Increased gut permeability and bacterial translocation after chronic chlorpyrifos exposure in rats

Abstract

The epithelium's barrier function is crucial for maintaining homeostasis and preventing the passage of food antigens and luminal bacteria. This function is essentially subserved by tight junctions (TJs), multiprotein complexes located in the most apical part of the lateral membrane. Some gastrointestinal disease states are associated with elevated intestinal permeability to macromolecules. In a study on rats, we determined the influence of chronic, daily ingestion of chlorpyrifos (CPF, a pesticide that crosses the placental barrier) during pre- and postnatal periods on intestinal permeability and TJ characteristics in the pups. Fluorescein isothiocyanate (FITC)-dextran was used as a marker of paracellular transport and mucosal barrier dysfunction. Pups were gavaged with FITC-dextran solution and blood samples were collected every 30 min for 400 min and analyzed spectrofluorimetrically. At sacrifice, different intestinal segments were resected and prepared for analysis of the transcripts (qPCR) and localization (using immunofluorescence) of ZO-1, occludin and claudins (scaffolding proteins that have a role in the constitution of TJs). In rats that had been exposed to CPF in utero and after birth, we observed a progressive increase in FITC-dextran passage across the epithelial barrier from 210 to 325 min at day 21 after birth (weaning) but not at day 60 (adulthood). At both ages, there were significant changes in intestinal TJ gene expression, with downregulation of ZO-1 and occludin and upregulation of claudins 1 and 4. In some intestinal segments, there were changes in the cellular localization of ZO-1 and claudin 4 immunostaining. Lastly, bacterial translocation to the spleen was also observed. The presence of CPF residues in food may disturb epithelial homeostasis in rats. Changes in TJ protein expression and localization may be involved in gut barrier dysfunction in this model. Uncontrolled passage of macromolecules and bacteria across the intestinal epithelium may be a risk factor for digestive inflammatory diseases.

Figure 1. Body mass (g) (A) and body length (cm) (B) of control rats (CPF0, open bars) or CPF-exposed pups (CPF1, 1 mg/kg/day, grey bars) at birth (D0), at weaning (D21) and as young adults (D60).

Values are quoted as the mean ± SEM. D0: n = 41, CPF1 n = 37; D21: CPF0 n = 28, CPF1 n = 24; D60: CPF0 n = 21, CPF1 n = 13. ** p<0.01.

Figure 2. The time course of the appearance of FITC-dextran in blood (µg/µL) in control rats (CPF0, grey line) or CPF-exposed pups (CPF1, 1 mg/kg/day, black line) at weaning (D21) and as young adults (D60).

Values are quoted as the mean ± SEM. D21: CPF0 n = 12, CPF1 n = 17; D60: CPF0 n = 14, CPF1 n = 13. * p<0.05.

Figure 3. Gene expression (mRNA) of markers of intestinal maturation in the ileum and colon of control rats (CPF0, open bars) or CPF-exposed pups ((CPF1, 1 mg/kg/day, grey bars) at weaning (D21) and as young adults (D60), expressed as 2^−DCt.

Values are quoted as the mean ± SEM. D21: CPF0 n = 28, CPF1 n = 24; D60: CPF0 n = 21, CPF1 n = 13. * p<0.05. (A) ZO-1; (B) occludin; (C) claudin1; (D) claudin4.

Figure 4. Immunofluorescent staining of ZO1 in the ileum and colon of CPF-exposed (1 mg/kg/day, CPF1) rats and control (CPF0) rats at weaning (D21) and as young adults (D60). Magnification: x 630.

D21: CPF0 n = 5, CPF1 n = 5; D60: CPF0 n = 5, CPF1 n = 5. ZO-1 detection at D21 and D60 in CPF0rats (panels A and C, respectively) and in CPF1rats (panels, B and D respectively).

Figure 5. Percentage of rats contaminated by bacterial translocation (mean ± SEM) to the spleen at weaning (D21) and as young adults (D60).

Control groups (CPF0, open bars) and CPF-exposed groups (CPF1: 1 mg/kg/day, grey bars). i, p<0.10; * p<0.05; ** p<0.01; *** p<0.001. D21: CPF0 n = 10, CPF1 n = 10; D60: CPF0 n = 10, CPF1 n = 10.