-in silico data and physiologically based kinetic (PBK) modeling. The first endpoint used to calculate the REPPANO to PA was the ratio of the area under the concentration-time curve of PA resulting from an oral dose of PA-N-oxide divided by that from an equimolar dose of PA (Method 1). The second endpoint was the ratio of the amount of pyrrole-protein adducts formed under these conditions (Method 2). REPPANO to PA values appeared to decrease with increasing dose, with the decrease for Method 2 already starting at lower dose level than for Method 1. At dose levels as low as estimated daily human intakes, REPPANO to PA values amounted to 0.92, 0.81, 0.78, and 0.68 for retrorsine N-oxide, seneciphylline N-oxide, riddelliine N-oxide and senecivernine N-oxide, respectively, and became independent of the dose or fraction bioactivated, because no GSH depletion, saturation of PA clearance or PA-N-oxide reduction occurs. Overall, the results demonstrate the strength of using PBK modeling in defining REPPANO to PA values, thereby substantiating the use of the same approach for other PA-N-oxides for which in vivo data are lacking.

Toxicokinetics of the Antidepressant Fluoxetine and Its Active Metabolite Norfluoxetine in Caenorhabditis elegans and Their Comparative Potency.

The nematode Caenorhabditis elegans is a valuable model for ecotoxicological research, yet limited attention has been given to understanding how it absorbs, distributes, metabolizes, and excretes chemicals. This is crucial for C. elegans because the organism is known to have strong uptake barriers that are known to be susceptible to potential confounding effects of the presence of Escherichia coli as a food source. One frequently studied compound in C. elegans is the antidepressant fluoxetine, which has an active metabolite norfluoxetine. In this study, we evaluated the toxicokinetics and relative potency of norfluoxetine and fluoxetine in chemotaxis and activity tests. Toxicokinetics experiments were conducted with varying times, concentrations of fluoxetine, and in the absence or presence of E. coli, simulated with a one-compartment model. Our findings demonstrate that C. elegans can take up fluoxetine and convert it into norfluoxetine. Norfluoxetine proved slightly more potent and had a longer elimination half-life. The bioconcentration factor, uptake, and elimination rate constants depended on exposure levels, duration, and the presence of E. coli in the exposure medium. These findings expand our understanding of toxicokinetic modeling in C. elegans for different exposure scenarios, underlining the importance of considering norfluoxetine formation in exposure and bioactivity assessments of fluoxetine.

Acetylcholinesterase Inhibition in Rats and Humans Following Acute Fenitrothion Exposure Predicted by Physiologically Based Kinetic Modeling-Facilitated Quantitative In Vitro to In Vivo Extrapolation.

Worldwide use of organophosphate pesticides as agricultural chemicals aims to maintain a stable food supply, while their toxicity remains a major public health concern. A common mechanism of acute neurotoxicity following organophosphate pesticide exposure is the inhibition of acetylcholinesterase (AChE). To support Next Generation Risk Assessment for public health upon acute neurotoxicity induced by organophosphate pesticides, physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed in this study, with fenitrothion (FNT) as an exemplary organophosphate pesticide. Rat and human PBK models were parametrized with data derived from in silico predictions and in vitro incubations. Then, PBK model-based QIVIVE was performed to convert species-specific concentration-dependent AChE inhibition obtained from in vitro blood assays to corresponding in vivo dose-response curves, from which points of departure (PODs) were derived. The obtained values for rats and humans were comparable with reported no-observed-adverse-effect levels (NOAELs). Humans were found to be more susceptible than rats toward erythrocyte AChE inhibition induced by acute FNT exposure due to interspecies differences in toxicokinetics and toxicodynamics. The described approach adequately predicts toxicokinetics and acute toxicity of FNT, providing a proof-of-principle for applying this approach in a 3R-based chemical risk assessment paradigm.

Predicting acute paraquat toxicity using physiologically based kinetic modelling incorporating in vitro active renal excretion via the OCT2 transporter.

