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Diallyl trisulfide attenuated n-hexane induced neurotoxicity in rats by modulating P450 enzymes
Chemico-Biological Interactions 265 (2017) 1e7
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Diallyl trisulfide attenuated n-hexane induced neurotoxicity in rats by modulating P450 enzymes Shuo Wang a, Ming Li a, Xujing Wang a, Xianjie Li a, Hongyin Yin b, Lulu Jiang a, Wenting Han a, Gleniece Irving c, Tao Zeng a, Keqin Xie a, * a b c
Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong 250012, China Jinan Municipal Center for Disease Control & Prevention, Jinan, Shandong Province, China North East Regional Health Authority, Ocho Rios, Jamaica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2016 Accepted 19 January 2017 Available online 20 January 2017
Chronic exposure to n-hexane can induce serious nerve system impairments without effective preventive medicines. Diallyl trisulfide (DATS) is a garlic-derived organosulfur compound, which has been demonstrated to have many beneficial effects. The current study was designed to evaluate whether DATS could restrain n-hexane induced neurotoxicity in rats and to explore the underlying mechanisms. Rats were treated with n-hexane (3 g/kg, p.o.) and different doses of DATS (10, 20 and 30 mg/kg, p.o.) for 8 weeks. Behavioral assessment showed that DATS could inhibit n-hexane induced neurotoxicity, demonstrated by the improvement of the grip strength and decline of gait scores. Toxicokinetic analysis revealed that the Cmax and AUC0-t of 2,5-hexanedione (product of n-hexane metabolic activation) and 2,5-hexanedione protein adducts in serum were significantly declined in DATS-treated rats, and the levels of pyrrole adducts in tissues were significantly reduced. Furthermore, DATS activated CYP1A1 and inhibited n-hexane induced increased expression and activity of CYP2E1 and CYP2B1. Collectively, these findings indicated that DATS protected the rats from n-hexane-induced neurotoxicity, which might be attributed to the modulation of P450 enzymes by DATS. © 2017 Elsevier B.V. All rights reserved.
Keywords: n-Hexane Diallyl trisulfide Neurotoxicology CYP450 2,5-Hexanedione Pyrrole adducts
1. Introduction The organic solvent n-hexane had been implicated in a multitude of occupational and intentional inhalation exposure settings. Chronic exposure to n-hexane could produce centraleperipheral axonopathy in humans and experimental animals, which was characterized by paranodal giant axonal swellings and demyelination [1]. It has been demonstrated that 2,5-hexanedione (2,5-HD), a gediketone metabolite of n-hexane oxidization by cytochromes P450, was responsible for its neuropathy [2,3]. Once formed, 2,5HD might bind to lysine ε-amine groups of proteins and form the 2,5-dimethylpyrrole (2,5-DMP) adducts, and then yielded secondary autoxidative reactions forming pyrrole dimers which produce
Abbreviations: DATS, diallyl trisulfide; 2,5-HD, 2,5-hexanedione; 2,5-DMP, 2,5dimethylpyrrole; CYP, cytochromes; NADPH, nicotinamide adenine dinucleotide phosphate; FID, flame ionization detector; Cmax, maximum concentration; Tmax, the time to peak plasma concentration; AUC(0-t), the area under the serum concentrationetime curve; t1/2, the half-life time. * Corresponding author. E-mail address: [email protected] (K. Xie). http://dx.doi.org/10.1016/j.cbi.2017.01.013 0009-2797/© 2017 Elsevier B.V. All rights reserved.
intra- and inter-molecular cross-linkings in proteins. This kind of protein posttranslational modifications may eventually lead to critical protein function loss and were believed to responsible for the nerve system dysfunction [4e7]. Thus, one possible approach for the protection of n-hexane-induced neuropathy is to prevent 2,5-HD or even pyrrole adducts formation and oxidation. Garlic is a popular herbal product that has been applied for centuries for medicinal purposes [8]. It has a variety of pharmacological activities such as anticancer, antimicrobial, antiatherosclerotic, antidiabetic, and immunomodulatory effects [9]. Diallyl trisulfide (DATS) is the major phytochemical component contained in garlic and garlic products such as garlic essential oil [8,10]. Previous studies have revealed that DATS has excellent antioxidant capacity and can attenuate many chemicalseinduced oxidative damage in liver and also in nervous system [11e13]. Furthermore, DATS can also increases GSH levels in vivo. The preventive effects of GSH had been confirmed through the use of N-acetylcysteine (NAC), a precursor of GSH, in n-hexane induced neuropathy [14,15]. We herein hypothesized that DATS could prevent n-hexane induced neurotoxicity, and established n-hexane induced peripheral nerve injury models to test the hypothesis and investigate the
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possible mechanisms.
