Cellular and molecular etiology of hepatocyte injury in a murine model of environmentally induced liver abnormality

Abstract

Exposures to a wide variety of environmental substances are negatively associated with many biological cell systems both in humans and rodents. Trichloroethane (TCE), a ubiquitous environmental toxicant, is used in large quantities as a dissolvent, metal degreaser, chemical intermediate, and component of consumer products. This increases the likelihood of human exposure to these compounds through dermal, inhalation and oral routes. The present in vivo study was aimed to investigate the possible cellular and molecular etiology of liver abnormality induced by early exposure to TCE using a murine model. The results showed a significant increase in liver weight. Histopathological examination revealed a TCE-induced hepatotoxicity which appeared as heavily congested central vein and blood sinusoids as well as leukocytic infiltration. Mitotic figures and apoptotic changes such as chromatin condensation and nuclear fragments were also identified. Cell death analysis demonstrates hepatocellular apoptosis was evident in the treated mice compared to control. TCE was also found to induce oxidative stress as indicated by an increase in the levels of lipid peroxidation, an oxidative stress marker. There was also a significant decrease in the DNA content of the hepatocytes of the treated groups compared to control. Agarose gel electrophoresis also provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation in the liver cells, indicating oxidative stress as the cause of DNA damage. These results suggest the need for a complete risk assessment of any new chemical prior to its arrival into the consumer market.

Keywords: Apoptosis; DNA damage; Environmental toxicant; Liver; Oxidative stress.

Fig. 1

Representative light photomicrograph of hepatic tissue sections from control and TCE-treated mice (H&E 100X). (a) Hepatic tissues of sham control mice with normal hepatic lobules and hepatocytes. (b) Hepatic tissues of 100 μg/kg TCE-treated mice with mild steatosis and inflammatory infiltrates. (c) Hepatic tissues of 400 μg/kg TCE-treated mice with severe steatosis and inflammatory infiltrates. Fibrotic and hepatocyte alterations can be seen in the TCE-treated tissues. The arrows in panel a, b, and c indicate a cell with vacuolated and pale-staining cytoplasm and alterations in nuclear morphology respectively. n=6. Scale bar = 250 μm.

Fig. 2

Quantification of hepatocytes with fragmented DNA. (a) Representative photomicrographs of hepatic tissues from control and TCE-treated mice (H&E 400X). Control mice nucleus showed no or little histopathological changes (normal nuclear appearance). Hepatocytes were partly lost and some cells exhibited necrotic characteristics after TCE exposure (arrows). In TCE-treated mice, the nuclei also exhibited typical apoptotic morphology as condensed and fragmented (arrows). Scale bar = 30 μm. (b) Quantification of hepatic cell death in control and TCE-treated mice. Data are expressed as the mean ± SEM (n = 6). #Significantly different from control (P < 0.05).

Fig. 3

DNA biomarker for hepatocytes (DNA content, quality and integrity) in control and TCE-treated mice. (a) Agarose gel electrophoresis of DNA isolated from liver of control and TCE-treated mice; sham control (lane1), vehicle control (lane2), 100 μg/kg TCE-treated mice (lane3) and 400 μg/kg TCE-treated mice (lane4). Almost no or little DNA degradation was detected in controls. DNA ladder with internucleosomal DNA fragmentation fragments appeared with a smear pattern in TCE treatment groups. (b) Quantification of DNA concentration in control and TCE-treated groups. Data are presented as the mean ± SEM. (c) Quantification of DNA concentration in sham and vehicle groups. Data are presented as the mean ± SEM (n = 6). *Significantly different from sham control (P ≤ 0.05). #Significantly different from 100 μg/kg TCE-treated group (P ≤ 0.05).

Fig. 4

Levels of oxidative stress biomarker MDA (nmol/ml) in the murine livers. (a) Quantification of levels of MDA (nmol/ml) in the livers of control and TCE-treated groups. (b) Quantification of levels of MDA (nmol/ml) in the livers of sham and vehicle groups. The values are presented as means ± SEM (n = 6).