Propiconazole is a triazole fungicide previously shown to induce triglyceride accumulation in human liver HepaRG cells, potentially via activation of the Pregnane X Receptor (PXR). However, whether propiconazole can disrupt hepatic and whole-body metabolism in vivo is currently unknown. Therefore, we aimed to examine the metabolic effects of propiconazole in the context of metabolic dysfunction-associated steatotic liver disease (MASLD), obesity, and insulin resistance. To this end, male C57BL/6J mice were fed a high-fat diet for 20 weeks. During the last 10 weeks, mice additionally received vehicle, 0.04, 30, or 100 mg/kg body weight (bw)/day propiconazole via oral gavage. High-dose propiconazole, but not low or intermediate dose, reduced body weight gain and adipose tissue weight in obese mice. Mice receiving high-dose propiconazole displayed improved glucose tolerance and reduced levels of plasma triglycerides and cholesterol. Propiconazole dose-dependently increased liver weight and triglyceride levels and at high dose caused signs of hepatic inflammation. RNA sequencing on the liver revealed that propiconazole mainly induced PXR target genes. At intermediate and high dose, propiconazole induced pathways related to cell-cell interactions and inflammation, while oxidative phosphorylation was repressed by propiconazole. Comparison of gene regulation in wildtype and PXR knockout primary hepatocytes as well as gene reporter assays confirmed the activation of PXR by propiconazole. All in all, our data underscore the capacity of propiconazole to activate PXR in the liver and thereby promote the development of hepatic steatosis in vivo.

Department of Pharmacology and Toxicology Faculty of Pharmacy Charles University Ak Heyrovského 1203 50005 Hradec Králové Czech Republic

Division of Nutritional Sciences Cornell University Ithaca NY 14853 USA

Nutrition Metabolism and Genomics Group Division of Human Nutrition and Health Wageningen University Wageningen The Netherlands

Research Unit of Biomedicine and Internal Medicine Medical Research Center Oulu University of Oulu and Oulu University Hospital Oulu Finland

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Aleksunes LM, Klaassen CD (2012) Coordinated regulation of hepatic phase I and II drug-metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARα-, and Nrf2-null mice. Drug Metab Dispos 40:1366–1379 PubMed DOI PMC

Allen JW, Wolf DC, George MH et al (2006) Toxicity profiles in mice treated with hepatotumorigenic and non-hepatotumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol 34:853–862 PubMed DOI

Arena M, Auteri D, EFSA et al (2017) Peer review of the pesticide risk assessment of the active substance propiconazole. EFSA J 15:04887

Attema B, Janssen AWF, Rijkers D et al (2022) Exposure to low-dose perfluorooctanoic acid promotes hepatic steatosis and disrupts the hepatic transcriptome in mice. Mol Metab 66:101602 PubMed DOI PMC

Bhat VS, Hester SD, Nesnow S, Eastmond DA (2013) Concordance of transcriptional and apical benchmark dose levels for conazole-induced liver effects in mice. Toxicol Sci 136:205–215 PubMed DOI

Bitter A, Rümmele P, Klein K et al (2015) Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. Arch Toxicol 89:2089–2103 PubMed DOI

Blumberg B, Sabbagh W, Juguilon H et al (1998) SXR, a novel steroid and xenobioticsensing nuclear receptor. Genes Dev 12:3195–3205 PubMed DOI PMC

Bourgon R, Gentleman R, Huber W (2010) Independent filtering increases detection power for high-throughput experiments. Proc Natl Acad Sci U S A. https://doi.org/10.1073/pnas.0914005107 PubMed DOI PMC

Cai X, Young GM, Xie W (2021) The xenobiotic receptors PXR and CAR in liver physiology, an update. Biochimica Et Biophysica Acta (BBA)-Mole Basis Dis 1867:166101 DOI

Calkin AC, Tontonoz P (2012) Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR. Nat Rev Mol Cell Biol 13:213–224 PubMed DOI PMC

Carazo A, Dusek J, Holas O et al (2018) Teriflunomide is an indirect human constitutive androstane receptor (CAR) activator interacting with epidermal growth factor (EGF) signaling. Front Pharmacol 9:993 PubMed DOI PMC