Including active renal excretion in physiologically based kinetic (PBK) models can improve their use in quantitative in vitro- in vivo extrapolation (QIVIVE) as a new approach methodology (NAM) for predicting the acute toxicity of organic cation transporter 2 (OCT2) substrates like paraquat (PQ). To realise this NAM, kinetic parameters Vmax and Km for in vitro OCT2 transport of PQ were obtained from the literature. Appropriate scaling factors were applied to translate the in vitro Vmax to an in vivo Vmax. in vitro cytotoxicity data were defined in the rat RLE-6TN and L2 cell lines and the human A549 cell line. The developed PQ PBK model was used to apply reverse dosimetry for QIVIVE translating the in vitro cytotoxicity concentration-response curves to predicted in vivo toxicity dose-response curves after which the lower and upper bound benchmark dose (BMD) for 50% lethality (BMDL50 and BMDU50) were derived by applying BMD analysis. Comparing the predictions to the in vivo reported LD50 values resulted in a conservative prediction for rat and a comparable prediction for human showing proof of principle on the inclusion of active renal excretion and prediction of PQ acute toxicity for the developed NAM.

On the Role of ROS and Glutathione in the Mode of Action Underlying Nrf2 Activation by the Hydroxyanthraquinone Purpurin.

Purpurin is a major anthraquinone present in the roots of Rubia cordifolia (madder). Purpurin is known to activate Nrf2 (Nuclear transcription factor erythroid 2-related factor 2) EpRE (electrophile responsive element) mediated gene expression as a potential beneficial effect. This study aimed to elucidate the balance between the electrophilicity or pro-oxidant activity of purpurin underlying the Nrf2 induction. For this, Nrf2 activation with modified intracellular glutathione (GSH) levels was measured in an Nrf2 CALUX reporter gene assay. In addition, both cell-free and intracellular ROS formation of purpurin with modified (intracellular) GSH levels at different pH were quantified using the DCF-DA assay. GSH adduct formation was evaluated by UPLC and LC-TOF-MS analysis. GSH and GSSG levels following purpurin incubations were quantified by LC-MS/MS. We show that Nrf2 induction by purpurin was significantly increased in cells with buthionine sulfoximine depleted GSH levels, while Nrf2 induction was decreased upon incubation of the cells with N-acetylcysteine being a precursor of GSH. In cell-free incubations, ROS formation increased with increasing pH pointing at a role for the deprotonated form of purpurin. Upon incubations of purpurin with GSH at physiological pH, GSH adduct formation appeared negligible (<1.5% of the added purpurin). The addition of GSH resulted in conversion of GSH to GSSG and significantly reduced the ROS formation. Together these results demonstrate that Nrf2 induction by purpurin originates from intracellular ROS formation and not from its electrophilicity, which becomes especially relevant when intracellular GSH levels can no longer scavenge the ROS. The present study demonstrated that the efficiency of intracellular Nrf2 activation by purpurin and related anthraquinones will depend on (i) their pKa and level of deprotonation at the intracellular pH, (ii) the oxidation potential of their deprotonated form and (iii) the intracellular GSH levels. Thus, the Nrf2 induction by purpurin depends on its pro-oxidant activity and not on its electrophilicity.

Integrating In Vitro Data and Physiologically Based Kinetic Modeling to Predict and Compare Acute Neurotoxic Doses of Saxitoxin in Rats, Mice, and Humans.

Current climate trends are likely to expand the geographic distribution of the toxigenic microalgae and concomitant phycotoxins, making intoxications by such toxins a global phenomenon. Among various phycotoxins, saxitoxin (STX) acts as a neurotoxin that might cause severe neurological symptoms in mammals following consumptions of contaminated seafood. To derive a point of departure (POD) for human health risk assessment upon acute neurotoxicity induced by oral STX exposure, a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed. The PBK models for rats, mice, and humans were built using parameters from the literature, in vitro experiments, and in silico predictions. Available in vitro toxicity data for STX were converted to in vivo dose-response curves via the PBK models established for these three species, and POD values were derived from the predicted curves and compared to reported in vivo toxicity data. Interspecies differences in acute STX toxicity between rodents and humans were found, and they appeared to be mainly due to differences in toxicokinetics. The described approach resulted in adequate predictions for acute oral STX exposure, indicating that new approach methodologies, when appropriately integrated, can be used in a 3R-based chemical risk assessment paradigm.

Physiologically based kinetic modeling of senecionine N-oxide in rats as a new approach methodology to define the effects of dose and endpoint used on relative potency values of pyrrolizidine alkaloid N-oxides.

