Comparison of the cytotoxicity induced by different exposure to sodium arsenite in two fish cell lines
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.
References (45)
- et al.
Arsenic-selenium interactions determined with cultured fish cells
Toxicol. Lett.
(1989) - et al.
Evidence for oxidative damage to red blood cells in mice induced by arsine gas
Toxicology
(1990) - et al.
Cytotoxicity of metals, metal–metal and metal–chelator combinations assayed in vitro
Toxicology
(1986) - et al.
Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants
Toxicol. Lett.
(2002) - et al.
Human studies do not support the methylation threshold hypothesis for the toxicity of inorganic arsenic
Environ. Res.
(1993) - et al.
Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells
Mutat. Res.
(1985) - et al.
Effects of sodium arsenite on the cytoskeleton and cellular glutathione levels in cultured cells
Toxicol. Appl. Pharmacol.
(1992) - et al.
Arsenic trioxide-induced apoptosis through oxidative stress in cells of colon cancer cell lines
Life Sci.
(2002) - et al.
Disruption of microtubule assembly and spindle formation as a mechanism for the induction of aneuploid cells by sodium arsenite and vanadium pentoxide
Mutat. Res.
(1997) - et al.
Arsenite inhibits beta-oxidation in isolated rat liver mitochondria
Biochim. Biophys. Acta
(1979)