Exposure Cessation During Adulthood Did Not Prevent Immunotoxicity Caused by Developmental Exposure to Low-Level Trichloroethylene in Drinking Water

Abstract

Exposure to the water pollutant trichloroethylene (TCE) can promote autoimmunity in both humans and rodents. Using a mouse model we have shown that chronic adult exposure to TCE at 500 μg/ml in drinking water generates autoimmune hepatitis in female MRL+/+ mice. There is increasing evidence that developmental exposure to certain chemicals can be more toxic than adult exposure. This study was designed to test whether exposure to a much lower level of TCE (0.05 μg/ml) during gestation, lactation, and early life generated autoimmunity similar to that found following adult exposure to higher concentrations of TCE. When female MRL+/+ mice were examined at postnatal day (PND) 259 we found that developmental/early life exposure [gestational day 0 to PND 154] to TCE at a concentration 10 000 fold lower than that shown to be effective for adult exposure triggered autoimmune hepatitis. This effect was observed despite exposure cessation at PND 154. In concordance with the liver pathology, female MRL+/+ exposed during development and early life to TCE (0.05 or 500 μg/ml) generated a range of antiliver antibodies detected by Western blotting. Expression of proinflammatory cytokines by CD4+ T cells was also similarly observed at PND 259 in the TCE-exposed mice regardless of concentration. Thus, exposure to TCE at approximately environmental levels from gestational day 0 to PND 154 generated tissue pathology and CD4+ T cell alterations that required higher concentrations if exposure was limited to adulthood. TCE exposure cessation at PND 154 did not prevent the immunotoxicity.

Keywords: autoimmune; developmental exposure and adult disease; trichloroethylene.

FIG. 1

Level of direct TCE exposure in offspring. TCE exposure (μg/kg/d) from weaning at PND 21 to PND 154 was calculated in the offspring based on average body weight over time and average consumption of water containing either 500 or 0.05 μg/ml TCE. The offspring were also exposed to TCE during gestation and lactation due to the presence of TCE (500 or 0.05 μg/ml) in maternal drinking water.

FIG. 2

TCE exposure did not alter spleen cell percentages. Spleen cellularity in the dams 1 week after their offspring were weaned, and in the female offspring at PND 154 and PND 259 was determined by flow cytometry and represented as a percentage of spleen cells.

FIG. 3

Despite cessation, TCE exposure increased number of total and effector/memory CD4⁺ T cells. Total spleen cell numbers in the female offspring at PND 259 were measured (A). Based on these results the spleen cells percentages described in Figure 2 were represented as cell numbers/spleen (B). The percentage of effector/memory (CD62L^l°) CD4⁺ and CD8⁺T cells in the spleens were also measured, and are presented as percentages (C), and total cell numbers (D). *Statistically different from control values.

FIG. 4

Despite cessation, TCE exposure altered CD4⁺ T cell gene expression. Expression of several cytokine genes in CD4⁺ T cells isolated from the offspring of the female mice at PND 259 and activated in vitro was measured by qRT-PCR. *Statistically different from control values.

FIG. 5

Cessation does not prevent TCE-induced liver pathology. Even though TCE exposure ended at PND 154, liver pathology commensurate with AIH was observed at PND 259. Pairwise comparison of liver pathology scores were conducted using the nonparametric Wilcoxon-Mann-Whitney test. *P values from Wilcoxon-Mann-Whitney test are reported. Kidney pathology was not increased by TCE exposure under these conditions.

FIG. 6

Effects of TCE on other autoimmune markers. A, Serum levels of ANA (ssDNA) were measured at different time points. B, Pooled sera from 8 female mice collected at PND 259 following exposure to 0.05 or 500 μg/ml TCE until PND 154 reacted with liver protein separated by SDS-PAGE. Mouse mIgG was run in adjoining lanes and detected by a shared secondary antibody (HRP-labeled goat antimouse IgG) in order to normalize exposure times.

FIG. 7

TCE exposure increased antiliver antibody production. A, Sera from pairs of mice in each treatment group collected at PND259 was pooled. Three representative pairs of sera/group were reacted with liver protein by Western blotting as described in Figure 6B. B, Densitometric analysis of total antiliver antibodies was normalized to mouse mIgG run in adjoining lanes as described in Figure 6. The F test significance (P = .0046) of the 1-way ANOVA was followed by a Holm procedure in which pairwise contrasts were made relative to the control values. *Significant with corresponding Holm P values.