Elsevier

Toxicology in Vitro

Volume 18, Issue 5, October 2004, Pages 639-648
Toxicology in Vitro

Experimental survey of non-clonogenic viability assays for adherent cells in vitro

https://doi.org/10.1016/j.tiv.2004.02.001Get rights and content

Abstract

Results of rapid cell viability assays were experimentally compared in order to reveal the most suitable test for in vitro investigations of the combination of photodynamic therapy (PDT) with chemotherapeutic drugs. meso-Tetra(3-hydroxyphenyl)-chlorin (m-THPC) accumulating in cell membranes and meso-tetra(4-sulfonatophenyl)-porphin (TPPS4) accumulating in lysosomes were used as photosensitisers. Doxorubicin that localises, mainly, to nucleus and vincristine that binds to microtubules were used as cytostatic drugs. Two adherent rodent cell lines, baby hamster kidney (BHK-21) and murine hepatoma (MH-22A), were used to examine the contribution of a cell. We tested cytotoxicity assays of the main groups of fast (non-clonogenic) methods of cell viability measuring. Plasma membrane integrity was estimated by trypan blue exclusion and LDH leakage, metabolic activity was tested by [3H]-thymidine incorporation and MTT assay, loss of monolayer adherence was measured by staining with crystal violet and CyQUANT. The most sensitive test in each case was the assay related to the site of the direct damage, and measurement of the loss of monolayer adherence proved to be as sensitive assay as the damage-specific one. All the assays applied, except for the LDH release, revealed a higher effect of combination of m-THPC-mediated phototreatment and doxorubicin compared to either of the single treatments.

Introduction

Photodynamic therapy (PDT) is a new modality for treatment of cancer and some other diseases (for a review see Bonnett, 1999; Dolmans et al., 2003). PDT is based on the use of photosensitising compounds that localise quite selectively in neoplastic/hyperplastic tissues and become cytotoxic when exposed to light in the red region of the visible spectrum. Phototoxic effects induced by interaction between light and chemicals span from the cytotoxic-apoptotic effect to the induction of a variety of stress genes acting on the cell cycle and the fate of the cell (Meunier et al., 2002). The recent progress in PDT has stimulated attempts to establish protocols for combination of PDT with conventional modes of cancer treatment such as radiotherapy (Schaffer et al., 2002) or chemotherapy (Peterson et al., 1996). In vitro cell culture systems have proven useful for prediction of drug exposure effects on target organs (Davila et al., 1998). Searching for the optimal protocol of the combination, cytotoxicity analysis in vitro could be the starting point, especially having in mind that the acute lethal potencies of antitumour compounds could be closely associated with their cytotoxic effects in vitro since such chemicals mainly exert their toxicity through their effects on basal cellular functions (Fry et al., 1990; Garle et al., 1994).

The endpoint measurements and assays used in cytotoxicity tests vary considerably. The most commonly used include (i) cell morphology, (ii) membrane function, (iii) cell metabolism, (iv) DNA precursor incorporation, (v) clonogenicity (Cook and Mitchel, 1989; Tyson and Green, 1987; Weisenthal and Lipman, 1985). The use of in vitro cytotoxicity tests in general, and tests involving permanent cell lines in particular, is the subject of continuing controversy. A number of studies have shown good correlations between the results of cytotoxicity tests that have different endpoints, however, the sensitivities of the endpoint measurements and the particular assays used may differ markedly (Garle et al., 1994). Since the criteria for determining cell viability should be chosen to fit the needs of each particular study, different cellular targets of photosensitisation-based PDT and chemotherapeutic agents make a problem for choosing an adequate method of measuring cytotoxic effects of the combined treatment.

The aim of this study was to survey rapid cytotoxicity assays in order to reveal the most suitable test for in vitro investigations of the combination of PDT with the chemotherapeutic drugs. We compared results of cytotoxicity testing of cells, treated with a photosensitiser or/and a cytostatic drug. meso-Tetra(3-hydroxyphenyl)-chlorin (m-THPC) that induces damages to cellular membranes (Teiten et al., 2003), and meso-tetra(4-sulfonatophenyl)-porphin (TPPS4) that induces damage to lysosomes (Berg, 1996), were used as photosensitisers. Doxorubicin that accumulates in cell nucleus mainly (Weaver et al., 1991) and vincristine that binds to microtubules (Duflos et al., 2002) were used as cytostatics. The role of the cell line was considered by comparison between two adherent rodent cell lines of different structure and physiology, namely, fibroblastic baby hamster kidney (BHK-21) cells and epithelial murine hepatoma (MH-22A) cells. The short-term tests involving exposure to light for 10–30 min or chemicals for 4 h, were used mostly.

Since the mechanisms of action of photosensitisation and cytostatics are so different, we tested cytotoxicity assays of the main groups of currently available fast (non-clonogenic) methods of cell viability measuring.

Section snippets

Chemicals

Test photosensitisers were prepared as stock solutions using various solvents. m-THPC (kindly provided by Bonnett, Queen Mary's College, the University of London, UK) was dissolved in ethanol as 1 mg/ml stock solution and stored at −20 °C in the dark. TPPS4 (Porphyrin Products, USA) was made as 5 mg/ml stock with Dulbecco's phosphate buffered saline (DPBS) and stored at −20 °C in the dark. Pharmaceutical solutions of doxorubicin hydrochloride (Ebewe Arzneimittel, Austria, 2 mg/ml) and

Estimation of cytotoxic effect of light on photosensitised cells

BHK and MH cells were sensitised to light by incubation with either m-THPC (18 h) or TPPS4 (4 h). No cytotoxicity of photosensitisers without light exposure was registered. The m-THPC-photosensitised cells were exposed to light at λ>590 nm and fluence rate of 4 W/m2 for 10–30 min, reaching the fluence of 2.4–7.2 kJ/m2. The TPPS4-photosensitised cells were exposed to white light at fluence rate of 85 W/m2 for 5–30 min, reaching the fluence of 25.5–153 kJ/m2. The cytotoxicity was measured after

Discussion

The assessment of an effect on cellular viability may depend heavily on the specific assay used. In evaluating cytotoxic response to the combined action of two different treatments with different cellular targets, the problem of assay to use becomes even more acute.

Combination of PDT with cytostatic drugs involves two modes of cytotoxic action. Light-activated photosensitisers generates reactive oxygen species that oxidizes various biomolecules in close proximity to the site of localisation of

Acknowledgements

The authors wish to thank R. Bonnett for the generous gift of m-THPC, D. Kirvelis for kind assistance with statistical analysis and V. Piskarskiene for the excellent care of the cells. This work was partly supported by European Commission––Programme of Centres of Excellence, project CEBIOLA, ICA1-CT-2000-70027, and Lithuanian State Foundation for Science and Studies––Programme Light in Biomedicine, P-14/01.

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