Impact of the gut microbiome on hepatic cytochrome P450 3A4 (CYP3A4) in humanized pregnane X receptor-constitutive androstane receptor-CYP3A4/3A7 mice

The interaction between the gut microbiome and drug metabolism is bidirectional and can influence the pharmacokinetics of certain drugs. In mice, the gut microbiome has been shown to influence Cyp3a11. However, evidence for microbial regulation of human CYP3A4 is lacking. We aimed to bridge this gap by manipulating the microbiome of a humanized mouse model expressing CYP3A4, CYP3A7, PXR and CAR. Three groups of male and female humanized mice were studied: conventional (CV), germ-free (GF), and germ-free mice conventionalized using sex-matched pooled human fecal samples (GFCV). The presence of microbiome upregulated CYP3A4 expression by 7.6-fold in male CV mice ( p < 0.001) but downregulated CYP3A4 expression by 1.69-fold in female CV mice ( p = 0.012) compared to GF mice. The human fecal microbiome transplant to sex-matched GF mice resulted in decreased microbial diversity ( p < 0.05 in males and p < 0.01 in females) and was not effective in restoring CYP3A4 expression, suggesting complex underlying microbe-CYP3A4 interactions. We show that the hepatic CYP3A4 mRNA and protein expression were strongly correlated (R = 0.91; p = 2.6 x 10 -6 ). A total of 57 bacterial species from the mouse gut microbiome were identified to be significantly correlated with CYP3A4 protein expression ( p < 0.05). Five bile acids and no short chain fatty acids were correlated with CYP3A4 protein expression. In summary, alterations in the gut microbiome influenced hepatic CYP3A4 in humanized mice in a sex-dependent manner, with distinct microbes strongly correlating with this regulatory pattern. Significance Statement This study is the first to evaluate the expression of CYP3A4 under different microbial conditions in a humanized mouse model, including conventionalization of germ-free mice using pooled sex-matched human feces. Alterations in the gut microbiome influenced hepatic CYP3A4 in a sex-dependent manner and were strongly correlated with microbial species.