2.4. Toxicokinetics studies
2. Materials and methods
Six rats randomly selected from n-hexane group and 3 DATS plus n-hexane groups were used to determine the long-term effect of DATS on n-hexane toxicokinetics. The rats were fasted overnight but had free access to water. DATS (10, 20 or 30 mg/kg) were gavaged two hours before the administration of n-hexane (3 g/kg). Blood samples were collected from the jugular vein at 0, 2, 4, 6, 8, 10, 12, 14, 16, 20 and 22 h after n-hexane exposure. The plasma samples were obtained immediately after sampling by centrifugation at 1000 g for 5 min. Pyrrole adduct was analyzed immediately and the rest was stored at 20 C for 2,5-HD analysis.
2.1. Chemicals and reagents n-hexane, cyclohexanone and dichloromethane were purchased from Hongyan Chemical Reagent Factory (Tianjin, China). DATS (purity > 97%) was purchased from Jiangsu Chiatai Qingjiang Pharmaceutical CO., LTD (Huaian, China). 2,5-hexanedione, 2,5dimethylpyrrole, 4-dimethylaminobenzaldehyde, NADPH, ethoxyresorufi, methoxyrsorufin, pentoxyresorufin and resorufin were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Primary antibodies of anti-CYP1A1, anti-CYP1A2, anti-CYP2B1/2, antiCYP2E1 were provide by Santa Cruz Biotechnology (Santa Cruz, CA, United States) and Abcam (Cambridge, United Kingdom). BCA™ protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA). All other chemicals were of the highest grade commercially available. 2.2. Animals treatment Seven-week-old male Wistar rats, weighing 220e240 g, were provided by Experimental Animal Center of Shandong University. Drinking water and commercial animal feed were available ad libitum. The animal room was maintained at 22 ± 2 C and 50 ± 10% relative humidity with a 12-h-light/-dark cycle. After the acclimatization period, the animals were randomly divided into 6 groups, i.e. control group, n-hexane group, DATS group, and 3 DATS plus nHexane groups (n ¼ 12 in each group). Rats in n-hexane plus DATS groups were administrated with DATS (10, 20 or 30 mg/kg/day, orally) combined with n-hexane (3 g/kg/day, orally) seven times per week for consecutive 8 weeks. The rats in n-hexane group were treated with corn oil and n-hexane (3 g/kg/day, orally) and rats in DATS group were treated with DATS (30 mg/kg/day, orally) and corn oil. The rats in control group received equal volume of vehicle. All animal experimental procedures and protocols of the study were approved by the Ethics Committee for Animal Experiments of Shandong University Institute of Preventive Medicine (Permit Number: 20111231) in accordance with the NIH Guide for Care and Use of Laboratory Animals. 2.3. Behavioral assessment Forelimb and hind limb grip strength and gait scores were used to assess the effects of DATS on n-hexane induced peripheral neuropathy. Forelimb and hind limb grip strength were measured every two weeks with a grip strength meter (YLS-13A, Jinan Yiyan Technology and Development Co., Ltd., Jinan, China). Briefly the rats were gently lowered over the top of the grid to let its front and hind paws grip the smooth metal pull bar. And then with a uniform force drew the rats until complete the length of the grid. Each anima repeated for three times. Prior to the rats being sacrificed, gait scores were obtained [16,17]. They were placed in an open field, and were observed for 3 min. Following observation, a gait score was assigned from 1 to 4 where 1 ¼ a normal, unaffected gait; 2 ¼ a slightly abnormal gait (hindlimbs show uncoordinated placement, exaggerated or overcompensated movements, or are splayed slightly, walks on tiptoes); 3 ¼ moderately abnormal gait (obvious movement abnormalities characterized by markedly splaying hindlimbs, ataxia, swaying, rocking, lurching, stumbling); 4 ¼ severely abnormal gait (flat foot walk, hindlegs flat on surface, crawling, or unable to support weight).