Over 1,000 pyrrolizidine alkaloids (PAs) and their N-oxides (PA-N-oxides) occur in 3% of all flowering plants. PA-N-oxides are toxic when reduced to their parent PAs, which are bioactivated into pyrrole intermediates that generate protein- and DNA-adducts resulting in liver toxicity and carcinogenicity. Literature data for senecionine N-oxide in rats indicate that the relative potency (REP) value of this PA-N-oxide compared to its parent PA senecionine varies with the endpoint used. The first endpoint was the ratio between the area under the concentration-time curve (AUC) for senecionine upon dosing senecionine N-oxide or an equimolar dose of senecionine, while the second endpoint was the ratio between the amount for pyrrole-protein adducts formed under these conditions. This study aimed to investigate the mode of action underlying this endpoint dependent REP value for senecionine N-oxide with physiologically based kinetic (PBK) modeling. Results obtained reveal that limitation of 7-GS-DHP adduct formation due to GSH depletion, resulting in increased pyrrole-protein adduct formation, occurs more likely upon high dose oral PA administration than upon an equimolar dose of PA-N-oxide. At high dose levels, this results in a lower REP value when based on pyrrole-protein adduct levels than when based on PA concentrations. At low dose levels, the difference no longer exists. Altogether, the results of the study show how the REP value for senecionine N-oxide depends on dose and endpoint used, and that PBK modeling provides a way to characterize REP values for PA-N-oxides at realistic low dietary exposure levels, thus reducing the need for animal experiments.

Physiologically-Based Kinetic Modeling Predicts Similar In Vivo Relative Potency of Senecionine N-Oxide for Rat and Human at Realistic Low Exposure Levels.

SCOPE: This study aims to determine if previously developed physiologically-based kinetic (PBK) model in rat can be modified for senecionine (SEN) and its N-oxide (SENO), and be used to investigate potential species differences between rat and human in relative potency (REP) of the N-oxide relative to the parent pyrrolizidine alkaloid (PA). METHODS AND RESULTS: In vitro derived kinetic parameters including the apparent maximum velocities (Vmax ) and Michaelis-Menten constants (Km ) for SENO reduction and SEN clearance are used to define the PBK models. The rat model is validated with published animal data, and the toxicokinetic profiles of SEN from either orally-administered SENO or SEN are simulated. REP values of SENO relative to SEN amount to 0.84 and 0.89 in rat and human, respectively. CONCLUSION: The REP value can be dose- and species-dependent, with the values for rat and human being comparable at low realistic exposure scenarios. In summary, PBK modeling serves as a valuable New Approach Methodology (NAM) tool for predicting REP values of PA-N-oxides and may actually result in more accurate REP values for human risk assessment than what would be defined using in vivo animal experiments.

Differences in gut microbial fructoselysine degradation activity between breast-fed and formula-fed infants.

The Amadori product fructoselysine is formed upon heating of food products and is abundantly present in infant formula while being almost absent in breast milk. The human gut microbiota can degrade fructoselysine for which interindividual differences have been described for adults. The aim of this study is to compare functional differences in microbial fructoselysine degradation between breast-fed and formula-fed infants, in view of their different diets and resulting different fructoselysine exposures. First, a publicly available metagenomic dataset with metagenome-assembled genomes (MAGs) from infant fecal samples was analyzed and showed that query genes involved in fructoselysine degradation (frlD/yhfQ) were abundantly present in multiple bacterial taxa in the fecal samples, with a higher prevalence in the formula-fed infants. Next, fecal samples collected from exclusively breast-fed and formula-fed infants were anaerobically incubated with fructoselysine. Both groups degraded fructoselysine, however the fructoselysine degradation activity was significantly higher by fecal samples from formula-fed infants. Overall, this study provides evidence that infant formula feeding, leading to increased dietary fructoselysine exposure, seems to result in an increased fructoselysine degradation activity in the gut microbiota of infants. This indicates that the infant gut microbiota adapts towards dietary fructoselysine exposure.

Identification of phosphodiesterase type-5 (PDE-5) inhibitors in herbal supplements using a tiered approach and associated consumer risk.

