The Effects Of Garlic Oil On Lipid Peroxidation Induced By 2,5-Hexanedione And Metabolism Of N-Hexane

n-Hexane is widely used in industry as organic solvent.The pollutant mainly contained in industrial environment can penetrate unbroken skin,the epithelial linings of gastrointestinal tract and respiratory tract,and distributes in the organs lipid content of which is high,such as brain,liver,kidney,spleen,testicle,blood and so on.Although n-hexane belongs to non-toxic chemical in acute toxicity range,it is considered as high-risk substance because of its high volatility and the abilities of lipid-resolvability and cumulation.There are different performances in n-hexane acute and chronic toxicity: high-dose n-hexane mainly elicits acute encephalopathy(inhibition of central never system),irritation of skin and membranes,whereas prolonged exposure to low-dose n-hexane mainly induces neuropathy characterizing as the central-peripheral distal axonopathy.But until now there is not effective special therapy yet.It had been reported that exposure of n-hexane can obviously produce lipid peroxidative damage(LPD) of liver and nevous tissues of human and experimental animals showing the increasing concentrations of oxidative productions and the changing activities of oxidases and antioxidases.These suggested that LPD might be one of mechanisms of neuropathy induced by n-hexane.Garlic oil(GO) is a kind of mixture extracted from garlic.Its major compositions are a variety of sulfides which have strong reductive ability and can reduce peroxides and oxygen so as to enhance the activities of peroxidases and dicrease the contents of oxidative products.2,5-Hexanedione(2,5-HD),the active metabolite of n-hexane,is the most important media of n-hexane neurotoxicity.Whether or not garlic oil can cause antagonism of the 2,5-HD inducing peroxidation in rat nervous tissues,and at the same time mitigate its peripheral nerve toxicity.The problem has not been reported at home and abroad up to now.In the present study,we administrated the Wistar rats with 2,5-HD 300mg/kg.bw by intraperitoneal injection for six weeks in order to make a n-hexane chronic poisoning model,meanwhile the therapeutic rats were given 40mg/kg.bw or 80mg/kg.bw oral GO,respectively,four to six hours advanced n-hexane exposure every day.The neurobehavioral changes were measured at different time during the administration,including hindlimb support index,balance index,and gait score.At the end of the study,the oxidative status of rat brain,spinal cord and sciatic nerve, malondialdehyde(MDA) and glutathione(GSH) contents,total antioxidant capacity (T-AOC) and hydroxyl radical inhibition capacity,was determined.Therefore we hope to observe the role of GO on lipid peroxidation injury and neurotoxicity caused by 2,5-HD.n-Hexane polyneurotoxicity is mainly induced by its metabolite 2,5-HD,and the cytochrome P450(CYP450) plays an essential function in the process of n-hexane metabolism.It has been reported that GO can obviously inhibit the activity of CYP450 in some kinds of animal disease model,but the function of GO on the metabolism of n-hexane has not been investigated yet.Studies have shown that serum levels of 2,5-HD positively correlated with its content in the target organ-sciatic nerve and n-hexane neurotoxicity,thus we presume that decreasing the serum level of 2,5-HD would possibly decrease the occurrence of n-hexane poisoning,and the serum level of 2,5-HD may reflect the change of level of n-hexane metabolism.In order to meet the need of determining mouse serum 2,5-HD in this experiment,we make use of ethyl acetate as extractant and anhydrous potassium carbonate(K₂CO₃) to improve its extraction rate with HP-5 capillary gas chromatographic column and hydrogen flame ionization detector(FID) separating and detecting the serum 2,5-HD.On this basis,the test was designed to administrate Kunming mice with n-hexane 3000mg/kg.bw,and the intervening group mice were given 80mg/kg.bw GO two hours before n-hexane exposure.And the gas chromatographic method was used to measure serum 2,5-HD content at different time after n-hexane exposure.Meanwhile we determined 2,5-HD contents of different doses of GO (10mg/kg.bw,20mg/kg.bw,40mg/kg.bw,80mg/kg.bw) interventing groups and different time-before and after two hours of exposure-interventing groups six hours after exposed to n-hexane.The serum levels of 2,5-HD in different gender and week-old mice exposed to the same dose n-hexane were determined.So we investigated the effect of GO on n-hexane metabolizing to 2,5-HD and the impact of sex and age on the toxicity of n-hexane,so as to provide feasible experimental evidence for preventing the toxicity of n-hexane.Results1.Body Weight ChangesThe body weight of normal rats grew steadily.While model and GO groups' weight had a slow gain,and even appeared negative growth.The weight of the model group rats was significantly lower than that of the control rats(P＜0.01) from the third week,and GO low-dose group from the second week and GO high-dose group from the first week were significantly lower than model group(P＜0.01 or P＜0.05).2.Neurobehavioral changes(1) Gait score:GO group rats performed abnormal exercise at the forth week after exposed to 2,5-HD,in advance of one week than model group.Gait scores of the other groups were significantly higher(P＜0.01) than that of control group,and GO high-dose group was obviously higher(P＜0.05) than model group.(2) Hindlimb support index:Compared to the initial index,hindlimb support indexs of model group and GO groups increased by 44%,50%,49%at the end of the forth week, and were significantly higher than that of control group(P＜0.01),but there was no statistical difference among the three groups(P＞0.05).