2.5. Free 2,5-HD detection A 50 ml of 0.5 mg/ml of cyclohexanone (internal standard) and 0.2 g of NaCl were added to 100 ml plasma sample. After addition of 1.0 ml of dichloromethane, samples rotated vigorously and centrifuged at 3500 rpm for 10 min. The dichloromethane extraction solution was evaporated to about 0.1 ml with a nitrogen flow and then injected into a gas chromatography system [18]. The GC used was a Shimadzu GC-2010 (Kyoto, Japan) equipped with a flame ionization detector (FID) and an HP-5 column (30 m 0.32 mm 0.25 mm, Agilent Technologies). The injector temperature was set at 180 C and the detector temperature at 260 C. The oven was set at 80 C and the injection volume was 2 ml. The carrier gas was nitrogen at a flow rate of 1.5 ml/min and the split ratio was set at 3:1. 2.6. Pyrrole adducts detection The pyrrole adducts were measured spectrophotometrically after reaction of 0.1 ml of plasma with 0.1 ml guanidine hydrochloride (70%) and 0.1 ml of Ehrlich's reagent (3% 4dimethylaminobenzaldheyde in 40 ml of methanolic 14% boron trifluoride and 60 ml of ethanol) [19]. Absorption values were measured at 526 nm, using automatic microplate reader (Infinite® 200 PRO, TECAN Inc. Switzer). The calculations were based on standard curve prepared with different concentrations of 2,5dimethylpyrrole (5e80 nmol/ml). Liver, kidney, spinal cord and brain samples were homogenized with a homogenizer (10,000e15,000 rev./min, 10 s) at 4 C in physiological saline at 0.5 g wet weight/ml. A 0.5 ml of 0.7 g/ml of aqueous guanidine hydrochloride was added to 0.1 ml tissue homogenate. After digestion at room temperature for 30 min, the pyrrole adducts was determined by the above procedure [20]. Sciatic nerves were initially broken into powder with pestle in liquid nitrogen, and then homogenized in physiological saline at a concentration of 0.5 g wet weight/ml, followed by adding 0.3 ml of 2.5% trypsin. After digestion at 56 C for 1 h, the pyrrole adducts was determined by the above procedure. Values were expressed as nanomoles of 2,5-dimethylpyrrole per milliliter or gram of tissue. 2.7. Western blot analysis of CYP proteins in the liver Total protein extract was obtained as previously described [21]. Briefly, livers samples were homogenized in ice-cold RIPA lysis buffer (50 mM Tris-HCl PH7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 5 mM NaF, 50 mM TriseHCl) supplemented with 1 mM PMSF and 1% cocktail protein protease inhibitors. After a 20-min centrifugation at 12,000 g, the supernatant was collected and the protein concentration was determined using the BCA™ protein assay kit. Equal aliquots of samples were mixed with loading buffer, and then heated at 100 C
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for 10 min. The pre-treated protein samples (about 20e50 mg) were separated by 10% or 15% SDS/polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Blots were blocked with 5% skim milk in TBST for 1 h at room temperature. Then the membrane was incubated with the anti-CYP1A1, anti-CYP1A2, anti-CYP2B1/2, anti-CYP2E1 or anti-bactin antibodies overnight at 4 C. After washing off unbound primary antibodies, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated antibodies for 1 h at room temperature. The blots were then washed, visualized by enhanced chemiluminescence detection kit and exposed on x-ray film. The relative optical densities of the bands were quantified and normalized by b-actin, using Kodak Imaging Program and ImagePro Plus software (Eastman Kodak Company, New Haven, CT, USA).
