The Study Of Dichloromethane On Hepatotoxicity

【Objective】 To explore the hepatotoxicity of dichloromethane through the animal experiments.To explore the effect of Nrf2/ARE signaling pathways in the oxidative stress caused by dichloromethane.To provide the basis data for population contacting dichloromethane and for the clinical study of dichloromethane poisoning.【Methods】 1. The study of dichloromethane on hepatotoxicity.After one week of acclimatization,48 male Kunming mice of specific pathogen free(SPF) quality weighing between 20 and 25 g were randomly divided into four groups including three exposure groups and a control group of 12 mice each.In the exposure groups,the mice were exposed to dichloromethane by inhalation at dosages of 1.3 mg/L(1/50 LC50),6.4 mg/L(1/10 LC50),12.9 mg/L(1/5 LC50) daily for 28 d,2h/d.The control were not infected.All animals were observed for the general growth under the same feeding conditions.Measured the weight at l,7,14,28 d.After the last exposure,the mice were sacrificed.Take the serum from 6 mice to determine the activity of alanine aminotransferase(ALT),aspertate aminotransferase(AST)and alkaline phosphatase(ALP).Take the liver from another 6 mice to do pathological examination by HE.Take the serum and liver tissue homogenate to determine the activity of superoxide dismutase(SOD),and the levels of malondialdehyde(MDA),glutathione(GSH)andtotalantioxidant capacity(T-AO C).Take the liver tissue homogenate to determine the levels of reactive oxygen species(ROS) and cytochrome P450 2E1(CYP2E1) by enzyme-linked immunosorbent method(ELISA).2. The study of Nrf2/ARE signaling pathway on hepatotoxicity mechanism caused by dichloromethane in mice.After one week of acclimatization,24 male Kunming mice of SPF quality weighing between 20 and 25g were randomly divided into four groups of 6 mice each,including the blank control group,the solvent control group(12.9 mg/L+corn oil),the dichloromethane treated group(12.9 mg/L)and the tBHQ intervention group(12.9 mg/L+tBHQ).The mice were exposed to dichloromethane by inhalation for 28 days,2h/d.After 24 h,the solvent control group were given 0.2 ml corn oil and the tBHQ intervention group were given 0.2 ml corn oil that the concentration of tBHQ is 1.25 mg/ml by oral,6d/w,for28 d.The blank control were not infected. After the last exposure,the mice were sacrificed.Take the serum to determine the activity of ALT,AST and ALP.Take the liver to observe the expression of Nrf2 protein by immunohistochemistry.Take the liver tissue homogenate to determine the levels of reactive oxygen species(ROS),MDA,glutathion-S-transferase(GST)and hemoglobin oxidase 1(HO-1) by ELISA.【Results】 1. The effect of dichloromethane to the hepatotoxicity in mice.The activity of mice were unusual,hyperactivity and restlessness at the start of the experiment.And then they became dispirited and reduced activities like sleeping.In each time node,compared with control group,the infected mice had no obvious change in body weight(P>0.05).The liver wet weight of each dose group increased,but there was no statistically significant difference(P>0.05).The viscera coefficient of low and high dose group increased obviously,the difference was statistically significant(P<0.05).The effect of DCM to liver pathology in mice.The hepatic lobule was clear,hepatic cord arranged radially,liver cells and liver sinusoidal were normal in the control group.But in the treated group,the hepatic lobule and hepatic cord were unclear.Liver cells showed diffuseedema and congestion.It showed visible point necrosis associated with inflammatory cells invasion in the high dose group.The effect of DCM to hepatic function in mice.Compared with control group,the activity of AST,ALT and ALP increased significantly in high dose group.The activity of AST,ALT and ALP showed a trend of increase with the increasing of treated dose(r=0.829,r=0.932,r=0.589,P<0.01).2. Relationship between oxidative stress and hepatotoxicity of dichloromethane in mice.Compared with control group,the level of ROS in treated group of liver tissue homogenate increased significantly(P<0.05).The level of CYP2E1 in