Elsevier

Aquatic Toxicology

Volume 69, Issue 1, 30 July 2004, Pages 67-79
Aquatic Toxicology

Comparison of the cytotoxicity induced by different exposure to sodium arsenite in two fish cell lines

https://doi.org/10.1016/j.aquatox.2004.04.007Get rights and content

Abstract

Arsenic, a common environmental pollutant, is toxic to many mammalian cells. However, the arsenic-induced toxicity to aquatic animal species is unclear. This study attempted to compare the arsenic-induced cytotoxicity in various fish cells. Two fish cell lines, JF (fin cells of Therapon jarbua) and TO-2 cells (ovary cells of Tilapia), were treated with sodium arsenite in two ways to mimic acute and subacute exposure. The distinguishable alterations of cell morphology and microtubule network were observed in the cells treated by two arsenite exposure protocols. By the colony-forming assay, we demonstrated that the survival of both cell lines, treated with the high concentrations of arsenite (20–160 μM) for 2 h or with the low concentrations (0.125–10 μM) for 24 h, was decreased in a dose-dependent manner. The difference between the susceptibility of JF and TO-2 cells to arsenite was revealed by the factorial ANOVA to compare the survival rates of the arsenite-treated cells; JF cells were more sensitive than TO-2 cells (P=0.008 and 0.013 for the high-concentration and the low-concentration treatment, respectively). The possible mechanisms to provoke the cytotoxicity of arsenite in two cell lines were also addressed. Antioxidants, N-acetyl-cysteine and dithiothreitol, significantly prevented JF cells, but not TO-2 cells, from the arsenite-induced inhibition of survival. Additionally, apparent apoptosis of JF cells and a mitotic arrest of TO-2 cells in response to the treatment of arsenite were also demonstrated by the DNA-fragmentation analysis and the flow cytometric analysis of cell-cycle progression. The results indicate that sodium arsenite induces apoptosis in JF cells probably by causing oxidative stress and disturbs the cell cycle of TO-2 cells. These two fish cell lines can serve as the potential tools to in detail study the toxicity and the hazards of arsenic compounds to aquatic animals at molecular level in the future.

Introduction

Arsenic, a widely distributed metalloid in nature, is a common environmental pollutant released through industrial and agricultural processes into the aquatic environment (Ishinishi et al., 1986). Epidemiological studies have indicated that chronic exposure to inorganic arsenic is strongly associated with high risks of skin, lung, liver, and prostate cancers in the human population (IARC, 1980; Chen et al., 1985, Chen et al., 1992; Chen and Wang, 1990, Chiou et al., 1995). Inorganic arsenic compounds also induce many cytotoxic and genotoxic effects in a variety of cultured cells (Rein et al., 1979; Lee et al., 1985a, Lee et al., 1985b, Lee et al., 1988, Lee et al., 1989; Seymour and Mothersill, 1988, Li and Chou, 1992, Ramirez et al., 1997, Yih et al., 1997, Li and Broome, 1999).

Most studies to understand the toxicity of arsenic compounds were performed in mammalian cells. However, the study of the arsenic toxicity to the aquatic animal species, including fish, is limited. In vivo bioassays are often administered when studying the hazards of environmental chemicals to fish, but the whole-fish are inconvenient, time consuming, difficult to reproduce, and require sacrificing the organisms. For several decades, the permanent fish cell lines have been widely used in laboratory test systems to measure the cytotoxicity and the genotoxicity of single compounds or environmental samples (Castaño et al., 2003). Therefore, fish cell lines seem to be the potential surrogates for entire fish to serve as the tools for studying the toxicity of the water-soluble arsenic compounds.

Arsenite, an inorganic and trivalent compound of arsenic (As3+) with potent toxicity (Maitani et al., 1987, Hopenhayn-Rich et al., 1993), has a direct affinity to the sulfhydryl group (Sunderman, 1979, Scott et al., 1993) and also induces considerable accumulation of the reactive oxygen species in many animal cells (Liu and Huang, 1997, Wang et al., 1997). Cytoskeletal proteins, especially tubulin, contain abundant cysteine residues (Mellon and Rebhum, 1976), and certain sulfhydryl groups of the cysteine residues are important for microtubule polymerization (Kuriyama and Sakai, 1974). Therefore, microtubules could be the ideal targets for the damage induced by arsenite. Some studies have also shown that the treatment of arsenite can disturb the organization of microtubules in the cultured cells (Li and Chou, 1992, Ramirez et al., 1997). Many toxicants have unique toxic effects when they are applied to the cells with different doses and for different periods. Yih and Lee (1999) have also demonstrated that arsenite can induce various ratios of the kinetochore-positive and the kinetochore-negative micronuclei in the normal human fibroblasts treated according to the different exposure protocols. Their results further suggest that arsenite perhaps acts differently on animal cells under acute and subacute exposures.

Here, we compared the toxic effects induced by the acute and the subacute treatment of arsenite in two fish cell lines via monitoring the cell-survival rates and the alterations of general and cytoskeletal morphology. The possible mechanisms of the arsenite-induced cytotoxicity were also addressed through analyzing the progression of cell cycle and the DNA fragmentation of apoptosis. The results of our study reveal that the unique cytotoxic responses in the JF (fin cells of Therapon jarbua) and TO-2 (ovary cells of Tilapia) fish cell lines can be induced by the particular ways of arsenite treatment.

Section snippets

Materials

Sodium m-arsenite (NaAsO2), N-acetyl-cysteine (NAC), dithiothreitol (DTT), mouse anti-β-tubulin first antibody, and FITC-conjugated anti-mouse IgG secondary antibody were purchased from Sigma (St. Louis, MO). All tissue-culture products were from Gibco (Grand Island, NY).

Cell culture

JF cell line which mainly consists of fibroblast-like cells was established from the pectoral fins of Therapon jarbua, and the population doubling time of JF cells was about 72 h (Wen et al., 1990). TO-2 cell line which mainly

Effects of arsenite on general and cytoskeletal morphology of fish cells

For general morphology, the control JF and TO-2 cells attached firmly to the substratum with random orientations (Fig. 1A and B). Following the treatment of 80 μM arsenite for 2 h, JF cells retained their normal shapes and hardly had morphological alteration (Fig. 1C); they apparently retracted and detached from the substratum after the treatment of 5 μM arsenite for 24 h (Fig. 1E). In contrast, TO-2 cells showed severe retraction and more rounding following the treatment of 80 μM arsenite for 2 h (

Discussion

In the present study, the apparent and particular cytotoxicity of arsenite to two permanent fish cell lines was reported. Morphological alterations are considered to be the primary indications of cytotoxicity and its underlying mechanisms. Li and Chou (1992) have noted that the microtubule organization in Swiss 3T3 cells is markedly damaged by arsenite in high doses (≧20 μM), but not by arsenite in low doses (2.5–10 μM). Moreover, Yih and Lee (1999) have also determined that arsenite has

Acknowledgements

We are grateful to the support from the National Science Council (Grant No. NSC 91-2511-S-003-070), and the Council of Agriculture [Grant No. 92AS-1.2.1-FD-Z1(3)] affiliated to the Executive Yuan, Taiwan, R.O.C. Prof. Yung-Ruei Chen is also appreciated for his valuable advice during the course of this research and paper writing.

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