Predicting the in vivo developmental toxicity of fenarimol from in vitro toxicity data using PBTK modelling-facilitated reverse dosimetry approach.

In vitro methods are widely used in modern toxicological testing; however, the data cannot be directly employed for risk assessment. In vivo toxicity of chemicals can be predicted from in vitro data using physiologically based toxicokinetic (PBTK) modelling-facilitated reverse dosimetry (PBTK-RD). In this study, a minimal-PBTK model was constructed to predict the in-vivo kinetic profile of fenarimol (FNL) in rats and humans. The model was verified by comparing the observed and predicted pharmacokinetics of FNL for rats (calibrator) and further applied to humans. Using the PBTK-RD approach, the reported in vitro developmental toxicity data for FNL was translated to in vivo dose-response data to predict the assay equivalent oral dose in rats and humans. The predicted assay equivalent rat oral dose (36.46 mg/kg) was comparable to the literature reported in vivo BMD10 value (22.8 mg/kg). The model was also employed to derive the chemical-specific adjustment factor (CSAF) for interspecies toxicokinetics variability of FNL. Further, Monte Carlo simulations were performed to predict the population variability in the plasma concentration of FNL and to derive CSAF for intersubject human kinetic differences. The comparison of CSAF values for interspecies and intersubject toxicokinetic variability with their respective default values revealed that the applied uncertainty factors were adequately protective.

Detection of bisphenols in Indian surface water, tap water, and packaged drinking water using dispersive liquid-liquid microextraction: exposure assessment for health risk.

The prevalence of bisphenols (BPs) has been well documented in the aquatic environment of many countries, but such studies from India are quite limited. The present work aimed to determine the occurrence of BPs in surface water (n = 96), tap water (n = 172), and packaged drinking water (n = 42) and estimate their exposure to humans. For this, a simple, sensitive, cost-effective, and green analytical chemistry method based on dispersive liquid-liquid microextraction (DLLME) was employed. Bisphenol A (BPA) was found as the most prevalent bisphenol (mean concentration range = 980-6470 ng/L) in all the water samples, with a % detection frequency of 17-39%. Bisphenol S (BPS) and bisphenol Z (BPZ) were also detected in all types of water samples. The mean estimated daily intake (EDI) for total BPs (tap water and packaged drinking water) was found to be 474.37 ng/kg b.w./day in adults and 665.65 ng/kg b.w./day in children, respectively. This indicated that the total exposure to all the detected BPs obtained for adults and children was lower than the temporary tolerable daily intake (t-TDI) recommended by the European Food Safety Authority (EFSA) (4 µg/kg b.w./day), thereby posing no substantial risks to humans from consuming water from the tap and/or packaged drinking water.

Co-occurrence of mycotoxins: A review on bioanalytical methods for simultaneous analysis in human biological samples, mixture toxicity and risk assessment strategies.

Mycotoxins are the toxic chemical substances that are produced by various fungal species and some of these are harmful to humans. Mycotoxins are ubiquitous in nature and humans could be exposed to multiple mycotoxins simultaneously. Unfortunately, exposure to mixed mycotoxins is not very well studied. Various studies have demonstrated the capacity of mycotoxins to show synergistic effect in the presence of other mycotoxins, thus, increasing the risk of toxicity. Hence, it is important to monitor mixed mycotoxins in human biological samples which would serve as a crucial information for risk assessment. Through this review paper, we aim to summarize the mixture toxicity of mycotoxins and the various bio-analytical techniques that are being used for the simultaneous analysis of mixed mycotoxins in human biological samples. Different sample preparation and clean-up techniques employed till date for eliminating the interferences from human biological samples without affecting the analyses of the mycotoxins are also discussed. Further, a brief introduction of risk assessment strategies that have been or could be adopted for multiple mycotoxin risk assessments is also mentioned. To the best of our knowledge, this is the first review that focuses solely on the occurrence of multiple mycotoxins in human biological samples as well as their risk assessment strategies.

Cytochrome P450 isoforms contribution, plasma protein binding, toxicokinetics of enniatin A in rats and in vivo clearance prediction in humans.

