Exploratory Study Of Drug And Measures Against Paraquat Poisoning

1 BackgroundThe incidence of paraquat poisoning is increasing in recent years, following the incidence of organophosphorus pesticide poisoning,and the treatment of paraquat poisoning remains to be an unresolved medical problem. It is in the first place in the rank of pesticide poisoning mortality in recent years. Currently, due to the lack of specific antidote, two aspects are focused on the clinical treatments. One is to alleviate lung injury by drugs, the other is to purge toxins. Drug treatment is mainly concentrated on three aspects: hormones, immune inhibitors and antioxidants. Based on the knowledge of the mechanisms of paraquat toxicity, we know that antioxidants play an important role in the application of the drug for treatment of paraquat poisoning. While it has not achieved a satisfying clinical efficacy. As a non-protein sulfhydryl donor, 3-mercaptopropionic acid derivative(DZ01) obtains a good effect when applied to lung injury toxicants induced lung injury, coenzyme Q10(Co Q10) as a fat-soluble antioxidant, is widely used in cardiovascular diseases. According to the knowledge above, preliminary research was conducted on the feasibility of DZ01 and Co Q10 used as an antioxidant in the treatment of paraquat poisoning. Gastrointestinal tract cleaning and blood purification are the main methods of toxin removal in clinical practice. However, the toxin removal in the lung tissue, the target organ of paraquat poisoning, has not been studied yet. Whole lung lavage(WLL) is a mature technology used to clear causative agents in the lungs clinically. Hence, a preliminary research is needed to evaluate the feasibility of removing paraquat in lung tissue with WLL. 2 ObjectiveTo evaluate the feasibility of the applications of potential antioxidants(DZ01, Co Q10) and WLL in the treatment of paraquat poisoning based on the application of antioxidant drugs and toxin removal in the target organ. The changes of related redox indicators of the tissues and paraquat content variation trend data in related samples were separately used as evaluation indicators. The research was aimed at providing new methods to the clinical treatment of paraquat poisoning. 3 Methods 3.1 The Preliminary Study of the Treatment of Paraquat Poisoning by DZ01, Co Q10N-Acetyl-L-cysteine(NAC) was chosen as the positive control which is widespread used in clinical. 160 healthy male CD-1(ICR)(License Number: SCXK(Beijing) 2012-0001) mice weighted around 18~20g were randomly divided into two groups, one is the observation group of survival rate 64 mice and the other is the lung injury evaluation group were 96 mice. Then each group was randomly divided into eight sub-groups: normal control group(NC), PQ group(PQ-C), Co Q10 prevention group(Co Q10-P), Co Q10 treatment group(Co Q10-T), NAC prevention group(NAC-P), NAC treatment group(NAC-T), DZ01 prevention group(DZ01-P), DZ01 treatment group(DZ01-T). N-C group was given intraperitoneal injection of saline(0.1ml/10 g BW) twice at 1h interval. The rest of the groups were given intraperitoneal injection of PQ(50mg/kg, 0.1ml/10 g BW), of which the PQ-C group was given intraperitoneal saline(0.1ml/10 g BW) after 1h. Prophylaxis group was dosed before 30 min of exposure, the treatment group was dosed after 1h of exposure. The route and dose of administration were: Co Q10(tail vein injection, 0.125ml/20 g BW), NAC(intraperitoneal injection, 150mg/kg, 0.1ml/10 g BW), DZ01(intraperitoneal injection, 517 mg/kg, 0.1ml/10 g BW). The 7-day general condition, body weight change and death of the mice in the subgroups of the observation group of survival rate were observed and recorded. For the lung injury evaluation group, WLL was used for the odd numbered mice in the subgroups after 48 h exposure, with the BALF protein content measured. The whole-lung of even numbered mice were taken, with 10% homogenized lung tissue after wet weight prepared. The reagent box was used strictly in accordance with the operating instructions to measure the T-SOD activity, GSH-Px activity, GSH and MDA content in lung tissue. Origin Pro software was used to process these data. The quantitative data was recorded in the forms of "mean±standard deviation”, the groups were compared using ANOVA analysis. Logrank test was used in survival analysis, P<0.05 was considered statistically significant. 3.2 The Research on Using WLL to Remove Paraquat in Lung Tissue72 healthy male Wistar rats(License Number: SCXK(Beijing) 2012-0001) weighted around 180~200g were divided into 12 groups through randomized block method. These groups were divided based on the time, 0h group(i.e. no exposure), 1h group, 3h group, 6h group, 9h group, 12 h group, 15 h group, 18 h group, 24 h group, 48 h group, 72 h group and 168 h group, when WLL were used after exposure. All the groups were given intragastric toxin for one time with the PQ dose 60mg/kg. The simulate whole left-lung lavage was conducted in vitro lungs after blood sampling at the corresponding time points. "3ml saline injection- withdrawing three times" was counted as one time lavage. Three times of lavage were conducted and the fluids were stored separately. Left and right lung were prepared as 10% homogenized of lung tissue. The paraquat content of the plasma, the lavage fluids and the tissue samples were detected by Surface enhanced Raman spectroscopy method. The obtained quantitative data were processed by Origin Pro software and recorded in the forms of "mean±standard deviation". The groups were compared using ANOVA, P<0.05 was considered statistically significant. 