Eicosapentaenoic acid protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced hepatic toxicity in cultured rat hepatocytes

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and ubiquitous environmental contaminant. The health impact of TCDD exposure is of great concern to the general public. Recent reports have implied that eicosapentaenoic acid (EPA) might be a potential chemopreventive agent and influence hepatotoxicity. The aim of the current study was to explore the effectiveness of EPA in alleviating the toxicity of TCDD on primary cultured rat hepatocytes. EPA (5, 10 and 20 μM) was added to cultures alone or simultaneously with TCDD (5 and 10 μM). Rat hepatocytes were treated with TCDD and EPA for 48 h, and then cytotoxicity was detected by [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT) assay and lactate dehydrogenase (LDH) release, while total antioxidant capacity (TAC) and total oxidative stress (TOS) levels were determined to evaluate the oxidative injury. The DNA damage was also analyzed by liver micronucleus assay (LMN) and 8-oxo-2-deoxyguanosine (8-OH-dG). The results of MTT and LDH assays showed that TCDD but not EPA decreased cell viability. TCDD also increased TOS level and significantly decreased TAC level in rat hepatocytes in a clear dose dependent manner. On the basis of increasing doses, the dioxin caused significant increases of micronucleated hepatocytes (MNHEPs) and 8-OH-dG as compared to control culture. Whereas, in cultures treated with EPA alone, TOS level did not change and the level of TAC significantly increased. The presence of EPA with TCDD minimized the toxic effects of the dioxin on primary hepatocytes cultures. Noteworthy, EPA has a protective effect against TCDD-mediated DNA damages.

Fig. 1

MTT reduction in rat hepatocyte cultures maintained 48 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), eicosapentaenoic acid (EPA) and their combinations. TCDD 1: 5 μM TCDD; TCDD 2: 10 μM TCDD; EPA 1: 5 μM eicosapentaenoic acid; EPA 2: 10 μM eicosapentaenoic acid; EPA 3: 20 μM eicosapentaenoic acid; bars shown by the same letter are not significantly different from each other at a level of 5%, using Duncan’s test. Also, this is valid for the relative combinations of letters. For example, the bar shown by the letters bc is not different from b, c and ab bars, but different from a or dbars. In other words, the bars having the different letters are different from each other.Values are means ± standard deviation (n = 7)

Fig. 2

Extracellular level of lactate dehydrogenase (LDH) in rat hepatocyte cultures maintained 48 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), eicosapentaenoic acid (EPA) and their combinations. Abbreviations are as in Fig. 1

Fig. 3

A sample hepatocyte from 20 μM of eicosapentaenoic acid (EPA)-treated culture (healthy cell without MN)

Fig. 4

A sample hepatocyte from 10 μM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated culture (arrow shows MN formation in micronucleated cell)

Fig. 5

8-oxo-2-deoxyguanosine (8-OH-dG) adducts in rat hepatocyte cultures maintained 48 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), eicosapentaenoic acid (EPA) and their combinations. Abbreviations are as in Fig. 1