piperidines and spiroxamine

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Topics: Animals; Cell Differentiation; Cells, Cultured; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Homeobox Protein Nkx-2.5; Mice; Mixed Function Oxygenases; Morpholines; Mouse Embryonic Stem Cells; Myocytes, Cardiac; Myosin Heavy Chains; Piperidines; Retinoic Acid 4-Hydroxylase; Risk Assessment; Spiro Compounds; Time Factors; Toxicity Tests; Tubulin

Fusarium graminearum, the causal agent of wheat head blight, shows intrinsic resistance to amine fungicides. It is commonly accepted that the amines target sterol C-14 reductase and sterol Δ(8)-Δ(7) isomerase of ergosterol biosynthesis, encoded by the genes ERG24 and ERG2, respectively. Analysis of the genome sequence of F. graminearum revealed that the fungus contains two paralogous FgERG24 genes (FgERG24A and FgERG24B), which are homologous to the ERG24 of Saccharomyces cerevisiae. In this study, we disrupted FgERG24A and FgERG24B in F. graminearum. Compared to the wild-type strain HN9-1, FgERG24A and FgERG24B deletion mutants did not show recognizable phenotypic changes in mycelial growth on potato dextrose agar or in virulence on wheat heads. HPLC analysis showed that the amount of ergosterol in FgERG24A or FgERG24B deletion mutants was not significantly different from that in the wild-type strain. These results indicate that neither of the two genes is essential for growth, pathogenicity or ergosterol biosynthesis in F. graminearum. FgERG24B deletion mutants exhibited significantly increased sensitivity to amine fungicides, including tridemorph, fenpropidin and spiroxamine, but not to non-amine fungicides. In contrast, FgERG24A deletion mutants did not show changed sensitivity to any amine tested. The resistance of the FgERG24B deletion mutant to amines was restored by genetic complementation of the mutant with wild-type FgERG24B. These results indicate that FgERG24B controls the intrinsic resistance of F. graminearum to amines. The finding of this study provides new insights into amine resistance in filamentous fungi.

Topics: Amines; Drug Resistance, Fungal; Ergosterol; Fungicides, Industrial; Fusarium; Gene Deletion; Microbial Sensitivity Tests; Morpholines; Oxidoreductases; Piperidines; Plant Diseases; Spiro Compounds; Steroid Isomerases; Triticum