Sihuta, K. | Search bioRxiv

bioRxiv2026

The Nematicide Tioxazafen Disrupts Proteasome Function via Cytochrome P450 Bioactivation

Tioxazafen is an effective nematicide whose commercialization was halted because handlers reportedly developed rashes after working with seeds coated with a tioxazafen-laced cocktail. Here, we show that tioxazafen is bioactivated into toxic products by nematode and human cytochrome P450s. Through systematic analyses, we found that bioactivated tioxazafen disrupts proteasome function, leading to the accumulation of the NRF1 transcription factor ortholog SKN-1A in the nematode C. elegans, and a bounce-back transcriptional up-regulation of proteasome components. Genetic upregulation of the C. elegans proteasome supresses tioxazafens lethality, indicating that proteotoxicity is a key contributor to death. A survey of human P450s revealed that skin-expressed CYP1A1 toxifies tioxazafen and may account for the reaction to tioxazafen-coated seeds. We also found that rabbit CYP1A1 fails to bioactivate tioxazafen, which may explain the pre-market failure to detect robust adverse skin reactions. Our work highlights vulnerabilities in pre-market toxicological assays and a provides potential solution to prospectively identifying P450 toxication events. One-Sentence SummaryTioxazafen is bioactivated by cytochrome P450s into a proteasome disruptor.

pharmacology and toxicology↗

bioRxiv2026

A Cationic Amphiphilic Drug (CAD) Defense System in the Nematode Caenorhabditis elegans

Cationic Amphiphilic Drugs (CADs) severely disrupt lysosomal function, which leads to a cellular pathology characterized by excess phospholipids called phospholipidosis. Through a forward genetic screen and mining of published datasets, we discovered that CADs induce the expression of the CYP-35B family of cytochrome P450s and the PGP-13 p-glycoprotein pump via the nuclear receptors NHR-70 and NHR-107 in the nematode C. elegans. A pgp-13 fluorescent reporter revealed hundreds of human drugs that upregulate the CAD defense system in vivo. Chemoinformatic analyses indicate that the pgp-13 reporter may be useful in identifying CADs that have pathogenic potential in humans. Mutant analyses coupled to metabolomics and structural modeling show that the CYP-35Bs are necessary and sufficient for CAD metabolism, and that CYP-35B2 D311 is key in mediating electrostatic interactions with the positively charged CADs. We also show that CAD metabolites are effluxed via PGP-13 acting partially redundantly with PGP-14 and that an intact defense system is necessary to resist CAD-induced pathology. Finally, we demonstrate that bacteria that likely cohabitate with C. elegans in nature trigger the CAD defense system, providing a plausible explanation for why a pathway that protects against anthropogenic small molecules exists in nematodes.

pharmacology and toxicology↗

bioRxiv2024

Naturally occurring variation in a cytochrome P450 modifies thiabendazole responses independent of beta-tubulin

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35d1 and the nuclear hormone receptor nhr-176, identified by another mapping technique. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of nhr-176 and cyp-35d1 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive parental strain, and reciprocal allele replacement strains in both genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele is deleterious, but the lysine allele is selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance. Author SummaryBenzimidazoles (BZs) are the most common drug class used to control parasitic nematodes, but because of overuse, resistance is widespread. The known genetic causes of BZ resistance are associated with mutations in beta-tubulin and are the most well understood of any anthelmintic class. However, BZ response varies significantly and differential levels of resistance likely require mutations in genes independent of beta-tubulin. We used the free-living model nematode Caenorhabditis elegans to identify and characterize a novel cytochrome P450 gene, cyp-35d1, associated with natural resistance to the BZ drug thiabendazole (TBZ). We demonstrated that a lysine at position 267 confers TBZ resistance and is selected over multiple generations after TBZ treatment. This allele significantly increased the rate of TBZ metabolism in both C. elegans and yeast. In conclusion, we have characterized the role of variation in a cytochrome P450 that contributes to TBZ resistance, independent of mutations in beta-tubulin.

genetics↗