The Study Of Hepatotoxicity Of Candidate Compounds By High Content Analysis

Effectiveness and toxicity are the main reason for clinical failure of candidate compounds. Meanwhile, Toxicity is also listed to be the key reasons of the withdrawn of marketed drugs or their limit use.Liver is an important organ of drug metabolism, and also is the most important target organs of drug toxicity. Therefore, systematically study the hepatotoxicity of candidate compounds, especially in the early stage of drug discovery, is important to improve the quality of candidate compounds and increases the efficiency of drug development.High Content Analysis (HCA) is an high efficient drug screening technology based on the analysis of cell phenotype. HCA can balance sample specificity, toxicity, bio-soluble and bio-utilization, and make up shortages of the existing toxicological screening system in flux, testing of toxic mechanisms, revealing of comprehensive aspects of the potential toxicity, providing new efficient technologies for the research of discovery toxicology. Currently, HCA could dynamicly monitor the multiple toxicity parameters of liver cells in vitro, a number of important marker molecules associated with toxicological mechanisms, including cellular and subcellular morphology and quantity, integrity of cell membrane, changes in apoptosis, and in oxidative stress and DNA damage, Thereafter, HCA is also effective means in assessing the hepatotoxicity of drug candidates, and in predicting the liver toxicity of candidate compounds.The aims of the present work are as follows: (1) Evaluating the hepatoxicity of candidate compounds with HCA; (2) Initialzing the construction of phenotype database about compound structures and hepatotoxicity on the basis of previous work, to pave the way for realization the goal of predicting the liver toxicity of candidate compounds. This work was composed of four parts: The first part, the multi-parameter study of hepatoxicity of candidate compounds; the second part, multi-parameter study of compounds-induced apoptosis; the third part, the studies of compounds-induced liver cell oxidative stress and DNA damage; The fouth part ,the initial construction of cell phenotype database about chemical structure and toxicity.The first part, after acute (4h), sub-acute (24h) and chronic (72h) treatment with 9 candidate compounds that was synthesized based on four targets, changes on cells number, nuclear intensity, nuclear area, nuclear size, membrane integrity, mitochondrial membrane potential, cytochrome C release of HepaG2 cells were analyzed through the HCA with the Cellomics Multiparameter Cytotoxicity kit, based on the viability of candidate compounds to the liver cells(HepaG2) with MTT assay, the results showed that after treatment of HepaG2 cells with candidate compounds for 72h, the IC50 of inhibiting the proliferation of liver cell was (161.57±15.29), (101.08±17.58), (74.06±7.44) and (107.40±14.09)μM for C333H and P633H(peroxide enzyme proliferator-activated receptor (PPAR /γ) dual agonist), F3SM and F3SMDM(PPARβ/ agonist), respectively; the IC50 is over 300μM for C36 and C36D2(cleavage agent of advanced glycation end products (AGEs)), MJ07 and MJ08(cannabinoid receptor - 1 (CB1) antagonist), respectively, whereas it was (117.62±26.15)μM for MJ15. The multi-parameter results of different treatments on toxicity are as follows: the toxicity of candidate compounds C333H, P633H, F3SM, F3SMDM, MJ07, MJ08 and MJ15 on HepG2 cells displayed in a time and dose- dependent manner (toxicity-related parameters increased with the increasing of time and dose ). Mitochondrial dysfunction is the early cytotoxic response of C333H and P633H (decreased mitochondrial membrane potential), was the main cytotoxic response of F3SM and F3SMDM showed in the acute, subacute and chronic toxicity, also was the main reason of cytotoxicity of C333H, P633H, F3SM and F3SMDM.Mitochondrial dysfunction and membrane damage are the early response of MJ07and MJ08 -induced cytotoxicity, MJ07 was stronger than that of MJ08. Mitochondrial dysfunction, membrane damage and inhibiting cell proliferation are common cytotoxic responses of MJ15 -induced cytotoxicity, MJ07 and MJ15 also demonstrated the role of cell division arrest in subacute toxicity, whreas no significant cytotoxicity were observed when the concentration is under 100μM for C333H, 30μM for P633H, F3SM, MJ07, MJ08, 10μM for MJ15 and 3μM for F3SMDM. In the C36 and C36D2 treatment group, no significant cytotoxicity were observed between the experimental concentration range of 3-300μM.The second part, with the Cellomics multi-parameter apoptosis kit, after 30h treatment with candidate compounds, the number of cell nuclei and intensity, nuclear area, nuclear DNA content, F-actin short-long axis ratio, F-actin brightness and the mitochondrial membrane potential on HepG2 cells were observed, and the following reslults were found:Under 