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3.2. DATS co-treatment led to the decrease of plasma concentration of 2,5-HD and pyrrole adducts The mean plasma concentration-time profiles of 2,5-HD and pyrrole adducts after chronic oral exposure of n-hexane (3 g/kg) in the absence and presence of DATS (10, 20, 30 mg/kg) are depicted in Fig. 2. The toxicokinetic parameters are summarized in Table 1. Compared with the rats in n-hexane group, the AUC0-t value of 2,5HD in rats of DATS (10, 20 and 30 mg/kg) groups were decreased by 3.3%, 17.1% and 37.4% (P < 0.05), respectively, while the AUC0-t of pyrrole adducts were reduced by 6.9%, 7.0% and 26.5% (P < 0.05), respectively. The Cmax and Cmin of 2,5-HD and pyrrole adducts were
2.8. Isolation of liver microsomes The liver microsomal fractions were prepared by homogenization and differential centrifugation as previously described [22]. Briefly, liver tissue was homogenized in four volume ice-cold TMS buffer (50 mM Tris-HCL, 6.4 mM MgCl2, 0.2 M saccharose, pH ¼ 7.5). The resulting homogenate was centrifuged at 12,000 g for 15 min and the supernatants were further centrifuged at 105,000 g for 60 min. The final pellet was reconstituted in the TMS buffer. The protein concentrations were quantified using BCA™ protein assay kits. All the procedure was conducted at 4 C. 2.9. Determination of the activities of CYP1A1, CYP1A2, CYP2B1/2 and CYP2E1 The activity of CYP2E1 was measured with aniline as the substrate as previously described [22]. CYP1A1, CYP1A2 and CYP2B1/2 activities were detected by measurement of the dealkylation of the ethoxyresorufin, methoxyresorufin, and pentoxyresorufin using Hitachi fluorescence spectrophotometer (Hitachi HighTechnologies Corporation) [23]. 2.10. Statistical analysis Values are presented as the mean ± SD and analyzed statistically using the SPSS 13.0 software. For Forelimb and hind limb grip strength parameters, the data was analyzed by two-way repeatedmeasures analysis of variance, followed by one-way ANOVA coupled with LSD post hoc test for each two weeks' grip strength. For all other data, one-way ANOVA was adopted, followed with LSD for the multiple comparisons. The level of statistical significance was set at P < 0.05. The maximum concentration (Cmax) and the time to maximum concentration (Tmax) were obtained from the observed data. The area under the serum concentrationetime curve (AUC(0-t)) and the half-life time (t1/2) were calculated using DAS 3.0 software. 3. Results 3.1. Effect of DATS on n-hexane induced peripheral neuropathy n-Hexane exposure led to significant peripheral neuropathy, shown by the decrease of grip strength and increase of gait scores. Before n-hexane exposure, all rats exhibited normal, unaffected grip strength. By the end of 8 weeks' exposure, rats exposed to nhexane exhibited a significantly grip strength lost compared with the control group, which was significantly suppressed by DATS (20 and 30 mg/kg) co-treatment. Parallelly, 20 and 30 mg/kg DATS also inhibited the increase of gait scores induced by n-hexane. (Fig. 1).
Fig. 1. The changes of grip strength and gait score after DATS co-treatment on nhexane induced peripheral neuropathy. (A) Every two weeks limb grip strength of the n-hexane experiment; (B) Abnormalities of gait during n-hexane experiment. The data were expressed as mean ± S.D. (n ¼ 12 per group). Significant statistical difference was indicated by *P < 0.05, **P < 0.01, with respect to the control rats; #P < 0.05, ##P < 0.01, with respect to the model rats.
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Fig. 2. Mean plasma concentrations-time curves of 2,5-HD (A) and pyrrole adducts (B) in rats following oral administration of 3 g/kg of hexane alone (C) or coadministered with 10 mg/kg (B), 20 mg/kg (-) and 30 mg/kg (,) of DATS. Bars represent standard error (n ¼ 6).
Table 1 Toxicokinetic parameters of plasma 2,5-HD and pyrrole adducts following n-hexane administration after coadministration of DATS (n ¼ 6). Group 2,5-HD Hexane Hexane þ DATS(10 Hexane þ DATS(20 Hexane þ DATS(30 Pyrrole Adducts Hexane Hexane þ DATS(10 Hexane þ DATS(20 Hexane þ DATS(30
AUC0-t (nmol/ml$h)
mg/kg) mg/kg) mg/kg)
8231.2 7956.4 6824.2 5150.9
mg/kg) mg/kg) mg/kg)
770.4 717.4 716.1 566.5
± ± ± ±
± ± ± ±
2811.4 2049.5 553.4 1648.6*
79.3 69.8 91.6 180.8*
Tmax (h) 10.3 10.3 10.0 10.3
Cmax (nmol/ml) ± ± ± ±
0.8 0.8 0.0 1.5
10.9 ± 0.1 11.7 ± 2.3 11.0 ± 0.1 9.7 ± 2.9
727.5 642.6 601.1 507.2 41.5 37.1 37.6 30.8
± ± ± ±
± ± ± ±
248.5 196.4 85.5 153.8
3.6 5.1 7.1 9.5*
Cmin (nmol/ml) 138.6 ± 73.9 173.7 ± 77.3 123.0 ± 18.5 65.7 ± 22.7 31.6 29.9 29.7 23.1
± ± ± ±
4.7 3.0 1.8 7.2*
Values are presented as mean ± S.D., AUC0-t area under the curve, Tmax the time to peak plasma concentration, Cmax the peak plasma concentration, Cmin the minimum plasma concentration. * P < 0.05, **P < 0.01 with respect to rats in the n-hexane group.
also suppressed by the DATS co-treatment. 3.3. Tissue distribution of pyrrole adducts Tissue distribution of pyrrole adducts after repeated n-hexane and DATS administration were presented. As shown in Fig. 3, the nervous system had higher adducts levels after adjusting by protein concentration, compared with the pyrrole adducts levels in the liver and the kidney. DATS (20 and 30 mg/kg) co-administrate led to a significant decline of the pyrrole adducts levels in the brain, spinal cord and sciatic nerves.
