Flumioxazin metabolism in pregnant animals and cell-based protoporphyrinogen IX oxidase (PPO) inhibition assay of fetal metabolites in various animal species to elucidate the mechanism of the rat-specific developmental toxicity.

Flumioxazin, an N-phenylimide herbicide, inhibits protoporphyrinogen oxidase (PPO), a key enzyme in heme biosynthesis in mammals, and causes rat-specific developmental toxicity. The mechanism has mainly been clarified, but no research has yet focused on the contribution of its metabolites. We therefore conducted in vivo metabolism studies in pregnant rats and rabbits, and found 6 major known metabolites in excreta. There was no major rat-specific metabolite. The most abundant component in rat fetuses was APF, followed by flumioxazin and 5 identified metabolites. The concentrations of flumioxazin and these metabolites in fetuses were lower in rabbits than in rats. In vitro PPO inhibition assays with rat and human liver mitochondria showed that flumioxazin is a more potent PPO inhibitor than the metabolites. There were no species differences in relative intensity of PPO inhibition among flumioxazin and these metabolites. Based on the results of these in vivo and in vitro experiments, we concluded that flumioxazin is the causal substance of the rat-specific developmental toxicity. As a more reliable test system for research on in vitro PPO inhibition, cell-based assays with rat, rabbit, monkey, and human hepatocytes were performed. The results were consistent with those of the mitochondrial assays, and rats were more sensitive to PPO inhibition by flumioxazin than humans, while rabbits and monkeys were almost insensitive. From these results, the species difference in the developmental toxicity was concluded to be due to the difference in sensitivity of PPO to flumioxazin, and rats were confirmed to be the most sensitive of these species.

Differential expression and function of alternative splicing variants of human liver X receptor α.

The liver X receptor α (LXRα) is a nuclear receptor that is involved in regulation of lipid metabolism, cellular proliferation and apoptosis, and immunity. In this report, we characterize three human LXRα isoforms with variation in the ligand-binding domain (LBD). While examining the expression of LXRα3, which lacks 60 amino acids within the LBD, we identified two novel transcripts that encode LXRα-LBD variants (LXRα4 and LXRα5). LXRα4 has an insertion of 64 amino acids in helix 4/5, and LXRα5 lacks the C-terminal helices 7 to 12 due to a termination codon in an additional exon that encodes an intron in the LXRα1 mRNA. LXRα3, LXRα4, and LXRα5 were expressed at lower levels compared with LXRα1 in many human tissues and cell lines. We also observed weak expression of LXRα3 and LXRα4 in several tissues of mice. LXR ligand treatment induced differential regulation of LXRα isoform mRNA expression in a cell type-dependent manner. Whereas LXRα3 had no effect, LXRα4 has weak transactivation, retinoid X receptor (RXR) heterodimerization, and coactivator recruitment activities. LXRα5 interacted with a corepressor in a ligand-independent manner and inhibited LXRα1 transactivation and target gene expression when overexpressed. Combination of LXRα5 cotransfection and LXRα antagonist treatment produced additive effects on the inhibition of ligand-dependent LXRα1 activation. We constructed structural models of the LXRα4-LBD and its complexes with ligand, RXR-LBD, and coactivator peptide. The models showed that the insertion in the LBD can be predicted to disrupt RXR heterodimerization. Regulation of LXRα pre-mRNA splicing may be involved in the pathogenesis of LXRα-related diseases.