The use of herbal supplements for improved sexual performance is a common practice amongst the youth and some senior citizens in Ghana. These products are considered 'natural' and greatly preferred over synthetic alternatives due to the assurance of little to no adverse effects by producers. However, the high rate of adulteration often compromises their safety. Forty herbal supplements, of which 25 were previously shown to result in medium to high intake of phosphodiesterase type-5 (PDE-5) inhibitors using a PDE-Glo bioassay, were further investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to examine the reliability of the bioassay and whether the observed higher responses could be ascribed to inherent plant constituents or adulterants. Results showed significant amounts of vardenafil, tadalafil and especially sildenafil, in 2, 1 and 10 samples, respectively, with total concentration levels resulting in estimated daily intakes (EDIs) above 25 mg sildenafil equivalents with six supplements even having EDIs above 100 mg sildenafil equivalents. Only one sample contained a natural ingredient (icariin), but its concentration (0.013 mg g-1) was too low to explain the observed potency in the bioassay. The estimated concentrations of PDE-5 inhibitors in 35 supplements, according to the bioassay, were in line with those of the LC-MS/MS analysis. However, discrepancies were observed for five supplements. Further examination of one of the latter supplements using the PDE-Glo bioassay to select the positive fraction and further examination with LC-MS/MS and 1H-NMR revealed the presence of hydroxythiohomosildenafil, a sildenafil analogue not yet included in the liquid chromatography-mass spectrometry reference library. This study demonstrates the significance of applying a tiered approach, where the use of a bioassay is followed by chemical analysis of bioactive samples in order to identify unknown bioactive compounds.

Inter-individual variation in chlorpyrifos toxicokinetics characterized by physiologically based kinetic (PBK) and Monte Carlo simulation comparing human liver microsome and Supersome™ cytochromes P450 (CYP)-specific kinetic data as model input.

The present study compares two approaches to evaluate the effects of inter-individual differences in the biotransformation of chlorpyrifos (CPF) on the sensitivity towards in vivo red blood cell (RBC) acetylcholinesterase (AChE) inhibition and to calculate a chemical-specific adjustment factor (CSAF) to account for inter-individual differences in kinetics (HKAF). These approaches included use of a Supersome™ cytochromes P450 (CYP)-based and a human liver microsome (HLM)-based physiologically based kinetic (PBK) model, both combined with Monte Carlo simulations. The results revealed that bioactivation of CPF exhibits biphasic kinetics caused by distinct differences in the Km of CYPs involved, which was elucidated by Supersome™ CYP rather than by HLM. Use of Supersome™ CYP-derived kinetic data was influenced by the accuracy of the intersystem extrapolation factors (ISEFs) required to scale CYP isoform activity of Supersome™ to HLMs. The predicted dose-response curves for average, 99th percentile and 1st percentile sensitive individuals were found to be similar in the two approaches when biphasic kinetics was included in the HLM-based approach, resulting in similar benchmark dose lower confidence limits for 10% inhibition (BMDL10) and HKAF values. The variation in metabolism-related kinetic parameters resulted in HKAF values at the 99th percentile that were slightly higher than the default uncertainty factor of 3.16. While HKAF values up to 6.9 were obtained when including also the variability in other influential PBK model parameters. It is concluded that the Supersome™ CYP-based approach appeared most adequate for identifying inter-individual variation in biotransformation of CPF and its resulting RBC AChE inhibition.

In vitro and in silico study on consequences of combined exposure to the food-borne alkenylbenzenes estragole and safrole.

Potential consequences of combined exposure to the selected food-borne alkenylbenzenes safrole and estragole or their proximate carcinogenic 1'-hydroxy metabolites were evaluated in vitro and in silico. HepG2 cells were exposed to 1'-hydroxyestragole and 1'-hydroxysafrole individually or in equipotent combination subsequently detecting cytotoxicity and DNA adduct formation. Results indicate that concentration addition adequately describes the cytotoxic effects and no statistically significant differences were shown in the level of formation of the major DNA adducts. Furthermore, physiologically based kinetic modeling revealed that at normal dietary intake the concentration of the parent compounds and their 1'-hydroxymetabolites remain substantially below the Km values for the respective bioactivation and detoxification reactions providing further support for the fact that the simultaneous presence of the two carcinogens or of their proximate carcinogenic 1'-hydroxy metabolites may not affect their DNA adduct formation. Overall, these results point at the absence of interactions upon combined exposure to selected food-borne alkenylbenzenes at realistic dietary levels of intake.

