Computational Toxicological Study On Dose-effect Relationship Of Different Types Of NRF2 Activators Activating KEAP1-NRF2 Signaling Pathway

Objective:The number of chemicals in the environment is continually increasing,with over 200 million chemicals registered with the Chemical Abstracts Service（CAS）.However,traditional methods cannot meet the urgent need for assessing the health risks associated with this large number of chemicals.Furthermore,the use of animals in life science research is facing increasing scrutiny,with the US EPA reducing funding for research using mammals each year and the European Union and other international markets prohibiting the use of animal testing for cosmetics.Alternative toxicological methods,strategies,and technical systems are emerging globally,and China must take the initiative to establish a technical system and standard of toxicity testing with independent intellectual property rights,train and reserve alternative toxicology talents,and ultimately protect the international market access and import and export trade of Chinese products.This study utilized computational toxicology to analyze the nonlinear relationship between exposure dose and biological physiological response,focusing on the oxidative stress caused by exposure to environmental chemicals.The nuclear factor erythroid-derived 2-like 2（NFE2L2）pathway,which is activated under oxidative stress,is involved in important biological processes such as antioxidant response,proteasome homeostasis,mitochondrial respiration,inflammation,lipid metabolism,and cell differentiation.Regulation of the KEAP1-NRF2 pathway has become a promising strategy for treating chronic diseases characterized by oxidative stress.To further understand the activation mechanism and systematically quantify the NRF2 pathway,this study utilized different types of NRF2 activators to obtain important quantitative information such as the half-life,maximum activation amount,response time,dose-response sensitivity,and threshold interval of each protein.This research will contribute to the systematic biology of the KEAP1-NRF2 pathway and provide vital quantitative data for chemical health risk assessment and drug design.Methods:For this study,a"bottom-up"approach was taken to calculate toxicological toxicity pathways.A network and mathematical model of molecular pathways were constructed using basic molecular components such as KEAP1,NRF2,Keap1-Nr F2complexes,and local reaction relationships.This allowed for a systematic and quantitative investigation of KEAP1-NRF2 signaling pathways.The different mechanisms of activating NRF2 by Class_Ⅰ-Ⅴ NRF2 activators and Class_Ⅵ NRF2activators were examined.Class_Ⅰ-Ⅴ NRF2 activators modify various cysteine residues of KEAP1,whereas Class_Ⅵ NRF2 activators directly compete with NRF2 and bind to KEAP1.Consequently,two distinct molecular mechanisms were used to construct models and equations of ordinary differential equations.Dynamic parameters,such as steady-state concentration,half-life,formation and deionization rate,and binding and deionization constant of each protein in the KEAP1-NRF2 pathway under physiological and activated states,were also included and solved using Berkeley Madonna software Rosenbrock（stiff）method.Channel level quantification was then performed systematically.The time-effect and dose-effect nonlinear quantitative relationships of each protein in the two models were studied,and the values of sensitivity LRC_maxand n H were calculated to confirm the threshold interval of the dose-response curve.Lastly,the dose-effect changes of free NRF2 in the nucleus were compared after the addition of Class_Ⅰ-ⅤNRF2 activators and Class_Ⅵ NRF2 activators.Results:1.Quantitative analysis of the time-effect of activation of NRF2 by Class_Ⅰ-Ⅴshowed that the concentration of total NRF2 in the nucleus was the highest after homeostasis,followed by the concentration of free NRF2 in the nucleus,total NRF2 in the whole cell and total NRF2 in the cytoplasm,and the concentration of free NRF2 in the cytoplasm was the lowest.2.By dose-response quantification of NRF2 activated by Class_Ⅰ-Ⅴ,the Hill coefficient n H of sensitivity to free NRF2 in the nucleus was 1.15,and the local maximum reaction coefficient was 0.31.It was found that the dose-response sensitivity of free NRF2 in the nucleus was highest when the KEAP1 level in the nucleus was 500-750 n M.3.The influence of different initial concentrations of NRF2_totnucleus on the dose-response relationship shows that the sensitivity of the dose-response relationship is the best when the initial basic concentration of NRF2 is 5-25n M under the action of Class_Ⅰ-Ⅴ activators.4.Under the action of Class_ⅥNRF2activators,the concentration of total NRF2 in the nucleus was the highest,followed by the concentration of free NRF2 in the nucleus,the concentration of total NRF2 in the whole cell and the concentration of total NRF2 in the cytoplasm.The concentration of free NRF2 in the cytoplasm was the lowest.5.The dose-effect quantitative study on the activation of NRF2 by Class_Ⅵ chemicals showed that the Hill coefficient of sensitivity to nucleus with free NRF2 was 1.09,and the maximum local reaction coefficient was0.46.When the level of KEAP1 was 500～750 n M,the sensitivity to the dose effect of free NRF2 was the highest,and the curve was the most feasible to study.6.The sensitivity of initial concentration of different NRF2_totnucleus to dose-response relationship was analyzed.When the basic concentration of NRF2 was 5-25 n M,the sensitivity of dose-response relationship was the highest.7.When Class_Ⅵ activators of NRF2 were fully activated,the amount of available NRF2 was greater compared to the amount generated by Class_Ⅰ-Ⅴ NRF2 activators.Conclusion:1.Mechanistically,KEAP1 has a dual effect on NRF2:firstly,it readily captures free NRF2 molecules with a high affinity;secondly,it mediates ubiquitination and degradation of NRF2,leading to a low concentration of free NRF2 in the cytoplasm.2.In terms of the dose-response relationship,Class_Ⅰ-Ⅴ NRF2 activators exhibit a threshold concentration,allowing easy activation of NRF2 below this threshold.For precise control of NRF2 activation,Class_Ⅵ agonists are preferred.