4 Results 4.1 The preliminary findings of DZ01,Co Q10 in the treatment of paraquat poisoning 4.1.1 Survival rate observationsThe survival rate of each exposure group was significantly lower than N-C group(P<0.01). In the antioxidant intervention group, the survival rate of DZ01-P and DZ01-T groups was higher than that of the PQ-C group and the median survival time of DZ01-P was prolonged. While, the differences above were not statistically significant(P>0.05). The preventive and therapeutic administration Co Q10 and NAC for PQ exposed mice showed no improvement in survival rate. 4.1.2 Results of Evaluation of Lung InjuryCompared to N-C group, PQ-C group had significantly higher level of the lung coefficient, BALF protein and MDA content(P values were 0.001, 0.0009, and 0.02 separately); T-SOD activity, GSH-Px activity, GSH levels were decreased, but no significant difference was demonstrated(P>0.05). Compared to PQ-C Group, the lung coefficient and BALF protein content in DZ01-P group were decreased, GSH content and T-SOD activity were increased but the difference was not significant(P> 0.05), MDA content were decreased significantly(P=0.0001). Compared to PQ-C Group, BALF protein content, MDA of DZ01-T group were decreased significantly(P values were 0.01 and 0.003), Lung coefficient was decreased and GSH content was increased with no significant difference demonstrated(P>0.05). Compare to PQ-C group, lung coefficient and BALF protein content were reduced in Co Q10-P and Co Q10-T groups, so was the MDA content, GSH content was increased, but the differences were not statistically significant(P>0.05). Compared to NAC-P group, the MDA was significantly decreased in DZ01-P group(P=0.018). No significant difference was found in other indicators. As DZ01 T group, the protein content in BALF was significantly decreased compared with NAC-T group(P=0.007), no significant difference was found in other indicators. 4.2 Results of WLL for the removal of Paraquat in Lung Tissue 4.2.1 Changes of paraquat content in the BALFParaquat could be detected in each exposure group of all the three times. High concentration was detected in the group of 1h, 3h and 6h(1573.9±310.13ng/ml, 994.6±147.28 ng/ml and 322.4±47.17ng/ml respectively) for the first time, which was significantly different from the following groups(P<0.01). As to the following groups, the groups of 15 h and 18 h had a higher concentration than others, but there was no statistical significance(P>0.5). 4.2.2 Changes of paraquat content in the bilateral lung tissueFor the left lung undergoing the WLL while the right lung not, the former had a lower concentration at each time, there was a statistical significance in the comparison of the left and right lung in each group(P<0.01). Paraquat concentration reached the peak at 12h(13642.2±1242.66ng/g), which is 19.15 times of the plasma concentration at the same time point. The clearance rate of WLL was(40±12.0)%, calculated by the formula “clearance rate=(paraquat concentration in the right lung-paraquat concentration in the left lung)/ paraquat concentration in the right lung×100%”. 4.2.3 Changes of the paraquat content in the plasmaThe concentration of paraquat in the group of 1h reached a high level(8926.3±1586.47ng/ml), which is significantly different with the subsequent time points(P<0.001). The paraquat concentration decreased rapidly( more than 90%) within 6 hours and maintained a high level in 24 hours, and it could still be detected at 168h(3.0±0.72ng/ml). 5 Conclusions1.With the administration of DZ01 and COQ10 at the given dose, there was no statistical significance of the 7-day survival rate of mice exposed to paraquat(intraperitoneal injection, 50mg/kg)(P>0.05). The prophylaxis of DZ01 could prolonged the median survival time of mice exposed to paraquat, but the difference was not statistically significant(P>0.05).2.The DZ01 could alleviate acute lung injury in the early phase and lipid peroxidation caused by the paraquat poisoning. There was experimental basis as an antioxidant used in the treatment of paraquat poisoning.3.There was no effect of Co Q10 against oxidative stress under the given dosage form and dose in this study.4.The change of the trends of paraquat in rat is similar to that in human, so the rat can be served as a good animal model in the study of the target organ therapy.5. WLL could remove paraquat at a clearance rate(40±12.0)% in the lung tissue of the rat. The first and second peak of the clearance was reached at 1h and 15 h after exposure. Certain amounts of paraquat can be removed in 168 hours. So WLL could be an effective way in the treatment of paraquat poisoning.