Physiologically based kinetic modelling predicts the in vivo relative potency of riddelliine N-oxide compared to riddelliine in rat to be dose dependent.

Pyrrolizidine alkaloids (PAs) are toxic plant constituents occurring often in their N-oxide form. This raises the question on the relative potency (REP) values of PA-N-oxides compared to the corresponding parent PAs. The present study aims to quantify the in vivo REP value of riddelliine N-oxide compared to riddelliine using physiologically based kinetic (PBK) modelling, taking into account that the toxicity of riddelliine N-oxide depends on its conversion to riddelliine by intestinal microbiota and in the liver. The models predicted a lower Cmax and higher Tmax for the blood concentration of riddelliine upon oral administration of riddelliine N-oxide compared to the Cmax and Tmax predicted for an equimolar oral dose of riddelliine. Comparison of the area under the riddelliine concentration-time curve (AUCRID) obtained upon dosing either the N-oxide or riddelliine itself revealed a ratio of 0.67, which reflects the in vivo REP for riddelliine N-oxide compared to riddelliine, and appeared to closely match the REP value derived from available in vivo data. The models also predicted that the REP value will decrease with increasing dose level, because of saturation of riddelliine N-oxide reduction by the intestinal microbiota and of riddelliine clearance by the liver. It is concluded that PBK modeling provides a way to define in vivo REP values of PA-N-oxides as compared to their parent PAs, without a need for animal experiments.

A new approach methodology (NAM) for the prediction of (nor)ibogaine-induced cardiotoxicity in humans.

The development of non-animal-based new approach methodologies (NAMs) for chemical risk assessment and safety evaluation is urgently needed. The aim of the present study was to investigate the applicability of an in vitro-in silico approach to predict human cardiotoxicity of the herbal alkaloid ibogaine and its metabolite noribogaine, which are promising anti-addiction drugs. Physiologically based kinetic (PBK) models were developed using in silico-derived parameters and biokinetic data obtained from in vitro liver microsomal incubations and Caco-2 transport studies. Human induced pluripotent stem cell-derived cardiomyocytes combined with a multi-electrode array (MEA) assay were used to determine in vitro concentration-dependent cardiotoxicity reflected by prolongation of field potential duration, which was subsequently translated to in vivo dose-dependent prolongation of the QTc (heart rate corrected duration from ventricular depolarization to repolarization) using PBK modeling-based reverse dosimetry. Results showed that the predictions matched well with in vivo kinetic data and QTc data for ibogaine and noribogaine available in the literature, indicating a good performance of the NAM. Benchmark dose analysis of the predicted dose response curves adequately predicted the onset of in vivo cardiotoxicity detected by QTc prolongation upon oral exposure to ibogaine and noribogaine. The present study provides an additional proof-of-principle of using PBK modeling-based reverse dosimetry as a NAM to predict human cardiotoxicity.

Physiologically based kinetic modelling based prediction of in vivo rat and human acetylcholinesterase (AChE) inhibition upon exposure to diazinon.

The present study predicts in vivo human and rat red blood cell (RBC) acetylcholinesterase (AChE) inhibition upon diazinon (DZN) exposure using physiological based kinetic (PBK) modelling-facilitated reverse dosimetry. Due to the fact that both DZN and its oxon metabolite diazoxon (DZO) can inhibit AChE, a toxic equivalency factor (TEF) was included in the PBK model to combine the effect of DZN and DZO when predicting in vivo AChE inhibition. The PBK models were defined based on kinetic constants derived from in vitro incubations with liver fractions or plasma of rat and human, and were used to translate in vitro concentration-response curves for AChE inhibition obtained in the current study to predicted in vivo dose-response curves. The predicted dose-response curves for rat matched available in vivo data on AChE inhibition, and the benchmark dose lower confidence limits for 10% inhibition (BMDL10 values) were in line with the reported BMDL10 values. Humans were predicted to be 6-fold more sensitive than rats in terms of AChE inhibition, mainly because of inter-species differences in toxicokinetics. It is concluded that the TEF-coded DZN PBK model combined with quantitative in vitro to in vivo extrapolation (QIVIVE) provides an adequate approach to predict RBC AChE inhibition upon acute oral DZN exposure, and can provide an alternative testing strategy for derivation of a point of departure (POD) in risk assessment.

