The Study On The High Throughput Screening Methods To Detect Acute Toxicity And Genotoxicity In Vitro

As the number of lead compounds continues to expand, there is a growingneed for toxicity testing. Historically, toxicity testing often involves the use ofanimal models, and the traditional animal-based methods are too slow, labourintensive and far too costly for the present day demands. To save the time andthe effort, the tests should be performed in vitro and can be applied easily andfast. Utilization of High Throughput Screening (HTS) for toxicity in vitro isnecessary for an effective screening strategy. Although HTS only began in the 1990s, the speed of screening hasincreased dramatically during the past decade. This phenomenal progress hasbeen achieved through assay miniaturization, parallel processing andinnovations in hardware and assay technologies. The most common detectionsystems in HTS are fluorescence and luminescence. All these techniquesprovide excellent sensitivity (to facilitate miniaturization) and enableultra-high-speed measurements (using luminescence or fluorescence imagingdevices). As a compound moves further down the drug-discovery pipeline, the costsassociated with its failure increase dramatically. So now the pharmaceuticalcompanies spend more money on compounds that subsequently fail becauseof their toxicity than any other area. The role of in vitro cytotoxicity testing istherefore being seriously evaluated and developed by the pharmaceuticalindustry. Despite the cytotoxicity, genotoxicity is an important aspect inassessing compounds. LD50 is an criterion in vivo to assess the toxicity of compounds. In this study,a high throughput in vitro toxicity screen has been developed and validated toidentify compounds that have a high potential to be acutely toxic in vivo. This 6screen is based on treating HepG2 cells with test compounds for 24h and thendetermining the degree of cytotoxicity by the neutral red uptake, LDH release,MTT formation and resazurin binding. Forty-seven structurally unrelatedcompounds were chosen that spanned a range of acute LD50 values andmechanisms of toxicity. Experimentally derived in vitro IC30, IC35, IC40, IC45 andIC50 results were compared to the LD50 values for each compound and eachmethod. The results of our study indicate that neutral red uptake is the mostsensitive and the correlation between IC45 and the LD50 has the most statisticalsignificance (R2=0.7793, neutral red uptake). However, due to the scatter ofthe data points, it was considered not appropriate to rank compoundsaccording to their degree of in vivo toxicity on the basis of the in vitro results. Inour study, by defining limit value concentrations for both the in vivo (LD50) andthe in vitro (IC45) values, it was possible to identify compounds that had a highpotential to be acutely toxic in vivo (LD50=50μmol/kg) by using the in vitroresult (IC45=10μM). This screen is highly specific (91%) and has a sensitivityof approximately 80%. This is deemed acceptable for a first tier toxicity screenat an early stage in the drug discovery process and the safety assessment ofcompounds. The single-cell gel electrophoresis (SCGE) assay, also called the cometassay or the microgel electrophoresis assay, is a rapid, reliable, and sensitivemethod of detecting DNA damage in individual cells induced by variousgenotoxic agents. The aim of this study was to increase the throughput ofcompounds tested and to minimize the amount of test compound needed foran assay. We modified the standard protocol and designed an experimentalprocedure suitable for 96-well plates so four compounds can be parallel testedon a single microplate. It was reported that non-genotoxins at cytotoxicconcentration may induce DNA migration in the Comet assay, we select 20compounds (including 8 genotoxic agents and 12 non-genotoxic agents) forfurther analysis on the basis of the cytotoxicity screening. By using ametabolically competent human cell line (HepG2 cells), we can detect the...