Elsevier

Toxicology Letters

Volume 160, Issue 2, 5 January 2006, Pages 171-177
Toxicology Letters

In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride

https://doi.org/10.1016/j.toxlet.2005.07.001Get rights and content

Abstract

The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events.

Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0–300 μM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay.

In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl2 for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl2 exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl2, toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay.

In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.

Introduction

Cytotoxicity assays are widely used in in vitro toxicology studies. The LDH leakage assay, a protein assay, the neutral red and the MTT assay are the most common employed for the detection of cytotoxicity or cell viability following exposure to toxic substances.

The LDH leakage assay is based on the measurement of lactate dehydrogenase activity in the extracellular medium. Reliability, speed and simple evaluation are some of the characteristics of this assay (Decker and Lohmann-Matthes, 1988). It has been employed as an indicator of cytotoxicity in HepG2 cells following exposure to cadmium chloride (Dong et al., 1998) as well as in toxicity studies using rat renal proximal tubular cells (Fukumoto et al., 2001). The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage.

The MTT assay is another cell viability assay often used to determine cytotoxicity following exposure to toxic substances. It has been used in HepG2 cells (Tully et al., 2000) and in rat lung epithelial cells after exposure to cadmium chloride (Hart et al., 1999) as well as in oligodendrocytes to assess cell viability (Almazan et al., 2000).

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water soluble tetrazolium salt, which is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by succinate dehydrogenase within the mitochondria. The formazan product is impermeable to the cell membranes and therefore it accumulates in healthy cells. The MTT assay was tested for its validity in various cell lines (Mossmann, 1983). More recent evidence suggests that reduction of MTT can also be mediated by NADH or NADPH within the cells and out of mitochondria (Berridge and Tan, 1992). Further modification of the initial protocol described by Mossmann (1983) were proposed (Denizot and Lang, 1986, Hansen et al., 1989) in order to improve the repeatability and the sensitivity of the assay.

The neutral red assay is also used to measure cell viability. It has been used as an indicator of cytotoxicity in cultures of primary hepatocytes (Fautz et al., 1991) and other cell lines (Morgan et al., 1991). Living cells take up the neutral red, which is concentrated within the lysosomes of cells.

Finally, the protein assay is an indirect measurement of cell viability since it measures the protein content of viable cells that are left after washing of the treated plates.

The aim of our study was to evaluate the sensitivity of the four cytotoxicity assays following exposure of HTC and HepG2 cells that were used as the test system, to cadmium chloride.

Section snippets

Materials

All the materials were from Sigma Aldrich unless otherwise stated.

Cell cultures and treatments

HTC cells (rat hepatoma cell line) and HepG2 cells (human hepatoma cell line) were obtained from the European collection of cell cultures. Cells were grown in DMEM (Dulbecco's modified Eagle's medium) (HTC cells) or MEM (modified Eagle's medium) (HepG2 cells) supplemented with 10% fetal bovine serum, 1% MEM non essential amino acid solution, and 1% penicillin-streptomycin solution (10,000 units of penicillin and 10 mg of streptomycin in 0.9% NaCl) in a humidified atmosphere of 5% CO2, 95% air at

Results

HTC cells were exposed to CdCl2 (0–2000 μM) for 3, 5, 8 and 24 h and cytotoxicity was determined with the LDH leakage assay, the protein assay, the neutral assay and the MTT assay. The EC50 values obtained by the four assays for these incubations times are shown in Table 1. EC50 values were obtained only with the neutral red assay when HTC cells were exposed to CdCl2 for 3, 5 and 8 h whereas the LDH leakage assay, the MTT and the protein assay provide EC50 value only after 24 h incubation period.

Discussion

The results obtained from the cytotoxicity assays indicate that there are differences between the two cell lines concerning their sensitivity to CdCl2. HepG2 cells appear to be more sensitive as indicated by the LDH leakage assay. Therefore, rupture of the cell membrane occurs when lower concentrations of cadmium chloride or shorter incubation times are employed in HepG2 cells than in HTC cells. This difference could be due to different uptake mechanisms of CdCl2 by the two cell lines. Studies

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