Incorporating renal excretion via the OCT2 transporter in physiologically based kinetic modelling to predict in vivo kinetics of mepiquat in rat.

The present study aimed at incorporating active renal excretion via the organic cation transporter 2 (OCT2) into a generic rat physiologically based kinetic (PBK) model using an in vitro human renal proximal tubular epithelial cell line (SA7K) and mepiquat chloride (MQ) as the model compound. The Vmax (10.5 pmol/min/mg protein) and Km (20.6 µM) of OCT2 transport of MQ were determined by concentration-dependent uptake in SA7K cells using doxepin as inhibitor. PBK model predictions incorporating these values in the PBK model were 6.7-8.4-fold different from the reported in vivo data on the blood concentration of MQ in rat. Applying an overall scaling factor that also corrects for potential differences in OCT2 activity in the SA7K cells and in vivo kidney cortex and species differences resulted in adequate predictions for in vivo kinetics of MQ in rat (2.3-3.2-fold). The results indicate that using SA7K cells to define PBK parameters for active renal OCT2 mediated excretion with adequate scaling enables incorporation of renal excretion via the OCT2 transporter in PBK modelling to predict in vivo kinetics of mepiquat in rat. This study demonstrates a proof-of-principle on how to include active renal excretion into generic PBK models.

Estragole DNA adduct accumulation in human liver HepaRG cells upon repeated in vitro exposure.

Accumulation of N2-(trans-isoestragol-3'-yl)-2'-deoxyguanosine (E-3'-N2-dG) DNA adducts derived from the alkenylbenzene estragole upon repeated dose exposure was investigated since the repair of this adduct was previously shown to be inefficient. To this end human HepaRG cells were exposed to repeating cycles of 2 h exposure to 50 µM estragole followed by 22 h repair to mimic daily exposure. The E-3'-N2-dG DNA adduct levels were quantified by LC-MS/MS after each cycle. The results show accumulation of E-3'-N2-dG DNA adducts at a rate of 17.53 adducts/108 nts/cycle. This rate at the dose level calculated by physiologically based kinetic (PBK) modeling to result in 50 µM was converted to a rate expected at average human daily intake of estragole. The predicted time estimated to reach adduct levels reported at the BMD10 of the related alkenylbenzene methyleugenol of 10-100 adducts /108 nts upon average human daily intake of estragole amounted to 8-80 (in rat) or 6-57 years (in human). It is concluded that the persistent nature of the E-3'-N2-dG DNA adducts may contribute to accumulation of substantial levels of DNA adducts upon prolonged dietary exposure.

Defining in vivo dose-response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach.

Aristolochic acid I (AAI) is a well-known genotoxic kidney carcinogen. Metabolic conversion of AAI into the DNA-reactive aristolactam-nitrenium ion is involved in the mode of action of tumor formation. This study aims to predict in vivo AAI-DNA adduct formation in the kidney of rat, mouse and human by translating the in vitro concentration-response curves for AAI-DNA adduct formation to the in vivo situation using physiologically based kinetic (PBK) modeling-based reverse dosimetry. DNA adduct formation in kidney proximal tubular LLC-PK1 cells exposed to AAI was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Subsequently, the in vitro concentration-response curves were converted to predicted in vivo dose-response curves in rat, mouse and human kidney using PBK models. Results obtained revealed a dose-dependent increase in AAI-DNA adduct formation in the rat, mouse and human kidney and the predicted DNA adduct levels were generally within an order of magnitude compared with values reported in the literature. It is concluded that the combined in vitro PBK modeling approach provides a novel way to define in vivo dose-response curves for kidney DNA adduct formation in rat, mouse and human and contributes to the reduction, refinement and replacement of animal testing.

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.

Molecular Dynamics and In Vitro Quantification of Safrole DNA Adducts Reveal DNA Adduct Persistence Due to Limited DNA Distortion Resulting in Inefficient Repair.

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.