Emerging mycotoxins, such as enniatin A (ENNA), are becoming a worldwide concern owing to their presence in different types of food and feed. However, comprehensive toxicokinetic data that links intake, exposure and toxicological effects of ENNA has not been elucidated yet. Therefore, the present study investigated the in vitro (rat and human) and in vivo (rat) toxicokinetic properties of ENNA. Towards this, an easily applicable and sensitive bioanalytical method was developed and validated for the estimation of ENNA in rat plasma. ENNA exhibited high plasma protein binding (99%), high hepatic clearance and mainly underwent metabolism via CYP3A4 (74%). The in-house predicted hepatic clearance (54 mL/min/kg) and observed in vivo rat clearance (55 mL/min/kg) were comparable. The predicted in vivo human hepatic clearance was 18 mL/min/kg. ENNA underwent slow absorption (Tmax = 4 h) and rapid elimination following oral administration to rats. The absolute oral bioavailability was 47%. The toxicokinetic findings for ENNA from this study will help in designing and interpreting toxicological studies in rats. Besides, these findings could be used in physiologically based toxicokinetic (PBTK) model development for exposure predictions and risk assessment for ENNA in humans.

Label-free plasma proteomics for the identification of the putative biomarkers of oral squamous cell carcinoma.

Mass spectrometry-based label-free proteomics is becoming the analytical tool of interest to identify and quantitate the biomarkers for cancer. The oral squamous cell carcinoma (OSCC) which is one of the leading cancers worldwide, lacks biomarkers for the early prognosis and diagnosis. The present study profiled plasma proteome of the Indian OSCC human patients using a label-free mass spectrometry proteomics approach. The study first time utilized the three most widely used data analysis software MaxQuant (MQ), Proteome discoverer (PD), and Trans proteomic pipeline (TPP) together for label-free quantitation of the proteins. The study identified 16 proteins which can be used as a signature protein panel for OSCC. The pathway analysis showed predominant involvement of the immune system, hemostasis as the major pathways that are indicative of the disease progression. The network analysis showed maximum interaction for the Fibronectin and C-reactive protein. The study demonstrates that plasma proteins contain signatures that can be used as putative biomarkers for OSCC. Based on the label-free quantitation and the mechanistic analysis C-reactive protein, Carbonic anhydrase-1, and Fibronectin are identified as putative biomarkers of OSCC. Once these findings are validated in the large cohorts these protein signatures can be used as a biomarker for OSCC. SIGNIFICANCE: The oral squamous cell carcinoma (OSCC) is the eighteenth most prevalent malignancy in the world and ranks second in India. There are no biomarkers that could be indicative of the diseased state. Various studies have been carried out on saliva and tumors of OSCC patients in India, but none of the studies have profiled the plasma. We utilized the label-free approach for the first time on the Indian population in generating the panel of plasma proteins which could be indicative of the diseased state. The study identified Carbonic anhydrase 1, C-reactive protein, and Fibronectin proteins as putative biomarkers for the OSCC. The study obtained the signature panel by utilizing the 3 most widely used software for the label-free quanatitation (LFQ) namely MaxQuant, Proteome Discoverer, and Trans proteomic pipeline. The utilization of 3 software for the LFQ reduced the bias and provided a comprehensive list of proteins. All the differential proteins were mechanistically studied for their biological relevance and the pathway and network analysis were carried out. The study helps us in increasing the understanding of the proteins which are involved in the progression of the diseases. Studying the panel of proteins from all biofluids along with plasma in large cohorts of the population will help in understanding the disease in greater depth and help in identifying the relevant biomarkers for the OSCC.

Determination of Bisphenol Analogues in Infant Formula Products from India and Evaluating the Health Risk in Infants Asssociated with Their Exposure.

