In Vitro-in Vivo Toxicity Correlations Of Pesticides And Typical Organic Pollutants And Toxicity Mechanisms Of Acetochlor

With the development of modern industrialization,the amount of newly synthesized compounds were continuously increasing around the world.Over 100 million chemicals were registered in Chemical Abstracts Service.These compounds play important roles in various fields,which promoted the development of corresponding industries.However,the on-going release of organic pollutants into the natural environment posed serious pollution,which brought about severe environmental risk to animals and human being via bio-concentration and food chain,multiple ecosystem can be polluted.Pesticides,frequently detected in the environment as organic pollutants,are one of the main sources of environmental pollution.Human being can be exposed to pesticides in many ways,and those pesticides can be uptake into human body through bioaccumulation.It was reported that pesticide residues are detected in some foods,which directly poses a threat to human health.Therefore,it is urgent to evaluate the toxicity of these pollutants and determine their potential adverse effects on wildlife and human beings.Among all of pesticide compounds,the potential risk of herbicides has been underestimated because they were believed to be less toxicity to animals,there are relatively few studies on cells.Thus,it is of great importance to study the correlation between toxicities in vitro and in vivo,and understand the toxicity effect and mechanism of organic pollutants.This study is of great significance,not only in reducing the number of animals in traditional toxicology,but in supporting the validation of alternative toxicology methods.This process lays a sound theoretical foundation for the assessment of ecological risk,as well as the protection of environment and reduction of human health risk.It has not only theoretical significance,but also practical application value.The traditional method of safety assessment for organic pollutants is through acute toxicity test in vivo.This procedure will consume a lot of animals in a cruel way.This method has been widely criticized by all parties from the perspective of morality and science.Among many alternative test methods,cytotoxicity test stands out because of a series of advantages.This study aims to verify the feasibility whether in vitro cytotoxicity data can be used to predict the toxicity of compounds to in vivo experiments.To this end,this study deeply explored the correlation between cytotoxicity and in vivo toxicity,as well as the influence factor,based on statistical analysis of large databases of both in vivo and in vitro toxicity data.In addition,the relationship between the modes of action of organic pollutants on mice and cells was discussed.On this base,this study evaluated the toxicity of acetochlor,a widely used herbicide around the world,and explored the potential toxic mechanism of acetochlor on cells via cell culture technology.Furthermore,zebrafish embryos were used as a toxicological model to verify if acetochlor share same toxicity mechanism in cells and fish,this provides a strong basis for in vivo-in vitro correlation study.The main results are as follows:?1?The correlation models of cell cytotoxicity versus human,rodent and fish toxicity was developed.Three theoretical models?bio-uptake equilibrium,kinetic theory and mode of action?were then used to investigate the in vivo-in vitro toxicity correlations and the influencing factors.In term of bio-uptake equilibrium theory,the critical body concentration?CBR?in fish was closely related to the bioconcentration?BCF?,meanwhile,the bioconcentration process in fish and cells were very similar,that maybe the reason why there is a significant correlation between fish and cell toxicity?R² = 0.70?;Moreover,the BCF was closed to the hydrophobicity,this is the reason why hydrophobic compounds are more toxic to humans and fish than reactive or specifically-acting compounds to cells.However,as for rodent,the CBR is not only related to BCF,but also to the absorption,distribution,metabolism and excretion in vivo,therefore,the kinetic theory can well explain why there is a very poor relationship between rodent and cell toxicity?R² = 0.44?.Inclusion of descriptors representing hydrophobicity,ionization,acidity and absorption into the correlation equations can significantly improve the correlations of cytotoxicity with human and fish toxicity?R² changed from 0.7 to 0.8?,but not with rodent toxicity?R² = 0.49?.These descriptors reflect the differences of the toxicodynamics and toxicokinetics between cells and organisms.?2?A large number of toxicity data of different chemicals in both 3T3 cells and mice were collected,and the regression analysis was carried out in this study,the results showed that the relationship of toxicities between mice and 3T3 cells was not strong?R² = 0.41?.Inclusion of molecular descriptors?e.g.ionization,pKa?improved the regression to R² = 0.56,indicating that this relationship is influenced by kinetic processes of chemicals or specific toxic mechanisms associated to the compounds.However,to determine if we were able to discriminate modes of action?MOAs?in mice using the toxicities generated from 3T3 cells,compounds were first classified into “baseline” and “reactive” guided by the toxic ratio?TR?for each compound in mice.Sequence,binomial and recursive partitioning analyses provided strong predictions of MOAs in mice based upon toxicities in 3T3 cells.The correct classification of MOAs based on these methods was 86%.Nearly all the baseline compounds predicted from toxicities in 3T3 cells were identified as baseline compounds from the TR in mice.The incorrect assignment of MOAs for some compounds is hypothesized to be due to experimental uncertainty that exists in toxicity assays for both mice and 3T3 cells.Conversely,lack of assignment can also arise because some reactive compounds have MOAs that are different in mice compared to 3T3 cells.?3?In this study,HepG2 cells and zebrafish embryos were used as toxicological models to assessment the toxicity mechanism of acetochlor.Experiments were conducted at concentrations ranging 0-800 ?M acetochlor over a 12 to 48 h period to quantify underlying toxicity mechanisms of acetochlor in human liver carcinoma cells?HepG2?.Our data indicate that acetochlor suppressed HepG2 cell proliferation in both a concentration-and time-dependent manner.Acetochlor induced reactive oxygen species?ROS?generation more than 700% with exposure to 400 ?M acetochlor,and acetochlor decreased the activities and levels of antioxidant responses?superoxide dismutase,glutathione?following exposure to 400?M acetochlor.Acetochlor also induced HepG2 cell damage through apoptotic-signaling pathways,enhanced intracellular free Ca²⁺concentration?>400%?.Notably,cell cycle progression was blocked at G0/G1 phase in HepG2 cells when treated for 24 h with 400 ?M acetochlor.Acetochlor decreased mitochondrial transmembrane potential?⁷7%?,and reduced ATP levels?⁶5%?following exposure to 400 ?M acetochlor compared to untreated cells,mitochondrial dysfunction may be the toxicity mechanism of acetochlor in cells.In order to verify whether acetochlor has the same toxicity mechanism in fish,the respiratory rate of mitochondria of zebrafish embryos after exposure was measured.The results showed that 125 ?M acetochlor significantly reduced the basic respiratory rate,ATP synthesis and the highest respiratory rate of zebrafish embryos,revealing that the toxic mechanism of acetochlor is related to mitochondrial dysfunction.