MC-LR detoxication in human and rats: inter-organ and inter-species differences

The transition from animal-based chemical risk assessment to Next-Generation Risk Assessment requires the integration of human-relevant New Approach Methodologies and mechanistic kinetic data to support physiologically based kinetic (PBK) modelling for quantitative in vitro-to-in vivo extrapolation. This study investigated isoform-specific and extrahepatic glutathione (GSH) conjugation of Microcystin-LR (MC-LR), a widespread cyanobacterial toxin of emerging concern in food and feed safety. MC-LR detoxification occurs via spontaneous GSH conjugation or catalysed by glutathione-S-transferases (GSTs). A panel of recombinant human GSTs and pooled human and rat intestinal and kidney cytosols was incubated with 1-60 µM MC-LR to characterise kinetic parameters and intrinsic clearance (Cli). The tested GST isoforms catalysed MC-LR conjugation with efficiencies spanning a 15-fold range, with P1 and T1 showing the highest activity. Isoforms exhibited distinct kinetic behaviours, including positive cooperativity, influencing their relative contribution across substrate concentrations. Human intestinal and renal cytosols showed GST-mediated metabolic efficiencies comparable to hepatic data, attributable to the high expression of GSTP1 and T1. However, when scaling to whole-organ Cli, the hepatic enzymatic conjugation exceeded extrahepatic one. In rats, intestine and kidney detoxification capacity was low with respect to the hepatic one, the latter resulting 3.3-fold more efficient than the human GST-mediated reaction. Although the spontaneous conjugation in vitro accounted for 70-80% of total conjugate formation, when the reaction rate scaled to the total hepatic cytosolic volume, its relative contribution was lower than the GST-mediated one. In addition the enzymatic reaction became dominant even in vitro under GSH depletion, at low MC-LR concentrations, typical of long term exposure. These findings underscore the relevance of GST isoform distribution, polymorphism, and GSH availability in driving inter-organ, interspecies, and inter-individual variability in MC-LR detoxification. Overall, this study fills critical data gaps on MC-LR kinetics and provides quantitative parameters to inform PBK model development.