64%, 274% and 79%, respectively (P < 0.05). In DATS group, the activities of CYP2E1 were decrease by 47%, while the activities of CYP1A1 and CYP2B1 were increased by 44% and 114%. The changes of protein levels of the above four phase I enzymes were paralleled with those of the activities (Fig. 4). The protein level of CYP2E1 showed a dose-dependent decrease in rats treated with 10, 20 and 30 mg/kg DATS. The expression changes of CYP2B1 induced by nhexane were suppressed by mega dose DATS co-treatment. The expression of CYP1A1 was upregulated after DATS co-treatment but CYP1A2 was not changed significantly. Meanwhile, DATS cotreatment also significantly inhibited the increases of the activities of CYP2E1 and CYP2B1 and increased the activity of CYP1A1.
3.4. Effects of DATS and n-hexane on the protein levels of hepatic cytochrome P450s in rats
4. Discussion
As shown in Table 2, n-hexane exposure resulted in the dramatic increase of the activities of CYP1A1, CYP1A2, CYP2B1 and CYP2E1. Compared with the control group, the activities of CYP1A1, CYP1A2, CYP2B1 and CYP2E1 in n-hexane group were increased by 113%,
The current study demonstrated that DATS, a garlic-derived organosulfur compound, could attenuate chronic n-hexane induced peripheral neurotoxicity. Treatment with DATS restrained the lesions of n-hexane on peripheral neuropathy. Through
S. Wang et al. / Chemico-Biological Interactions 265 (2017) 1e7
Fig. 3. Tissue distribution of pyrrole adducts of n-hexane group or coadministration group (10 mg/kg, 20 mg/kg or 30 mg/kg) 24 h later after the last administration. Data were shown as mean ± S.D (n ¼ 12). Compared with the n-hexane group, *P < 0.05, **P < 0.01.
toxicokinetic studies and tests of related P450 enzymes, the possible mechanism may be connected to the modulatory effects of DATS on hepatic cytochrome P450s which disturbed the bioactivation of n-hexane. The exact mechanism of toxicity of n-hexane neurotoxicity remains obscure. However, previous studies have shed some light on this issue. Generation of 2,5-HD, the ultimate toxicant of n-hexane, is thought to play an important role in n-hexane toxicity [24]. It is formed via sequential hydroxylation of n-hexane at the 2- and 5carbon atoms by CYP2E1 and CYP2B1/2, followed by oxidation of the hydroxyl functions by alcohol dehydrogenase [25,26]. The critical roles of CYP2E1 in the bioactivation of n-hexane and the consequent neurotoxicity have been highlighted. One previous study showed that continuous daily n-hexane exposure resulted in increased urinary level of 2,5-HD in wild-type mice but not in CYP2e1(/) mice [2]. CYP2E1 inducer phenobarbital and competitive inhibitors methyl ethyl ketone could significantly affect the 2,5-HD formation in rats administered n-hexane [27,28]. The current study demonstrated that DATS administration inhibited the expression and activity of CYP2E1, which might at least partially account for the protection of DATS against n-hexaneinduced neuropathy. In addition, result of the current study also suggested that co-treatment with DATS could decrease the protein level and activity of CYP2B1/2. DATS was known as an activator of CYP2B1/2 [29], and was manifested by the present study in DATS group. However, the antagonistic actions of CYP2B1 in coadministrate group might be a subsidiary effect of CYP2E1 inhibition. As the total amount of bioactive n-hexane decrease, activation
