Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells.: II Mechanistic assays on NAD(P)H, ATP and DNA contents
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:
- (1)
Alamar BlueTM (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).
- (2)
ATP-LiteTM is a luminescent measure for the intracellular ATP levels (Germain et al., 2003).
- (3)
Cyto-LiteTM is a luminescent measure for the intracellular NADH levels (Chan et al., 2001).
- (4)
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).
Section snippets
Materials
All compounds and reagents were of analytical grade. Most of the toxic compounds were ordered by Sigma–Aldrich (St. Louis, USA). AB was obtained from Sero-Tec (Oxford, UK), Cyto-Lite and ATP-Lite from Perkin–Elmer (Groningen, The Netherlands), Hoechst 33342 from Boehringer (Mannheim, FRG), trypsin from Flow Laboratories (Irvine), Dulbecco’s Modified Eagles medium, Nutrient Mixture F-12 (DMEM/HAM F12 medium in a ratio of 1:1) with phenol-red from Gibco (Invitrogen LifeTechnologies, Breda, The
General
The energy status of the cell was measured by means of three assay types, i.e. AB, Cyto-Lite and ATP-Lite, while the DNA quantification was measured with Hoechst 33342. These assays were performed with Hep G2, HeLa, ECC-1 and CHO cells for 60 different chemical and pharmaceutical toxicants (Table 1). From these compounds 33 were also present in a MEIC study. This analysis was performed in a concentration range of 3.2 × 10−8 up to 3.2 × 10−5 M to demonstrate possible changes in toxicity between cells
Discussion
The AB, Cyto-Lite, ATP-Lite and Hoechst 33342 assays with human Hep G2, HeLa and ECC-1 and CHO cell lines showed in general similar patterns of toxicity. The long term incubation for 72 h showed its sensitivity in the dose range of 3.2 × 10−8 and 3.2 × 10−5 M. Sometimes the AB and Cyto-Lite assays and sometimes the Hoechst coloration is more sensitive for the assessment of the toxicity effects. ATP-Lite was slightly less effective in determining these effects. Chlorprothixene citrate, cytarabine,
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