Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance

• Published in: Toxicology (Amsterdam) 2024-06, Vol.505, p.153828-153828, Article 153828
• Main Authors: Goettel, Manuela, Werner, Christoph, Honarvar, Naveed, Gröters, Sibylle, Fegert, Ivana, Haines, Corinne, Chatham, Lynsey R., Vardy, Audrey, Lake, Brian G.
• Format: Article
• Language: English
• Subjects: androstanes; Animals; Constitutive Androstane Receptor; cytochrome P-450; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; DNA replication; DNA Replication - drug effects; dose response; Dose-Response Relationship, Drug; enzymes; epidermal growth factor; fatty acids; Female; females; Fluxapyroxad; fungicides; Fungicides, Industrial - toxicity; hepatocytes; Hepatocytes - drug effects; Hepatocytes - metabolism; Hepatocytes - pathology; Humans; hypertrophy; liver; Liver - drug effects; Liver - metabolism; Liver - pathology; liver neoplasms; Liver Neoplasms - chemically induced; Liver Neoplasms - metabolism; Liver Neoplasms - pathology; Liver Neoplasms, Experimental - chemically induced; Liver Neoplasms, Experimental - metabolism; Liver Neoplasms, Experimental - pathology; Male; males; mechanism of action; Microsomes, Liver - drug effects; Microsomes, Liver - metabolism; Organ Size - drug effects; phenobarbital; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Replicative DNA synthesis; sodium; Sodium phenobarbital, Mode of action for liver tumour formation; subfamily
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/j.tox.2024.153828

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50–3000 ppm fluxapyroxad for 7 days and 250–3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250–3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1–100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1–100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1–16 μM fl