Bisphenol A (BPA) is a well-recognized endocrine disruptor, and considering its adverse effects its use in infant bottles has been banned in many countries. Growing concern on the use of BPA has led to its replacement with its analogues in numerous applications. Present is the first report determining the occurrence of seven bisphenols (BPs: BPA, BPAF, BPC, BPE, BPFL, BPS, and BPZ) in Indian infant formula. A reliable and efficient UPLC-MS/MS method for their simultaneous determination was developed and validated in powdered infant formula (n = 68). The limit of quantification of the method was 0.19 ng/g for BPA, BPAF, BPE, BPS and BPZ and 0.78 ng/g for BPC and BPFL. The highest concentration was detected for BPA (mean = 5.46 ng/g) followed by BPZ and BPS. BPAF, BPFL, BPC and BPE were detected in none of the samples. The estimated daily intake (EDI) of total BPs in infants (0-12 months old infants) was determined to be 54.33-213.36 ng/kg b.w./day. BPA mainly contributed to the total intake (EDI = 92.76 ng/kg b.w./day). The dietary exposure to total BPs evaluated in the present study was approximately 1 order of magnitude lower than the reference value of BPA set by EFSA (4 µg/kg b.w./day) and, thus, may not pose considerable risks to infants.

Investigating the glucuronidation and sulfation pathways contribution and disposition kinetics of Bisphenol S and its metabolites using LC-MS/MS-based nonenzymatic hydrolysis method.

Despite showing serious health consequences and widespread exposure, the toxicokinetic information required to evaluate the health risks of BPS is insufficient. Thus, we aim to describe the comprehensive toxicokinetics of BPS and its glucuronide (BPS-G) and sulfate (BPS-S) metabolites in rats. Simultaneous quantification of BPS and its metabolites (authentic standards) was accomplished using UPLC-MS/MS method. BPS displayed rapid absorption, extensive metabolism and fast elimination after oral administration. Following intravenous administration, BPS exhibited CL (8.8 L/h/kg) higher than the rat hepatic blood flow rate suggesting the likelihood of extrahepatic clearance. The CL value differed from those reported previously (sheep and piglets) and the probable reason could be attributed to dose- and/or interspecies differences. BPS was extensively metabolized and excreted primarily through urine as BPS-G (∼56%). BPS and BPS-S exhibited a high protein binding capacity in comparison to BPS-G. In in vitro metabolic stability study, BPS was predominantly metabolized through glucuronidation. The predicted in vivo hepatic clearance of BPS suggested it to be a high and intermediate clearance chemical in rats and humans, respectively. The significant interspecies difference observed in the clearance of BPS between rats and humans indicated that toxicokinetics of BPS should be considered for health risk assessment in humans.

Pesticide interactions induce alterations in secondary structure of malate dehydrogenase to cause destability and cytotoxicity.

Environmental exposure to pesticides increases the risk of neurotoxicity and neurodegenerative diseases. The mechanism of pesticide-induced toxicity is attributed to the increased reactive oxygen species, mitochondrial dysfunction, inhibition of key cellular enzymes and accelerated pathogenic protein aggregation. The structural basis of pesticide-protein interaction is limited to pathogenic proteins such as α-synuclein, Tau and amyloid-beta. However, the effect of pesticides on metabolic proteins is still unexplored. Here, we used rotenone and chlorpyrifos to understand the interaction of these pesticides with a metabolic protein, malate dehydrogenase (MDH) and the consequent pesticide-induced cytotoxicity. We found that rotenone and chlorpyrifos strongly bind to MDH, interferes with protein folding and triggers alteration in its secondary structure. Both pesticides showed high binding affinities for MDH as observed by NMR and LCMS. Rotenone and chlorpyrifos induced structural alterations during MDH refolding resulting in the formation of cytotoxic conformers that generated oxidative stress and reduced cell viability. Our findings suggest that pesticides, in general, interact with proteins resulting in the formation of cytotoxic conformers that may have implications in neurotoxicity and neurodegenerative diseases.

Plasma protein binding, metabolism, reaction phenotyping and toxicokinetic studies of fenarimol after oral and intravenous administration in rats.

