Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells.

Abstract

In this study the focus is on the comparison of fluorometric assays, using Alamar Blue (AB) and Hoechst 33342 coloration, and luminometric assays, using Cyto-Lite and ATP-Lite, for toxicity measurements. With AB, ATP-Lite and Cyto-Lite the energy status of the cell is measured and with Hoechst 33342 the amount of DNA. These assays were carried out with different dosages of several toxic compounds with the following permanent cell lines: human liver (Hep G2), human endometrium (ECC-1), human cervix (HeLa) and Chinese hamster ovary (CHO) cells.

In these assays toxicity of 110 compounds was assessed in Hep G2 cells. With 60 of those, toxicity was assessed in Hela, ECC-1 and CHO cells. These compounds were non-narcotic antitussives, nasal decongestants, narcotic analgesics, hypnotics, vasodilators, specific cellular energy blockers, cellular proliferation inhibitors, ion channel blockers, estrogens, antiestrogens, androgens, progestagens and others.

The outcome of this study is that all four cell lines were responsive to the same set of 60 drugs with a comparable indication of toxicity. Hep G2 cells appear slightly more sensitive, as compared to the other three cell lines. Evaluation up to dosages of 3.2 × 10⁻⁴ or even 3.2 × 10⁻³ M for some of the compounds for these four assays in Hep G2 cells demonstrated toxicity for 45 of the 60 (75%) reference compounds with known toxicity in these assays. With a new set of 50 compounds, among which there were estrogens, androgens, progestagens and antiestrogens, 18 (36%) were identified as toxic up to a concentration of 3.2 × 10⁻⁵ M.

In conclusion, many of the 60 tested reference compounds gave similar dose and toxicity effects on these permanent cell lines. Therefore, all these cell lines can be used for toxicity screening with AB, ATP-Lite, Cyto-Lite and Hoechst 33342. However, species specific cell lines may reveal species specific effects, as shown with digoxin.

Introduction

An approach to reduce the cost aspects of analyzing large numbers of synthetic compounds within the pharmaceutical industry is the introduction of medium and high throughput in vitro screening. Animal studies can be largely reduced in this way. Within toxicology this medium and high throughput screening is only in its infancy. Further development of this area may lead to an earlier prediction of toxic effects of compounds in cellular assays. Due to the high attrition rate of toxicity of drug candidates in development, i.e. 50% of the compounds, interest is raised within the pharmaceutical industry to examine compounds at earlier instance and with relatively small quantities of compound. For a first glance on toxicity simple assays are needed to identify general aspects of cellular toxicity. Interference with normal cell physiology, such as for instance energy metabolism and cellular proliferation, can be simple cell toxicity markers.

In the present study the focus is on these more old-fashioned cellular indications of cytotoxicity. The aspects of energy metabolism, cell viability and cellular proliferation are studied with:

• Alamar Blue^TM (AB) is a major indicator of NADH conversion and a minor indicator of NADPH, FADH and/or FMNH reduction. AB is taken up in cells by passive diffusion and reduced in cytosol, mitochondria and/or microsomes (Gonzalez and Tarloff, 2001). This reduction signal can be measured by fluorescence (Andrews et al., 1997, Nakayama et al., 1997, Slaughter et al., 1999, O’Brien et al., 2000).

• ATP-Lite^TM is a luminescent measure for the intracellular ATP levels (Germain et al., 2003).

• Cyto-Lite^TM is a luminescent measure for the intracellular NADH levels (Chan et al., 2001).

• Hoechst 33342 is a fluorescent dye used for the quantification of DNA and cellular proliferation (Lydon et al., 1980, Richards et al., 1985, Blaheta et al., 1991).

The present study deals with three human and one rodent permanent cell line, although toxicologists prefer the analysis with primary cells above those with permanent cell lines. The choice for two of the human cell lines was based on experience of the multicenter evaluation of in vitro cytotoxicity (MEIC) (Ekwall and Sandström, 1978a, Ekwall and Sandström, 1978b, Ekwall and Johansson, 1980, Clemedson et al., 1996, Clemedson and Ekwall, 1999). In these MEIC studies a human liver hepatocyte cell line (Hep G2) (Thabrew et al., 1997) and a human cervix cell line (HeLa) (Ekwall, 1980a) were commonly used. The cell specificity in these studies appeared less important than the species specificity (Clemedson and Ekwall, 1999). The toxic effects of 50–250 compounds were demonstrated with Hep G2 and/or HeLa cells during 24 h and seven day experiments. The evaluation of approximately 250 toxic compounds on these cell lines led to a reliable comparison between these in vitro tests as well as towards in vivo data for a smaller set of compounds (Ekwall, 1980b, Ekwall, 1980c, Ekwall et al., 1989, Clothier et al., 1987, Bondesson et al., 1989, Jover et al., 1992, Barile et al., 1994, Pondosa et al., 1997, Scheers et al., 2001). From these studies the validity of cellular cytotoxicity testing with respect to in vivo toxicity analysis confirmed the correlation between serum levels and cytotoxic effects.

In the present study Hep G2 and HeLa cell lines were combined with human endometrium (ECC-1) and Chinese hamster ovary (CHO) cell lines. In this way a comparison can be made between four different cell lines with tissue selectivity and species specificity for these assay types. A selection of 33 compounds was made from the MEIC studies, while 27 other reference compounds were selected on their pharmacological profile. All these 60 compounds were tested on four different cell lines in the present study. Another set of 50 pharmacological compounds were selected and included for the assays on Hep G2 cells only. In a previous study the same compounds were already tested for ROS formation, glutathione depletion and calcein uptake (Schoonen et al., 2005).