(3) Balance index:Excluding the control group,the other three groups' balance indexs reduced in varying degrees.To the forth 2,5-HD exposure week,the indexs of model and GO groups decreased by 30%,45%,68%,and were lower than that of control group (P＜0.05 or 0.01).Especially there was obvious difference(P＜0.05) between GO high-dose group and model group.3.The oxidative level changes of nerve tissues(1) Changes of MDA content:The MDA contents,in brain,spinal cord and sciatic nerve, of model group,comparing with these of control group,increased by 26%,25%and 17% (P＜0.05 or 0.01),respectively.While two GO groups reduced by 32%～43%(P＜0.01) and 32%～52%(P＜0.01) than model group,respectively. (2) Changes of T-AOC:Comparing with T-AOC of control group,that of nerve tissues of model group rats increased,but there was no statistical difference among each group (P＞0.05).(3) Changes of GSH content:In rat nerve tissues,GSH contents of model group were lower than these of control group.The contents of GO groups were higher than these of model group in each nerve tissues,with the exception of the content of GO low-dose group in sciatic nerve was lower,but all of these changes were not statistically significant (P＞0.05).(4) Changes of hydroxyl radical inhibition capacity:Compared with control group,the capacity of model group in brain,spinal cord and sciatic nerve was weakened by 34%, 16%and 28%(P＜0.05 or 0.01),respectively.But GO had antagonized this trend in different degrees in the study,and the low-dose GO enhanced the capacity by 22%,28% and 28%(P＜0.01),respectively,and simultaneously the high-dose GO enhanced by 50%, 46%and 40%(P＜0.01),respectively,compared with that of model group.4.Changes of mice serum 2,5-HD level(1) Metabolism of n-hexane after once exposure:After the mice given 4000 mg/kg.bw n-hexane,the serum 2,5-HD content increased continuously until the tenth hour when the peak appeared,and then declined gradually.At the twenty hour it could almost not be determined.(2) Changes of serum 2,5-HD content after different doses of n-hexane exposure:Serum 2,5-HD concentrations increased as n-hexane doses increasing.It could not be detected in normal mice serum,but the serum contents of 2000,4000 and 6000mg/kg.bw groups reached 8.04,16.68 and 22.38μg/ml,respectively,eight hours after exposed to n-hexane. The doses of n-hexane and the concentrations of 2,5-HD in serum showed an obvious dose-effect relationship.(3) Impact of GO on n-hexane metabolism:It showed the same metabolic trend as 4(1) in GO group which was given n-hexane 3000mg/kg.bw by gavage,increasing with time after administration of n-hexane and then reducing until to zero.But the concentrations of serum 2,5-HD at different time were lower than that of simple n-hexane mice,and they were significantly lower at four,six,eight,ten hours after n-hexane exposure(P＜0.05 or 0.01).(4) Effects of different doses of GO on serum 2,5-HD content:The Kunming mice were administrated with different doses of GO two hours before n-hexane exposure,and 2, 5-HD concentrations in serum decreased by 16.2%,20.8%,22.8%(P＜0.05) and 32.1% (P＜0.01) comparing with the simple poisonning group.And the doses of GO and the levels of serum 2,5-HD showed an obvious dose-effect relationship.(5) Effects of time of giving GO on serum 2,5-HD content:Two hours before and after n-hexane exposure,respectively,the mice were given 80mg/kg GO,and then their serum 2, 5-HD concentrations decreased significantly comparing to simple group(P＜0.05). Furthermore pre-treatment group was lower in the concentration than post-treatment group, but there is no significant difference between the two groups(P＞0.05).(6) Effect of age on serum 2,5-HD content:Serum 2,5-HD levels of different week-old mice were different(P＜0.05) eight hours after exposure to the same dose of n-hexane:the five-week-old group(22.83μg/ml) was higher than the four-week-old(19.59μg/ml) and six-week-old groups(16.42μg/ml).(7) Effect of gender on serum 2,5-HD content:The serum 2,5-HD levels were different in different genders after once n-hexane administration,and the female mice's content (13.22μg/ml) was significantly higher than the male's(10.34μg/ml),increasing by 27.9% (P＜0.05).5.Determination of mouse serum 2,5-HD by gas chromatography(1) Selection and specificity of methodology:Comparison of extraction rate showed: dichloromethane＞ethyl acetate＞ether,and we simultaneously considered the volatility and toxicity.Finally ethyl acetate was selected as extractant in the method.Comparing extraction rate when we added anhydrous Na₂SO₄,anhydrous K₂CO₃ or no any salt to the serum samples,we found that salts could significantly increase the extraction efficiency of ethyl acetate on mice serum 2,5-HD,but anhydrous K₂CO₃ was better.Finally the new method could completely separate ethyl acetate(Rt=2.26min) and 2,5-HD(Rt=3.33min), moreover n-hexane(Rt=2.266min) did not interfere the determination of 2,5-HD. (2) Detection of methodology:The minimum detectable concentration of this method was 0.03μg/ml,and its linear range was 0.03μg/ml～80μg/ml,extraction rate 83.8～99.9%, the relative standard deviation(RSD) within one day 3.3%～5.2%and that among days 3.3%～8.0%.And the samples can be stored for 24 hours under 4℃,and one week under -20℃.Conclusion(1) GO can antagonize the 2,5-HD induced LPD in rat nerve tissues,but can not improve the nerve dysfunction.It suggests that oxidative damage may be not the main mechanism of 2,5-HD toxic nerve injury.(2) GO can significantly inhibit the production of 2,5-HD with the level of serum 2, 5-HD decreasing,and the inhibiting ability relates to the dose and the giving time of GO, so that GO may result in the weakness of the toxicity of n-hexane.Age and gender have a certain impact on the metabolism of n-hexane to 2,5-HD.All of these provide strong experimental evidences for us to prevent and reduce the toxicity of n-hexane.(3) The new gas chromatography method established in the experiment is accurate and reliable with good reproducibility,and can be used for the determination of serum 2,5-HD.