Fenarimol (FNL), an organic chlorinated fungicide, is widely used in agriculture for protection from fungal spores and fungi. Despite being an endocrine disruptor, no toxicokinetic data is reported for this fungicide. In the present work, we determined the plasma protein binding, metabolic pathways and toxicokinetics of FNL in rats. In vitro binding of FNL to rat and human plasma proteins was ∼90%, suggesting that FNL is a highly protein bound fungicide. The predicted in vivo hepatic clearance of FNL in rats and humans was estimated to be 36.71 and 14.39 mL/min/kg, respectively, indicating it to be an intermediate clearance compound. Reaction phenotyping assay showed that CYP3A4 mainly contributed to the overall metabolism of FNL. The oral toxicokinetic study of FNL in rats at no observed adverse effect level dose (1 mg/kg) showed maximum plasma concentration (C max) of 33.97 ± 4.45 ng/mL at 1 h (T max). The AUC0-∞ obtained was 180.18 ± 17.76 h*ng/mL, whereas, the t 1/2 was ∼4.74 h. Following intravenous administration, FNL displayed a clearance of 42.48 mL/min/kg which was close to the predicted in vivo hepatic clearance. The absolute oral bioavailability of FNL at 1 mg/kg dose in rats was 45.25%. FNL at 10 mg/kg oral dose exhibited non-linear toxicokinetics with greater than dose-proportional increase in the systemic exposure (AUC0-∞ 8270.53 ± 1798.59 h*ng/mL).

Species differences between rat and human in vitro metabolite profile, in vivo predicted clearance, CYP450 inhibition and CYP450 isoforms that metabolize benzanthrone: Implications in risk assessment.

Benzanthrone (BNZ) is a polycyclic aromatic hydrocarbon found in industrial effluent causing skin, respiratory, gastrointestinal, genitourinary, nervous and hemopoietic toxicity. While its toxicity has been well studied, its metabolism in humans has not been investigated. The aim of this study was to characterize species differences in the in vitro metabolism of BNZ in rat and human liver microsomes and to identify the CYP isoforms involved in its metabolism. Upon incubation in liver microsomes, BNZ was found to be a direct substrate of phase I metabolism in both rat and human, undergoing oxidation and reduction. The Km in rat, 11.62 ± 1.49 µM, was two-fold higher than humans (5.97 ± 0.83 µM) suggesting higher affinity for human CYPs. Further, incubation with human rCYPs, BNZ was found to be substrate of multiple CYPs. The predicted in vivo hepatic clearance was 63.55 and 18.91 mL/min/kg in rat and human, respectively, indicating BNZ to be a high clearance compound. BNZ was found to be a moderate inhibitor of human CYP1A2. BNZ metabolism by multiple CYPs indicates that single enzyme genetic polymorphism is unlikely to have profound effect on the toxicokinetics of BNZ and default uncertainty factor of 3.16 might be sufficient to capture the intraspecies kinetic variability.

Simultaneous determination of multiclass pesticide residues in human plasma using a mini QuEChERS method.

Blood is one of the most assessable matrices for the determination of pesticide residue exposure in humans. Effective sample preparation/cleanup of biological samples is very important in the development of a sensitive, reproducible, and robust method. In the present study, a simple, cost-effective, and rapid gas chromatography-tandem mass spectrometry method has been developed and validated for simultaneous analysis of 31 multiclass (organophosphates, organochlorines, and synthetic pyrethroids) pesticide residues in human plasma by means of a mini QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. We have adopted a modified version of the QuEChERS method, which is primarily used for pesticide residue analysis in food commodities. The QuEChERS method was optimized by use of different extraction solvents and different amounts and combinations of salts and sorbents (primary-secondary amines and C18) for the dispersive solid-phase extraction step. The results show that a combination of ethyl acetate with 2% acetic acid, magnesium sulfate (0.4 g), and solid-phase extraction for sample cleanup with primary-secondary amines (50 mg) per 1-mL volume of plasma is the most suitable for generating acceptable results with high recoveries for all multiclass pesticides from human plasma. The mean recovery ranged from 74% to 109% for all the analytes. The limit of quantification and limit of detection of the method ranged from 0.12 to 13.53 ng mL-1 and from 0.04 to 4.10 ng mL-1 respectively. The intraday precision and the interday precision of the method were 6% or less and 11% or less respectively. This method would be useful for the analysis of a wide range of pesticides of interest in a small volume of clinical and/or forensic samples to support biomonitoring and toxicological applications. Graphical Abstract Pesticide residues analysis in human plasma using mini QuEChERS method.