100μM, C333H, P633H, F3SM, F3SMDM induced karyopyknosis reaction, induced the increase of mitochondrial membrane potential under 300μM; 30μM for MJ07 and MJ08,10μM for MJ15 induced karyopyknosis reaction, induced the increase of mitochondrial membrane potential, individually at 3μM, 30μM, 10μM. the strength in inducing apoptosis is similar between P633H and C333H, but non-apoptosis cytotoxicity of P633H is relatively stronger. the concentration inhibiting cell proliferation of F3SM and F3SMDM were lower than that induced apoptosis; the concentration of inducting apoptosis MJ07, MJ08, MJ15 is lower than, equal to, higher than the concentration of the inhibition of cell proliferation respectively. C36 and C36D2 have no significant change on induction of apoptosis phenotype between the experimental concentration range of 3-300μM. The third part, with the Millipore MnSOD and Histone H2A.X Phosphorylation Kit of high content analysis specific, the changes of HepG2 cells caused by compounds in acute, subacute, chronic treatment, impacting on MnSOD and pHH2A.X were observed. The following reslults were found:C333H and P633H generated oxidative stress (MnSOD increase) and DNA damage (increase the expression of pHH2A.X) at 300μM, 300μM F3SM and 200μM F3SMDM induced oxidative stress (MnSOD increases) in the cells, this damage mainly occurred in the treatment of acute and subacute phase; When the concentration is above 100μM concentration, MJ07 induced oxidative stress (MnSOD increase), but not DNA damage; MJ08 slightly decreased MnSOD expression in the concentration below 100μM, had no significant effect on oxidative stress, but had the role of DNA damage (pHH2A.X increased expression) over the 100μM concentration; MJ15 slightly decreased MnSOD expression from 0.30μM to 10μM, above 30μM concentration the cells were induced significant oxidative stress and DNA damage.The fourth part, the part use hepatoxicity data summary that obtained by HCA cytotoxicity, apoptosis and oxidative stress and DNA damage of 9 candidate compounds, calculated IC50 or EC50, Initialzing the construction of phenotype database about compound-induced hepatotoxicity, and using NCSS Statistical and Data Analysis 2007 to cluster analysis (minimum distance), clustering results show that 9 compounds can be divided into 3 categories, The first category is F3SM \ F3SMDM, C333H, MJ08, C36 \ C36D2; second category is P633H and MJ07; third category of MJ15.From the experiments of 4 part, the following conclusions of the research were found:1)PPAR /γagonist C333H and P633H①Toxic effects on HepG2 cells is displayed in a time and dose- dependent manner. Mitochondrial dysfunction is the early damage response, that is one of the main cytotoxic reasons of two compounds. C333H below 100μM, P633H below 30μM had no significant cytotoxicity. The cytotoxicity of P633H was stronger than C333H;②Both induce apoptosis phenomenon in HepG2 cells at 100μM, P633H-induced apoptosis is similar to the strength of C333H, but the non- apoptosis cytotoxicity was stronger than C333H;③both have the oxidative stress and DNA damage at 300μM, C333H in the 1-100μM and P633H in the 0.3-30μM had no significant effect of oxidative stress and DNA damage, those are safe dose.2)PPAR / agonist F3SM and F3SMDM①Toxic effects on HepG2 cells is displayed in a time and dose- dependent manner. Mitochondrial dysfunction is the main facter in the acute, subacute and chronic toxicity of two candidate compounds-induced. F3SM below 30μM, F3SMDM below 3μM have no significant cytotoxicity. Cytotoxicity of F3SMDM was stronger than F3SM's;②Both induced HepG2 cell apoptosis at 100μM;③F3SM at 300μM and F3SMDM at 200μM produced the oxidative stress, no DNA damage. Both had no the role of oxidation stress and DNA damage below 30μM.3)AGEs cleavage agent C36 and C36D2①Non-cytotoxic range of 3-300μM;②No induced HepG2 cell apoptosis below 300μM③Both no the oxidative stress and DNA damage below 300μM.4) Compounds targeted CB1 MJ07, MJ08, and MJ15①Toxic effects on HepG2 cells is displayed in a time and dose- dependent manner. Mitochondrial dysfunction and membrane integrity damage are one of the main cytotoxic reasons. Cytotoxicity of three compounds, MJ15 is the strongest, followed by MJ07, MJ08 in the last;②Three induce apoptosis in HepG2 cells at 30μM, when MJ08 and MJ15 significantly inhibited cell proliferation;③MJ07 induce oxidative stress in the concentration of 100μM and above, but no DNA damage, MJ08 have DNA damage in 100μM and above concentration, MJ15 have significant oxidative stress and DNA damage in and above the concentration of 30μM, MJ08 and MJ15 is more similar on the phenotype, and similar pharmacodynamic.5) The cytotoxicity phenotype data of 9 candidate compounds is obtained by using HCA, initialzing the construction of phenotype database about compound hepatotoxicity on the basis of previous work, and conducted toxicity phenotype cluster analysis.