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related-enzymes became less compared with the n-hexane group. n-Hexane could also be hydroxylated by other P450 isozymes including CYP1A1 and CYP1A2. However, this is a detoxification way, which produced 1-hexanol, 3-hexanol, 4,5-dihydroxy-2hexanone and other compounds [27,29]. In the present study, the protein level and activity of detoxification way related-enzymes CYP1A1 was upregulated. This might accelerate n-hexane to metabolize by the detoxification way. Accompanied with the P450 enzymes changes, the toxicokinetic parameters of 2,5-HD and pyrrole adducts in serum was also changed, manifested as a decreased Cmax and AUC0-t. Pyrrole adducts in the tissue, which reflected the accumulative total of toxic effect, were also parallel to neurobehavioral manifestation and enzymes changes. Particularly, the amount of pyrrole adducts in nerve system seemed to be the highest following the adjustment by the protein concentration. The results were seemed to be different from a previous study when pyrrole adducts were adjusted by the weight of tissues, which showed that liver and kidney contained more pyrrole adducts [18]. This might be because of the variations of tissues protein concentration. The reason for the nerve system to be more vulnerable to form pyrrole adducts might be longevity of the axonal proteins; neurofilaments are extremely stable and have an extremely long lifetime, facilitating continued pyrrole adduct formation [30]. This can also be the underlying reason why the nerve system is the target organ of n-hexane. Previous study has found that free 2,5-HD and pyrrole adducts in serum were related to exposures of n-hexane [31,32]. They might be served as biomarkers in prevention of n-hexane neurotoxicity. However, in present study the amount of free 2,5-HD was periodical variated, but serum pyrrole adducts was relative stable with continuous exposure. If using the pyrrole adducts reduced in DATS group as effect indicators, the point of departure (POD) of the protective effects of DATS is 9.14 mg/kg calculated by Benchmark Dose Software (EPA). DATS could also restrain the nerve system damage of n-hexane induced neurotoxicity by other mechanisms. As an organosulfur compound structurally containing three sulfur atoms, DATS is an exogenous donor of hydrogen sulfide (H2S) in vivo [33]. They might combine with the active 2,5-HD. Besides, DATS was also an effective antioxidant that might inhibit the oxidation of pyrrole adducts to reduce the extent of protein aggregation. Previous study with NAC, another organosulfur compound, had achieved good effects [15]. However, as the bioactive amount of n-hexane was changed in the present study, the mechanisms of GSH/cysteine conjugation and antioxidation in the present study are difficult to elucidate. Neuropathy built with 2,5-HD is a best choice and will be explored in the future study. In addition to modulated P450 enzymes, there are many other factors that can influence the bioavailability of n-hexane, for example, drug solubility, the gastrointestinal pH and
Table 2 Effect of n-hexane and DATS on hepatic CYP1A1, CYP1A2, CYP2B1 and CYP2E1 activities. Groups
CYP1A1 (mmol/min/mg microsomal proteins)
Control DATS n-Hexane Hexane þ DATS (10 mg/ kg) Hexane þ DATS (20 mg/ kg) Hexane þ DATS (30 mg/ kg)
0.62 0.89 1.32 1.43
± ± ± ±
0.13 0.17* 0.21** 0.25**
CYP1A2 (mmol/min/mg microsomal proteins) 2.39 2.27 3.93 3.96
± ± ± ±
CYP2B1 (mmol/min/mg microsomal proteins)
0.52 0.49 0.77** 0.67**
2.31 4.96 8.64 7.98
± ± ± ±
0.27 1.08** 1.53** 1.44**
CYP2E1 (nmol/min/mg microsomal proteins) 53.26 28.15 95.11 70.76
± ± ± ±
16.28 7.33** 29.30** 13.84#
1.63 ± 0.24**#
3.64 ± 0.68**
7.69 ± 1.76**
66.19 ± 12.70##
1.71 ± 0.29**##
2.79 ± 0.72**
6.64 ± 0.97**##
57.14 ± 16.05##
Compared with control group, *P < 0.05,
**
P < 0.01; compared with hexane group, #P < 0.05,
##
P < 0.01.
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Acknowledgments This research was supported by grants from National Natural Science Foundation of China (81373044, to K.X.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cbi.2017.01.013. References
Fig. 4. DATS effects on n-hexane induced activation of CYP1A1, CYP1A2, CYP2B1/2 and CYP2E1. Data were shown as mean ± SD (n ¼ 6). Representative blots (A) and the quantitative data (B) are shown; *P < 0.05, **P < 0.01, vs. control group; #P < 0.05, ## P < 0.01, vs. n-hexane group.
