Cytotoxicity assessment based on the AUC₅₀ using multi-concentration time-dependent cellular response curves

Abstract

Although many indices have been developed to quantify chemical toxicity, substantial shortcoming is inherent in most of them, such as observation time dependence, insufficient robustness, and no comparison with the negative control. To assess the extent of exposure of the tested substance, a cytotoxicity assay named AUC₅₀ was developed to describe the time and concentration-dependent cellular responses. By monitoring the dynamic cytotoxicity response profile of living cells via the xCELLigence real-time cell analysis high-throughput (RTCA HT) system, changes in cell number (named cell index, CI) were recorded and analyzed subsequently. A normalized cell index (NCI) is introduced to reduce the influence of inter-experimental variations. The log-phase of cellular growth is considered, which alleviates the cell's spontaneous effect. The area between the control line and the assessed time-dependent cellular response curve (TCRC) of the tested substance was calculated, and the corresponding exponential kill model (concentration–response curve) was developed along with exploiting the concept of AUC₅₀. The validation of the proposed method is demonstrated by exposing HepG2 cell line to seven chemical compounds. Our findings suggested that the proposed AUC-based toxicity assay could be an alternative to the traditional single time-point assay, and it has potential to become routine settings for evaluating the cell-based in vitro assay. Furthermore, the AUC₅₀ combined with RTCA HT assay can be used to achieve a high-throughput screening that conventional cellular assay cannot achieve.

Introduction

The ever-increasing number of chemical compounds produced by various industrial processes has prompted the development of methods for fast and efficient cytotoxicity screening to enhance environmental and health monitoring. An essential prerequisite for successfully identifying potential hazards is the continuous collection of accurate data used to assess toxicological properties. Conventional toxicity testing of environment has been performed by characterizing and quantifying specific substances before comparing them to the known regulatory guidelines. A wide range of analytical techniques have been used to achieve this goal. One of the traditional and efficient methods is to survey the effect of chemicals on living organisms (i.e., in vivo assays). Such approach can give detailed explanations on various classes of compound toxicity and provide detailed mechanistic understanding of the molecular targets of toxicity [1]. However, conducting this kind of biological experiments is expensive and time consuming, and the high throughput screening (HTS) cannot be achieved. Moreover, biological infection is a potential threat for operators.

Compared to in vivo assays, cell-based in vitro assays are attractive and powerful alternatives for the assessment of compound-induced cytotoxicity. These experiments are designed to assess functional responses related to the specific mechanisms [2], which have now become a key tool in many research fields such as disease modeling, compound screening and safety assessment [3]. Furthermore, the integration of mathematical and computational models with cell or molecular biology could improve hazard identification, prioritize chemicals required in regulatory risk assessment, and enhance toxicity testing required in toxicological risk assessment [4], [5]. The application of human-cell-based HTS assay for a wide range of concentrations is an important cellomic approach to assess and characterize the chemicals in the well established in vitro system [6], [7].

A cell-based HTS assay using a real-time cell analysis system (RTCA) has been developed and applied to general (basal) cytotoxicity testing [8], [9], [10]. The details of RTCA system has been described by Abassi [11] and Slanina et al. [12]. The RTCA system uses 96× or 384× well micro-electronic plates (E-Plates), with gold micro-electrode arrays integrated to glass substrates at the bottom of the wells, to measure cellular status in real time. Microelectrode electronic impedance directly measures the cellular population from which the cytotoxicity (cell inhibition, apoptosis and cell death), cellular proliferation, cell morphological changes, and cell attachment quality can be interpreted. This impedance-based high throughput technology provides an efficient screening tool to generate information-rich dataset including features of cellular response profiles, time kinetics, and wide range of concentrations for the fast identification of unknown chemicals.

When describing the cell-based in vitro toxicity of chemicals, most researchers use the toxicity indices such as half maximal inhibitory concentration (IC₅₀), 50% growth inhibition (GI₅₀), total growth inhibition (TGI), 50% lethal concentration (LC₅₀) [13], etc. However, these toxicity indices are largely dependent on the time of incubation. For example, the LC₅₀ for a 24-h incubation period would be different from 48-h incubation. This makes the end-point dependent toxicity indices much less reliable than generally anticipated. Compared with the end-point toxicity assay, KC₅₀ offers an alternative to evaluate toxicity, which is independent of both incubation and recovery time [14], [15]. The KC₅₀ uses an exponential model to describe toxicity and to calculate the concentration producing 50% lethal to proliferating fraction of cells. Although KC₅₀ represents the relationship of cytotoxic effect to concentrations without influence of incubation and recovery time, it neglects the negative control line. The negative control line could provide essential information about inter-experimental reproducibilities.

In order to quantify the growth and apoptotic effects of a cell line following toxic treatment, and to develop a mathematical model assaying the toxicity of chemicals, human hepatocellular carcinoma cells (HepG2) were treated in vitro with seven chemicals, each at 11 concentrations for up to 72 h exposure time. The area under the negative control line curve (AUC) was developed to evaluate the extent of exposure to chemicals. By integrating over time rather than looking at individual concentration measurements, a more accurate and robust estimate of the overall exposure to the compound is obtained, which can describe the intensity of the chemical toxic effect. The AUC-based cytotoxicity assay is capable of showing the distinct and important advantages over traditional end-point assays by providing a cellular dynamic information and quantifying the cytotoxicity of a given treatment.