Cave MC, Clair HB, Hardesty JE et al (2016) Nuclear receptors and nonalcoholic fatty liver disease. Biochimica Et Biophysica Acta (BBA)-Gene Regulat Mech 1859:1083–1099 DOI

Chávez-Talavera O, Tailleux A, Lefebvre P, Staels B (2017) Bile acid control of metabolism and inflammation in obesity, type 2 diabetes, dyslipidemia, and nonalcoholic fatty liver disease. Gastroenterology 152:1679–1694 PubMed DOI

Cui JY, Klaassen CD (2016) RNA-Seq reveals common and unique PXR-and CAR-target gene signatures in the mouse liver transcriptome. Biochimica Et Biophysica Acta (BBA)-Gene Regulat Mech 1859:1198–1217 DOI

Currie RA, Peffer RC, Goetz AK et al (2014) Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action. Toxicology 321:80–88 PubMed DOI

Dauwe Y, Mary L, Oliviero F et al (2023) Steatosis and metabolic disorders associated with synergistic activation of the CAR/RXR heterodimer by pesticides. Cells 12:1201 PubMed DOI PMC

EFSA (2015) Reasoned opinion on the review of the existing maximum residue levels (MRLs) for propiconazole according to Article 12 of Regulation (EC) No 396/2005. EFSA J 13:3975 DOI

EFSA, Anastassiadou M, Bernasconi G, et al (2021) Evaluation of confirmatory data following the Article 12 MRL review for propiconazole. EFSA J 19:e06405 PubMed

Elcombe CR, Peffer RC, Wolf DC et al (2014) Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: a case study with phenobarbital as a model constitutive androstane receptor (CAR) activator. Crit Rev Toxicol 44:64–82 PubMed DOI

Fragki S, Dirven H, Fletcher T et al (2021) Systemic PFOS and PFOA exposure and disturbed lipid homeostasis in humans: what do we know and what not? Crit Rev Toxicol 51:141–164 PubMed DOI

Frankish A, Diekhans M, Ferreira A-M et al (2019) GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res 47:D766–D773. https://doi.org/10.1093/nar/gky955 PubMed DOI

Ghisari M, Long M, Tabbo A, Bonefeld-Jørgensen EC (2015) Effects of currently used pesticides and their mixtures on the function of thyroid hormone and aryl hydrocarbon receptor in cell culture. Toxicol Appl Pharmacol 284:292–303 PubMed DOI

Goetz AK, Dix DJ (2009) Toxicogenomic effects common to triazole antifungals and conserved between rats and humans. Toxicol Appl Pharmacol 238:80–89 PubMed DOI

Grün F, Blumberg B (2006) Environmental obesogens: organotins and endocrine disruption via nuclear receptor signaling. Endocrinology 147:s50–s55 PubMed DOI

Gwag T, Meng Z, Sui Y et al (2019) Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis. J Hepatol 70:930–940 PubMed DOI PMC

He J, Gao J, Xu M et al (2013) PXR ablation alleviates diet-induced and genetic obesity and insulin resistance in mice. Diabetes 62:1876–1887 PubMed DOI PMC

Heindel JJ, Vom Saal FS, Blumberg B et al (2015) Parma consensus statement on metabolic disruptors. Environ Health 14:54. https://doi.org/10.1186/s12940-015-0042-7 PubMed DOI PMC

Heindel JJ, Blumberg B, Cave M et al (2017) Metabolism disrupting chemicals and metabolic disorders. Reprod Toxicol 68:3–33 PubMed DOI

Heise T, Schmidt F, Knebel C et al (2015) Hepatotoxic effects of (tri) azole fungicides in a broad dose range. Arch Toxicol 89:2105–2117 PubMed DOI

Hester SD, Wolf DC, Nesnow S, Thai S-F (2006) Transcriptional profiles in liver from rats treated with tumorigenic and non-tumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol 34:879–894 PubMed DOI

Jones SA, Moore LB, Shenk JL et al (2000) The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol Endocrinol 14:27–39 PubMed DOI

Kanehisa M, Furumichi M, Tanabe M et al (2017) KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45:D353–D361 PubMed DOI