gastrointestinal transit [34]. To reduce the effects of these, the administration of DATS and n-hexane was done at an interval for at least 2 h. We even had tried to establish an n-hexane inhalation neurotoxicity model (1000 ppm, 8 h/day) which lasted for two years but observed no signs of paralysis. And until now there was no reported n-hexane inhalation paralysis model of rats. P450 enzymes are easily affected in daily life and have polymorphisms among population. Take the CYP2E1, the main CYP450 isoform responsible for the metabolism of n-hexane, as an example. Alcohol is an inducer well known to enhance its activity [35]. Other factors such as gene, gender, age and smoking also change its status [36,37]. People with high CYP2E1 activities had greater risks of peripheral nerve damage. So this must be borne in mind of the occupational prevention of n-hexane. In summary, our current study showed that DATS protected the rat from n-hexane induced neurotoxicity. These effects could be associated with the modulation of P450 enzymes that decreased the internal 2,5-hexanedione and pyrrole adducts. These data collectively suggest that DATS as a measure of control n-hexane occupational disease and strengthened the role of garlic as a beneficial substance when included in the diet for prevention of toxication.
Conflict of interest The authors declare that they have no conflict of interest.
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Diallyl trisulfide attenuated n-hexane induced neurotoxicity in rats by modulating P450 enzymes Shuo Wang a, Ming Li a, Xujing Wang a, Xianjie Li a, Hongyin Yin b, Lulu Jiang a, Wenting Han a, Gleniece Irving c, Tao Zeng a, Keqin Xie a, * a b c
Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong 250012, China Jinan Municipal Center for Disease Control & Prevention, Jinan, Shandong Province, China North East Regional Health Authority, Ocho Rios, Jamaica
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2016 Accepted 19 January 2017 Available online 20 January 2017
Chronic exposure to n-hexane can induce serious nerve system impairments without effective preventive medicines. Diallyl trisulfide (DATS) is a garlic-derived organosulfur compound, which has been demonstrated to have many beneficial effects. The current study was designed to evaluate whether DATS could restrain n-hexane induced neurotoxicity in rats and to explore the underlying mechanisms. Rats were treated with n-hexane (3 g/kg, p.o.) and different doses of DATS (10, 20 and 30 mg/kg, p.o.) for 8 weeks. Behavioral assessment showed that DATS could inhibit n-hexane induced neurotoxicity, demonstrated by the improvement of the grip strength and decline of gait scores. Toxicokinetic analysis revealed that the Cmax and AUC0-t of 2,5-hexanedione (product of n-hexane metabolic activation) and 2,5-hexanedione protein adducts in serum were significantly declined in DATS-treated rats, and the levels of pyrrole adducts in tissues were significantly reduced. Furthermore, DATS activated CYP1A1 and inhibited n-hexane induced increased expression and activity of CYP2E1 and CYP2B1. Collectively, these findings indicated that DATS protected the rats from n-hexane-induced neurotoxicity, which might be attributed to the modulation of P450 enzymes by DATS. © 2017 Elsevier B.V. All rights reserved.
Keywords: n-Hexane Diallyl trisulfide Neurotoxicology CYP450 2,5-Hexanedione Pyrrole adducts
1. Introduction The organic solvent n-hexane had been implicated in a multitude of occupational and intentional inhalation exposure settings. Chronic exposure to n-hexane could produce centraleperipheral axonopathy in humans and experimental animals, which was characterized by paranodal giant axonal swellings and demyelination [1]. It has been demonstrated that 2,5-hexanedione (2,5-HD), a gediketone metabolite of n-hexane oxidization by cytochromes P450, was responsible for its neuropathy [2,3]. Once formed, 2,5HD might bind to lysine ε-amine groups of proteins and form the 2,5-dimethylpyrrole (2,5-DMP) adducts, and then yielded secondary autoxidative reactions forming pyrrole dimers which produce
Abbreviations: DATS, diallyl trisulfide; 2,5-HD, 2,5-hexanedione; 2,5-DMP, 2,5dimethylpyrrole; CYP, cytochromes; NADPH, nicotinamide adenine dinucleotide phosphate; FID, flame ionization detector; Cmax, maximum concentration; Tmax, the time to peak plasma concentration; AUC(0-t), the area under the serum concentrationetime curve; t1/2, the half-life time. * Corresponding author. E-mail address: [email protected] (K. Xie). http://dx.doi.org/10.1016/j.cbi.2017.01.013 0009-2797/© 2017 Elsevier B.V. All rights reserved.
