Effects of chlorpyrifos exposure on liver inflammation and intestinal flora structure in mice

Abstract

Chlorpyrifos (CPF) is an organophosphate insecticide commonly used to treat fruit and vegetable crops. CPF can cause severe adverse effects on body organs including the liver and central nervous system. This study investigated the CPF-induced inflammation in mice and explored the role of intestinal flora changes in liver inflammation. Adult C57BL/6 male mice were exposed to a CPF of 0.01-, 0.1-, 1- and 10-mg/kg bodyweight for 12 weeks. The mice in experimental group given CPF solution dissolved in corn oil vehicle by gavage, was administered by intraoral gavage for 5 days per week for 12 weeks. Histopathological examination and inflammatory factor detection were performed on mice liver tissue. Faeces were used for 16S ribosomal RNA high-throughput sequencing to explore the impact of CPF on intestinal flora structure and diversity. The results showed that 1- and 10-mg/kg CPF caused different degrees of liver focal inflammation. The structure of intestinal flora changed significantly in mice including the decreased beneficial bacteria (Akkermansia, Prevotella and Butyricimonas) and increased pathogenic bacteria (Helicobacter and Desulfovibrio). Meanwhile, the results of Q-RT-PCR showed that there was more total bacterial DNA in the liver tissue of the mice treated with 10-mg/kg groups. In conclusion, the imbalance of intestinal flora, the decreased abundance of beneficial bacteria and the increased abundance of pathogenic bacteria, as well as the increase of total bacterial DNA in the liver tissues, maybe associated with the liver focal inflammation induced by CPF.

Keywords: 16S rRNA; chlorpyrifos; intestinal flora; liver inflammation.

Figure 1

body weight (A) and the liver coefficient (B) of mice; each value is expressed as a mean ± SD, n = 10. ^*P < 0.05, ^**P < 0.01 vs. control (0 mg/kg).

Figure 2

histological evaluation of the livers of mice from control and CPF-exposed groups: (A) mice from control group; (B) mice treated with 0.01 mg/kg CPF; (C) mice treated with 0.1 mg/kg CPF; (D) mice treated with 1 mg/kg CPF and (E) mice treated with 10 mg/kg CPF; the red arrows indicate necrotic hepatocytes, and the photomicrographs were taken at ×200 magnification after H&E staining, n = 4.

Figure 3

the levels of TNF-a (A), IL-1β (B) and IL-6 (C) in the livers of mice were measured by ELISA; each value is expressed as a mean ± SD, n = 6. ^*P < 0.05, ^**P < 0.01.

Figure 4

effects of CPF on the gut microbiota composition of the fecal, n = 6; (A) Venn diagram of OTUs among each group, and each symbol represents one sample; (B) analysis of β diversity of intestinal flora in mice, and PCoA was performed to calculate the distances between fecal samples from the mice of control and CPF groups, and each point represents a sample. A clear separation is observed between the samples of control and CPF3 and CPF4 groups.

Figure 5

the composition of gut microbiota at the phylum level after exposure to CPF, n = 6.

Figure 6

(A) significantly differences of the gut microbiota induced by CPF between CPF3, CPF4 and control groups at genus level; (B) significantly differences of Helicobacter induced by CPF between CPF3, CPF4 and control groups. Data were showed as relative abundance (%) of genus in each group, and statistical analysis was performed by the Kruskal–Wallis H test, n = 6, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 7

the total bacterial load in the livers of mice from CPF3, CPF4 and control groups, and each value is expressed as a mean ± SD, n = 3, ^*P < 0.05, ^**P < 0.01.