Deletion of Constitutive Androstane Receptor Led to Intestinal Alterations and Increased Imidacloprid in Murine Liver

Abstract

Imidacloprid (IMI) is the most frequently detected neonicotinoid pesticide in the environment. Despite typically low toxicity in vertebrates, IMI exposure is associated with liver and gastrointestinal toxicity. The mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, such as the constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. This study examined the role of CAR in mediating IMI off-target toxicity. Female Car^-/- and wild-type (WT) mice were orally administered imidacloprid (50 mg/kg, twice daily) for 21 days, following which serum, liver, and intestinal tissues were collected. Liver tissue analysis indicated mild inflammation and induction of detoxification gene Cyp2b10 in IMI-exposed WT mice. The absence of CAR increased hepatic IMI accumulation. Microbiome analysis of ileal samples revealed IMI altered microbial diversity in a genotype-specific manner, with increased α-diversity in Car^-/- mice while decreased α-diversity in WT mice. We observed Car^-/- mice exhibit intestinal alterations with decreased CYP-P450 expression, blunted villi height, and increased small intestine length and weight independent of IMI exposure. Our results suggest that IMI is not overtly toxic. However, the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the villi structure and Cyp gene expression in the intestine.

Keywords: gastrointestinal tract; imidacloprid; microbiome; neonicotinoids; nuclear receptor; toxicity.

Figure 1.

Effect of imidacloprid on the liver. A, Mice were orally dosed with IMI (50 mg/kg BW) twice daily, for 21 days. Tissue samples were collected to examine influence of IMI toxicity. B–D, Serum aspartate transaminase (AST), alanine transaminase (ALT), and triglycerides levels were not induced upon this dose of IMI exposure. E, Representative images of hematoxylin and eosin–stained liver sections from control and IMI-fed mice. IMI-fed livers showed mild inflammatory pockets. F, Detoxification gene Cyp2b10 was induced in IMI-fed livers. Arrow indicates inflammatory cell cluster. Values are displayed as mean ± SD. Statistics were calculated using Student t test analysis. ***P < 0.001. N = 6 mice per group. Schematic was created with BioRender.com.

Figure 2.

Excess IMI accumulation in the Car^−/− liver does not alter hepatic Cyp450 gene expression. Serum and liver tissue was analyzed by mass spectrometry to measure IMI accumulation. Car^−/− mice accumulated more IMI than wild-type mice (A-B). Car^−/− and wild-type livers (control and IMI-fed) were examined to quantify expression of genes involved in detoxification and oxidative stress. Hepatic gene expression of phase I genes Cyp3a11 (C), Cyp2e1 (D), and phase II Sult2a1 (E) and antioxidant regulating transcription factor Nrf2 (F) was unaltered by IMI exposure. Values are displayed as mean ± SD. Statistics were calculated using two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05, ***P < 0.001, ****P < 0.0001. N = 5-8 mice per group.

Figure 3.

Absence of CAR drastically reduced ileal Cyp450 gene expression and alters villi length while IMI reduces mucus production in the ileum. Ileal tissues from Car^−/− and wild-type mice (control and IMI-fed) were examined to quantify expression of genes involved in detoxification and oxidative stress. Ileal expression of phase I genes Cyp3a11 (A), Cyp2e1 (B), Cyp2b10 (C), were unaltered by IMI exposure. However, Cyp3a11 (A) and Cyp2e1 (B) expression, was blunted in Car^−/− ileum. Expression of Nrf2 gene, which is a key regulator of antioxidant mechanismswas also unaltered by IMI exposure (D). Values are displayed as mean ± SD. N = 5-8 mice per group. Ileum tissue sections were analyzed for villi structure and mucin production by Alcian Blue/Periodic Acid-stain (E). Car^−/− villi had decreased length compared to wild-type control (F). Goblet cells were decreased in IMI-treated wild-type and Car^−/− mice compared to their respective control group (G). Statistics were calculated using two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05, **P < 0.01. N = 5 mice per group, 15 villi per mouse. 200 μm.

Figure 4.

Influence of Imidacloprid on ileum microbiota. Abundance heat map of bacterial families varied more by the genotype than treatment (A). Taxonomic abundance plots showed Erysipelotrichaceae spp more prevalent in Car^−/−, while Bacillaceae spp were present in higher levels in WT (B). Number of observed species based on amplicon sequence variant (ASV) present in each type of treatment (C). Shannon diversity (evenness and distribution) of bacterial species (D). Bray Curtis PCoA plot shows bacterial communities clustered more by genotype than treatment (E).