Karpale M, Käräjämäki AJ, Kummu O et al (2021) Activation of pregnane X receptor induces atherogenic lipids and PCSK9 by a SREBP2-mediated mechanism. Br J Pharmacol 178:2461–2481 PubMed DOI

Karpale M, Hukkanen J, Hakkola J (2022) Nuclear receptor PXR in drug-induced hypercholesterolemia. Cells 11:313 PubMed DOI PMC

Karpale M, Kummu O, Kärkkäinen O et al (2023) Pregnane X receptor activation remodels glucose metabolism to promote NAFLD development in obese mice. Mol Metab 76:101779 PubMed DOI PMC

Kersten S, Desvergne B, Wahli W (2000) Roles of PPARs in health and disease. Nature 405:421–424 PubMed DOI

Knebel C, Kebben J, Eberini I et al (2018a) Propiconazole is an activator of AHR and causes concentration additive effects with an established AHR ligand. Arch Toxicol 92:3471–3486 PubMed DOI

Knebel C, Neeb J, Zahn E et al (2018b) Unexpected effects of propiconazole, tebuconazole, and their mixture on the receptors CAR and PXR in human liver cells. Toxicol Sci 163:170–181 PubMed DOI

Knebel C, Buhrke T, Süssmuth R et al (2019a) Pregnane X receptor mediates steatotic effects of propiconazole and tebuconazole in human liver cell lines. Arch Toxicol 93:1311–1322 PubMed DOI

Knebel C, Heise T, Zanger UM et al (2019b) The azole fungicide tebuconazole affects human CYP1A1 and CYP1A2 expression by an aryl hydrocarbon receptor-dependent pathway. Food Chem Toxicol 123:481–491 PubMed DOI

Krammer J, Digel M, Ehehalt F et al (2011) Overexpression of CD36 and acyl-CoA synthetases FATP2, FATP4 and ACSL1 increases fatty acid uptake in human hepatoma cells. Int J Med Sci 8:599 PubMed DOI PMC

Lapinskas PJ, Brown S, Leesnitzer LM et al (2005) Role of PPARα in mediating the effects of phthalates and metabolites in the liver. Toxicology 207:149–163 PubMed DOI

Lasch A, Marx-Stoelting P, Braeuning A, Lichtenstein D (2021) More than additive effects on liver triglyceride accumulation by combinations of steatotic and non-steatotic pesticides in HepaRG cells. Arch Toxicol 95:1397–1411 PubMed DOI PMC

Law CW, Chen Y, Shi W, Smyth GK (2014) voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 15:R29. https://doi.org/10.1186/gb-2014-15-2-r29 PubMed DOI PMC

Le Maire A, Grimaldi M, Roecklin D et al (2009) Activation of RXR–PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Rep 10:367–373 PubMed DOI PMC

Lee SM, Muratalla J, Diaz-Ruiz A et al (2021) Rosiglitazone requires hepatocyte PPARγ expression to promote steatosis in male mice with diet-induced obesity. Endocrinology 162:bqab175 PubMed DOI PMC

Lee BP, Dodge JL, Terrault NA (2024) National prevalence estimates for steatotic liver disease and subclassifications using consensus nomenclature. Hepatology 79:666–673 PubMed DOI

Lemaire G, Mnif W, Pascussi J-M et al (2006) Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system. Toxicol Sci 91:501–509 PubMed DOI

Liu R, Holik AZ, Su S, et al (2015) Why weight? Modelling sample and observational level variability improves power in RNAseq analyses. Nucleic Acids Res 43:e97 PubMed DOI PMC

Luisier R, Lempiäinen H, Scherbichler N et al (2014) Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors. Toxicol Sci 139:501–511 PubMed DOI

Ma Y, Liu D (2012) Activation of pregnane X receptor by pregnenolone 16 α-carbonitrile prevents high-fat diet-induced obesity in AKR/J mice. PLoS ONE 7:e38734 PubMed DOI PMC

Marx-Stoelting P, Knebel C, Braeuning A (2020) The connection of azole fungicides with xeno-sensing nuclear receptors, drug metabolism and hepatotoxicity. Cells 9:1192 PubMed DOI PMC

Morton T, Camp J, Van Deusen B, Williams C (2019) US EPA propiconazole human health risk assessment-122101. US EPA

