| n-Hexane is one of the most important aliphatic compounds, which widely used in glue industry, paints, varnishes, printing inks and so on. n-Hexane has been demonstrated to be neurotoxic in experimental animal studies and in studies on human occupational exposure. The neurotoxicity observed during exposure to n-hexane is the result of the formation of the metabolite2,5-HD from the precursors2-hexanol and2,5-hexanodiol produced in live. It was reported that n-hexane is converted by cytochrome P-450dependent monooxygenases, mainly the CYP2E1and CYP2B6(the orthologous human form of rat2B1) isomers, and by ADH into2-hexanone and, subsequently, to2,5-HD. And CYP2E1was one of key enzymes in vivo formation of2,5-HD from n-hexane in the mice. However, whether the inhabitor of CYP2E1can prevent the neurotoxicity induced by n-hexane is nuclear.Garlic is an herb consumed in both foodstuffs and dietary supplements. Garlic has known antioxidant activity and has been hypothesized to be beneficial to human health, including cardio-protective and chemotherapeutic properties. Garlic oil is a kind of mixture extracted from garlic. Its major compositions are a variety of sulfides including diallyl trisulfide (DATS), diallyl disulfide (DADS) and diallyl sulfide (DAS), which have been considered to be the major biological agents. DAS can inhibit CYP2E1activity in vivo, and can induce hepatic mRNA levels of CYP1A,2B, and3A. GO have strong reductive ability and can reduce peroxides and oxygen so as to enhance the activities of peroxidases and decrease the contents of oxidative products, but It can not improve the function of the neurotoxicity induced directly by2,5-HD. The changes of CYP2B1and CYP2E1caused by GO are completely opposite, whether garlic oil can inhibit the formation of2,5-HD and antagonists neurotoxicity of hexane is unclear.The current study was designed to study the effects of GO on the formation of the metabolite2,5-HD from n-hexane, and to investigate the protective effect induced by garlic oil on the peripheral nerve injuries induced by n-hexane.Methods1.Animal treatmentAdult male Wistar rats, weighing230-260g, were provided by the Laboratory Animal Center of Shandong University. Rats were housed respectively, and drinking water and a commercial animal feed were available ad libitum. The animal room was maintained at approximately22oC and50%relative humidity with a12h light-dark cycle. They were kept in the Experimental Animal Center of Shandong University for7days for acclimatization, and randomly divided into five group i.e., the control, the GO control group, the n-hexane, the low dose GO, and the high dose GO group (n=10each group). The rats in the low and high doses of GO groups were pretreated with GO (40and80mg/kg bw) before exposed to n-hexane (i.g.,2000mg/kg bw) for continuous10weeks (six times per weeks), while the animals of the n-hexane group received equal volume of peanut oil and then the same dose of n-hexane. n-Hexane was dissolved in peanut oil and administered at4ml/kg body weght/per dose. The control group rats received an equivalent volume of peanut oil by gavage. The GO control group rats were treated with GO (80mg/kg bw). The serum was obtained for2,5-HD assay. The livers were quickly dissected and frozen in liquid nitrogen before storing them at-80℃. All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals.2. Body Weight measurement and Neurological TestingThe body weight was measured daily, and the dosage was varied accordingly. Neurological deficits were detected and quantified using gait score once a week. The measurement was chosen because it represents a sensitive and reliable index of neurotoxicant-induced changes in behavioral status. To measure gait abnormalities, rats were placed in a clear plexiglass box and were observed for3min. Following observation, a gait score was assigned from1to4, where1=a normal, unaffected gait;2=a slightly affected gait (foot splay, slight hindlimb weakness and spread);3=a moderately affected gait (foot splay, moderate hindlimb weakness, moderate limb spread during ambulation); and4=a severely affected gait (foot splay, severe hindlimb weakness, dragging hindlimb, inability to rear). A trained, blinded observer who was not involved in animal care or gavage administration performed the behavioral evaluation. Three successive measurements were averaged for rats.3.Preparation of the microsome samplesThe hepatic microsomes were prepared by homogenization and differential centrifugation. Fresh liver tissue was homogenized in four volume ice-cold TMS buffer (50mM Tris-HCL,6.4mM MgC12,0.2M saccharose, pH=7.5). The resulting homogenate was centrifuged at12,000g for15min and the supernatants were further centrifuged at105, OOOg for60min. The final pellet was reconstituted in the above buffer. The protein concentrations were quantified using BCATM protein assay kits. All the procedure was conducted at4℃and stored at-80℃.4.Determination of the activities of CYP2E1and CYP2B1The activity of CYP2E1was measured with aniline as the substrate as previously described. CYP2B1activity was detected by measurement of the dealkylation of the PROD using Hitachi fluorescence spectrophotometer (Hitachi High-Technologies Corporation; excitation λ=535nm, emission λ=586nm).5.Liver alcohol dehydrogenase activityADH activities were measured at37℃in a mixture containing5mM NAD and5mM ethanol in9mM glycine-120mM sodium pyrophosphate buffer, pH8.8. All the results were corrected for blank reactions run without the substrate. Specific activity was expressed as nmol ethanol (EtOH) metabolized min-1mg protein-1, calculated using the value of0.00622as the absorbance of1μmol NADH