middle and high dose group increased significantly(P<0.05).And it showed a trend of increase with the increasing of treated dose(r=0.935,r=0.869,P<0.01).Both content was positively correlated(r=0.858,P< 0.01).Compared with control group, the activity of SOD and the level of MDA in both serum and liver tissue homogenate increased significantly(P<0.05).And it showed a trend of increase with the increasing of treated dose.The level of GSH in serum decreased(P<0.05),and it showed a trend of decrease with the increasing of treated dose(r=-0.806,P<0.01).The level of GSH in liver tissue homogenate in high dose group increased significantly(P<0.05),and it showed a trend of increase with the increasing of treated dose(r=0.772,P<0.01).The level of T-AOC in serum in middle and high dose group decreased(P<0.05),and it showed a trend of decrease with the increasing of treated dose(r=-0.692,P<0.01).The level of T-AOC in liver tissue homogenate had no significant change(P>0.05).The content of MDA in serum and liver tissue homogenate were positively correlated(r=0.582,P < 0.05) by the correlation analysis.The activity of SOD were positively correlated(r=0.608,P<0.05).The content of GSH were negatively correlated(r=-0.611,P<0.05).The content of GSH were not correlated(r=0.02,P>0.05).The content of ROS in mice liver tissue homogenate and the activity of AST, ALT, ALP in serum were positively correlated(r=0.808,r=0.832,r=0.549,P<0.01).3. The study of Nrf2/ARE signaling pathway on hepatotoxicity mechanism caused by dichloromethane in mice.In the control group,Nrf2 expressed in a minority of liver cell cytoplasm but not in the nucleus.In solvent control group and dichloromethane treated group,the expression in cytoplasm increased and it had a small amount of expression in the nucleus.In tBHQ intervention group, the expression in cytoplasm decreased and it mainly expressed in the nucleus.Compared with blank control group,the level of ROS,MDA,GST and HO-1 in solvent control group and dichloromethane treated group increased significantly(P<0.05).The level of ROS and MDA had no significant change(P>0.05),but GST and HO-1 increased significantly in tBHQ intervention group(P<0.05).Compared with solvent control group and dichloromethane treated group, the level of ROS and MDA decreased significantly(P<0.05),but GST and HO-1 increased significantly in tBHQ intervention group(P<0.05).Compared with blank control group,the activity of AST,ALT and ALP in solvent control group and dichloromethane treated group increased significantly(P<0.05),but they had no significant change in tBHQ intervention group(P>0.05).Compared with dichloromethane treated group,they decreased significantly in tBHQ intervention group(P<0.05).Compared with solvent control group,the activity of AST and ALT increased significantly in dichloromethane treated group(P<0.05),ALT decreased significantly in tBHQ intervention group(P<0.05).【Conclusions】1. Under the experimental conditions,liver function and liver pathology changed aft er dichloromethane exposure.It indicated liver cells were damaged, inflammation and necrosis. Dichloromethane can cause damage to the structure and function of the liver.2. Oxidative stress play an important role in mice hepatotoxicity induced by dichl oromethane.The increase of body oxidative stress, lipid peroxidation and the decrease of antioxidant capacity lead to inflammation and liver cells damage.And oxidative stres s may be related to the increased of CYP2E1 in liver caused by the stimulation of dichloromethane.3. Dichloromethane can lead the oxidative stress to rise in mice.It can activate the antioxidant capacity of Nrf2/ARE signaling pathway.In vivo antioxidant system was depleted because the elevated ROS beyond the antioxidant capacity of Nrf2/ARE signa ling pathway.Accumulation of ROS did oxidative damage to liver cells.tBHQ is a kind of antioxidant and pathway activated agent.It can strengthen the antioxidant capacityof Nrf2/ARE signaling pathway to resist oxidative stress reaction.Then it can keep theox idation-antioxidant system in a state of equilibrium to reduce the liver damage caused by dichloromethane.