Nakamura K, Moore R, Negishi M, Sueyoshi T (2007) Nuclear pregnane X receptor cross-talk with FoxA2 to mediate drug-induced regulation of lipid metabolism in fasting mouse liver. J Biol Chem 282:9768–9776 PubMed DOI

Patro R, Duggal G, Love MI et al (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417–419. https://doi.org/10.1038/nmeth.4197 PubMed DOI PMC

Rinella ME, Lazarus JV, Ratziu V et al (2023) A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology 78:1966–1986 PubMed DOI

Ritchie ME, Phipson B, Wu D et al (2015) limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43:e47–e47. https://doi.org/10.1093/nar/gkv007 PubMed DOI PMC

Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25. https://doi.org/10.1186/gb-2010-11-3-r25 PubMed DOI PMC

Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140. https://doi.org/10.1093/bioinformatics/btp616 PubMed DOI

Smith U, Kahn BB (2016) Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids. J Intern Med 280:465–475 PubMed DOI PMC

Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:1–25. https://doi.org/10.2202/1544-6115.1027 DOI

Soneson C, Love MI, Robinson MD (2015) Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res 4:1521. https://doi.org/10.12688/f1000research.7563.1 PubMed DOI

Spruiell K, Richardson RM, Cullen JM et al (2014) Role of pregnane X receptor in obesity and glucose homeostasis in male mice. J Biol Chem 289:3244–3261 PubMed DOI

Stahlhut RW, van Wijngaarden E, Dye TD et al (2007) Concentrations of urinary phthalate metabolites are associated with increased waist circumference and insulin resistance in adult US males. Environ Health Perspect 115:876–882 PubMed DOI PMC

Sun G, Thai S-F, Tully DB et al (2005) Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver. Toxicol Lett 155:277–287 PubMed DOI

Takeuchi S, Iida M, Yabushita H et al (2008) In vitro screening for aryl hydrocarbon receptor agonistic activity in 200 pesticides using a highly sensitive reporter cell line, DR-EcoScreen cells, and in vivo mouse liver cytochrome P450–1A induction by propanil, diuron and linuron. Chemosphere 74:155–165 PubMed DOI

Wada T, Gao J, Xie W (2009) PXR and CAR in energy metabolism. Trends Endocrinol Metab 20:273–279 PubMed DOI

Ward WO, Delker DA, Hester SD et al (2006) Transcriptional profiles in liver from mice treated with hepatotumorigenic and nonhepatotumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol 34:863–878 PubMed DOI

Warrilow AG, Parker JE, Kelly DE, Kelly SL (2013) Azole affinity of sterol 14α-demethylase (CYP51) enzymes from Candida albicans and Homo sapiens. Antimicrob Agents Chemother 57:1352–1360 PubMed DOI PMC

Wei P, Zhang J, Egan-Hafley M et al (2000) The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407:920–923 PubMed DOI

Willson TM, Kliewer SA (2002) PXR, CAR and drug metabolism. Nat Rev Drug Discov 1:259–266 PubMed DOI

Wilson CG, Tran JL, Erion DM et al (2016) Hepatocyte-specific disruption of CD36 attenuates fatty liver and improves insulin sensitivity in HFD-fed mice. Endocrinology 157:570–585 PubMed DOI

Wu Q, Li G, Zhao C-Y, et al (2023) Association between phthalate exposure and obesity risk: a meta-analysis of observational studies. Environ Toxicol Pharmacol 102:104240 PubMed DOI

Xie W, Barwick JL, Downes M et al (2000) Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435–439 PubMed DOI

Younossi Z, Tacke F, Arrese M et al (2019) Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology 69:2672–2682 PubMed DOI

Zahn E, Wolfrum J, Knebel C et al (2018) Mixture effects of two plant protection products in liver cell lines. Food Chem Toxicol 112:299–309 PubMed DOI

Zhou J, Zhai Y, Mu Y et al (2006) A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J Biol Chem 281:15013–15020 PubMed DOI

Zhou J, Febbraio M, Wada T et al (2008) Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARγ in promoting steatosis. Gastroenterology 134:556–567 PubMed DOI

Zhou C, King N, Chen KY, Breslow JL (2009) Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice [S]. J Lipid Res 50:2004–2013 PubMed DOI PMC