at340nm. Proteins were estimated using BCATM protein assay kits.6.Western blot analysisFrozen liver tissue was homogenized in RIPA buffer(100mM Tris (pH=7.4),150mM NaCl,1%Triton X-100,1%sodium deoxycholate,0.1%sodium dodecyl sulphate (SDS),5 mM EDTA,1mM PMSF,5mg/ml aprotinin, leupeptin and pepstatin). Of the total protein,25mg (for CYP2E1) or30mg (for CYP2B1) was subjected to SDS-PAGE followed by electrical transfer to a polyvinylidene fluoride membrane for immunoblot analysis. The membranes were incubated with primary antibodies for CYP2E1and CYP2B1, or β-actin overnight at4℃. After incubation with horseradish peroxidase-conjugated secondary antibody for60min, the antigenic protein on the membrane was visualized by the chemiluminescence method using a Pierce ECL reagent kit. The results were expressed as the ratio of protein to β-actin.7.Analysis of2,5-HD in serum1.2ml ethyl acetate was dissolved in0.3ml serum containing0.03g anhydrous potassium carbonate. After40min of mixing on a shaker, all samples were centrifuged at3000rpm for20min. The solvent extract was withdrawn and2,5-HD concentrations in the serum were analyzed by gas chromatography. The concentrations of serum2,5-HD were determined using a GC2010gas chromatograph with flame ionization detection (GC-FID). For the separation of2,5-HD a capillary column DB-5(length30m, I.D.0.32mm, film thickness0.25μm, Agilent Technologies) was used. The detector and injector temperatures of the GC were250and230℃. The starting temperature of the GC oven was100℃for2min. Then, the temperature raised to250℃with10℃/min and was held for15min. Nitrogen was used as a carrier gas at a flow-rate of1.0ml/min. Then,1u1of the sample was injected at a split ratio of20:1. From the stock solution of2,5-HD, various standard solutions in a concentration range between10and160mg/1were prepared in blank serum. The standard solutions were used to draw the standard curve.Results1. Changes of body weight and clinical signsInitial body weights were similar in all groups, and weight gains during the study were similar in control and GO control groups. Rats in the n-hexane group failed to gain weight and started to lose weight at8weeks. In the low dose GO, and the high dose GO group, rats did not gain as much weight as control animals, but weighed significantly better than did those in the n-hexane groups. Simultaneously, intoxicated rats developed progressive gait abnormalities, about on4weeks of exposure, rats in the n-hexane exhibited unsteady gait and foot splay, and slight hindlimb weakness. With the time passing by, intoxication of n-hexane caused progressive development of gait abnormalities (3.6±0.70), which were less apparent in the GO-treated rats. In the last week of the test, n-hexane-treated rats exhibited a pronounced functional impairment consisting hindlimb paralysis and dragging of the feet along the floor. Compared with those of n-hexane group, rats in the low and high GO dose profoundly reduced these motor deficits (2.1±0.74) and (1.9±0.57), respectively.2. Alterations of the activities and protein levels of CYP2E1in liverThe activity of CYP2E1in GO control group was less than that in control group (p<0.01). The expression level of CYP2E1protein in GO control rats was also lower than that in the control rats (p<0.01). Conversely, the activity of CYP2E1was greater in n-hexane-treated rats than in control rats (p<0.01), and the protein levels was similar to it (p<0.01). Compared with control rats, in the low and high dose GO groups, the activities CYP2E1were decreased by27.4%and44.5%, respectively. Analogously, the expression levels of CYP2E1were significantly lower (by32.9%and39.1%) compared to the control rats (p<0.01).3. Alterations of the activities and protein levels of CYP2B1in liverThe activity of CYP2B1in GO control group was more than that in control group (p<0.05). The expression level of CYP2B1protein in GO control rats was also higher than that in the control rats (p<0.01). Similarly, the activity and protein levels of CYP2B1were greater in n-hexane-treated rats than in control rats (p<0.01). Compared with control rats, in the low and high dose GO groups, The activities CYP2B1were increased by13.1%(p>0.05) and52.6%(p<0.01), respectively. Analogously, the expression levels of CYP2B1were significantly higher (by3.3%and57.2%) compared to the control rats.4. Alterations of the activity and protein levels of alcohol dehydrogenase in liverAlthough, the expression levels of ADH protein in five groups were not significantly changed, the activities were distinct. Compared with control rats, the activity of ADH in n-hexane-treated group was increased by49.8%(p<0.01), but it in GO control group was not changed. The activities ADH were decreased by25.3%(p<0.01) and86%(p<0.01) than the n-hexane-treated group, respectively, in the low and high dose GO groups. 5. Changes of rats serum2,5-HD level2,5-HD was not detected by GC-FID in the serum from rats that did not receive n-hexane. The serum concentration of the metabolite increased in n-hexane-treated rats to μg/ml, but that in the low and high dose GO rats were decreased by47.7%(p<0.01) and78.7%(p<0.01), respectively.Conclusions1. The garlic oil could effectively attenuate n-hexane-induced peripheral nerve injury in rats and improve the neurobehavior.2. The metabolic activation of n-hexane to2,5-HD could be prevented efficiently by garlic oil, in the rats of chronic n-hexane poisoning.3. The garlic oil could significantly inhibit the activation and expression level of CYP2E1and inhibit the activation of ADH.This might be involved in the effect of garlic oil against n-hexane. |
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