Molecular And Cellular Analysis Of The Effects Of Cyclohexanol And Cyclohexanone On The Antioxidant System Of Earthworm

The leakage and discharge of chemical products and petroleum substances in the process of use and transportation have a serious impact on the organisms in water and soil.Cyclohexanol and cyclohexanone are common products of petroleum pollutants,important chemical raw materials and excellent solvents.At the same time,cyclohexanol is the raw material of industrial production of cyclohexanone,so they often appear at the same time as environmental pollutants.Cyclohexanol and cyclohexanone,as aliphatic compounds,are difficult to degrade naturally.They not only cause certain toxicity to animals and plants in the environment,but also produce carcinogenic and mutagenic effects in coordination with other substances in the body.At present,there are few researches on the toxicity of alicyclic compounds,and most of them focus on mammal,and pay little attention to the toxicity of soil biological.On the other hand,the toxicity of some low toxic industrial chemicals and petroleum substances,including cyclohexanol and cyclohexanone,to organisms often shows sublethal effect,and the commonly used method of using mortality as the standard to evaluate the toxicity of substances to soil organisms is particularly inadequate.Therefore,cyclohexanol and cyclohexanone were selected as the toxic substances in this paper,and their effects on the antioxidant system of common biological earthworms in the soil were studied from the cellular and molecular levels,which could provide new ideas and methods for the ecological risk assessment of soil chemical and petroleum pollution.This paper is divided into the following three parts:1.The body cavity cells of eisheng earthworm were selected as the research object.The cells were cultured and exposed in vitro for a short time.A number of indicators related to oxidative stress in the cells were measured.The cytotoxicity and oxidative stress induced by direct exposure of cyclohexanol and cyclohexanone to the cells were evaluated.The relationship between the dose of cyclohexanol and the cell viability showed a negative correlation.At the highest concentration of 10-2 mol/L,the relative cell viability was only 66.0%of that of the blank group;when the concentration of cyclohexanone was lower than 10-6 mol/L,it promoted the cell viability,which was 118%compared with the blank group,and decreased to 86.7%at the highest concentration of 10-2mol/L.The effect of cyclohexanol and cyclohexanone on cat and SOD activity was similar:SOD activity increased as a whole,CAT activity decreased as a whole.The relative level of MDA in the cells increased significantly,and the MDA in the cells exposed to cyclohexanone was significantly higher than that of cyclohexanol;the total antioxidant capacity first increased and then decreased,but it was still higher than that in the blank group,and the antioxidant capacity of the cells exposed to cyclohexanol was significantly higher than that of cyclohexanone.The results of cell experiments showed that both the exposure of cyclohexanol and cyclohexanone had toxic effects on the coelomic cells of earthworm.Comparatively speaking,the overall toxicity of cyclohexanol to cells was stronger than that of cyclohexanone,but the oxidative damage of cells caused by cyclohexanone was stronger,which indicated that the toxicity of cyclohexanol to coelomic cells of earthworm was more caused by the effects other than oxidative stress.2.CAT,a key antioxidant enzyme,was selected as the research object.The changes of molecular structure and function of CAT exposed to cyclohexanol and cyclohexanone were studied by means of various spectroscopy,enzyme activity detection and molecular simulation technology.Cyclohexanol changed the microenvironment of fluorescent group in CAT molecule,but did not affect its polarity;it had no significant effect on the structure of CAT polypeptide chain.The effect of cyclohexanone on the microenvironment of CAT skeleton and amino acid residues is similar to that of cyclohexanol.When the concentration reaches 10-2 mol/L,CAT skeleton becomes loose,and the polarity of fluorescence chromophore microenvironment is changed indirectly.The results of molecular simulation showed that the binding sites of two toxic substances and CAT were not in their active centers,which corresponded to the results of no significant change in enzyme activity test.In contrast,cyclohexanone has a greater influence on the molecular structure of CAT.3.SOD,a key antioxidant enzyme,was used to study the changes of molecular structure and function of SOD exposed to cyclohexanol and cyclohexanone.The exposure of two kinds of small molecules had an effect on the microenvironment of SOD fluorescent chromophore residues,and cyclohexanone had a greater effect,but neither of them changed its polarity.Cyclohexanol can slightly constrict the skeleton of SOD peptide chain,and it has similar effect when the concentration of cyclohexanone is low.When the concentration of cyclohexanone reaches 10-2 mol/L,the skeleton structure of cyclohexanone will be changed obviously and become loose.The results of molecular simulation showed that the binding sites of the two toxic substances and SOD were not in their active centers,and there was no significant change in SOD activity after exposure in the enzyme activity test.In contrast,cyclohexanone has a greater influence on the molecular structure of SOD.The reason why cyclohexanone has a stronger effect on the structure of the two proteins than cyclohexanol may be that cyclohexanone has a stronger dipole moment than cyclohexanl(1.80D for cyclohexanol,3.57D for cyclohexanone).In this paper,the toxic effects of cyclohexanol and cyclohexanone on the antioxidant stress system of earthworm were studied and compared from two levels of cell and molecule.A new method of cell culture and exposure in vitro was explored for the study of earthworm toxicology,which provided a way to evaluate the effects of petroleum pollutants and chemical pollution on soil organisms.