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.

DBS-platform for biomonitoring and toxicokinetics of toxicants: proof of concept using LC-MS/MS analysis of fipronil and its metabolites in blood.

A simple, sensitive and high throughput LC-MS/MS method was developed and validated for quantification of fipronil, fipronil sulfone and fipronil desulfinyl in rat and human dried blood spots (DBS). DBS samples were prepared by spiking 10 µl blood on DMPK-C cards followed by drying at room temperature. The whole blood spots were then punched from the card and extracted using acetonitrile. The total chromatographic run time of the method was only 2 min. The lower limit of quantification of the method was 0.1 ng/ml for all the analytes. The method was successfully applied to determine fipronil desulfinyl in DBS samples obtained from its toxicokinetic study in rats following intravenous dose (1 mg/kg). In conclusion, the proposed DBS methodology has significant potential in toxicokinetics and biomonitoring studies of environmental toxicants. This microvolume DBS technique will be an ideal tool for biomonitoring studies, particularly in paediatric population. Small volume requirements, minimally invasive blood sampling method, easier storage and shipping procedure make DBS a suitable technique for such studies. Further, DBS technique contributes towards the principles of 3Rs resulting in significant reduction in the number of rodents used and refinement in sample collection for toxicokinetic studies.