intra- and inter-molecular cross-linkings in proteins. This kind of protein posttranslational modifications may eventually lead to critical protein function loss and were believed to responsible for the nerve system dysfunction [4e7]. Thus, one possible approach for the protection of n-hexane-induced neuropathy is to prevent 2,5-HD or even pyrrole adducts formation and oxidation. Garlic is a popular herbal product that has been applied for centuries for medicinal purposes [8]. It has a variety of pharmacological activities such as anticancer, antimicrobial, antiatherosclerotic, antidiabetic, and immunomodulatory effects [9]. Diallyl trisulfide (DATS) is the major phytochemical component contained in garlic and garlic products such as garlic essential oil [8,10]. Previous studies have revealed that DATS has excellent antioxidant capacity and can attenuate many chemicalseinduced oxidative damage in liver and also in nervous system [11e13]. Furthermore, DATS can also increases GSH levels in vivo. The preventive effects of GSH had been confirmed through the use of N-acetylcysteine (NAC), a precursor of GSH, in n-hexane induced neuropathy [14,15]. We herein hypothesized that DATS could prevent n-hexane induced neurotoxicity, and established n-hexane induced peripheral nerve injury models to test the hypothesis and investigate the
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possible mechanisms.
2.4. Toxicokinetics studies
2. Materials and methods
Six rats randomly selected from n-hexane group and 3 DATS plus n-hexane groups were used to determine the long-term effect of DATS on n-hexane toxicokinetics. The rats were fasted overnight but had free access to water. DATS (10, 20 or 30 mg/kg) were gavaged two hours before the administration of n-hexane (3 g/kg). Blood samples were collected from the jugular vein at 0, 2, 4, 6, 8, 10, 12, 14, 16, 20 and 22 h after n-hexane exposure. The plasma samples were obtained immediately after sampling by centrifugation at 1000 g for 5 min. Pyrrole adduct was analyzed immediately and the rest was stored at 20 C for 2,5-HD analysis.
2.1. Chemicals and reagents n-hexane, cyclohexanone and dichloromethane were purchased from Hongyan Chemical Reagent Factory (Tianjin, China). DATS (purity > 97%) was purchased from Jiangsu Chiatai Qingjiang Pharmaceutical CO., LTD (Huaian, China). 2,5-hexanedione, 2,5dimethylpyrrole, 4-dimethylaminobenzaldehyde, NADPH, ethoxyresorufi, methoxyrsorufin, pentoxyresorufin and resorufin were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Primary antibodies of anti-CYP1A1, anti-CYP1A2, anti-CYP2B1/2, antiCYP2E1 were provide by Santa Cruz Biotechnology (Santa Cruz, CA, United States) and Abcam (Cambridge, United Kingdom). BCA™ protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA). All other chemicals were of the highest grade commercially available. 2.2. Animals treatment Seven-week-old male Wistar rats, weighing 220e240 g, were provided by Experimental Animal Center of Shandong University. Drinking water and commercial animal feed were available ad libitum. The animal room was maintained at 22 ± 2 C and 50 ± 10% relative humidity with a 12-h-light/-dark cycle. After the acclimatization period, the animals were randomly divided into 6 groups, i.e. control group, n-hexane group, DATS group, and 3 DATS plus nHexane groups (n ¼ 12 in each group). Rats in n-hexane plus DATS groups were administrated with DATS (10, 20 or 30 mg/kg/day, orally) combined with n-hexane (3 g/kg/day, orally) seven times per week for consecutive 8 weeks. The rats in n-hexane group were treated with corn oil and n-hexane (3 g/kg/day, orally) and rats in DATS group were treated with DATS (30 mg/kg/day, orally) and corn oil. The rats in control group received equal volume of vehicle. All animal experimental procedures and protocols of the study were approved by the Ethics Committee for Animal Experiments of Shandong University Institute of Preventive Medicine (Permit Number: 20111231) in accordance with the NIH Guide for Care and Use of Laboratory Animals. 2.3. Behavioral assessment Forelimb and hind limb grip strength and gait scores were used to assess the effects of DATS on n-hexane induced peripheral neuropathy. Forelimb and hind limb grip strength were measured every two weeks with a grip strength meter (YLS-13A, Jinan Yiyan Technology and Development Co., Ltd., Jinan, China). Briefly the rats were gently lowered over the top of the grid to let its front and hind paws grip the smooth metal pull bar. And then with a uniform force drew the rats until complete the length of the grid. Each anima repeated for three times. Prior to the rats being sacrificed, gait scores were obtained [16,17]. They were placed in an open field, and were observed for 3 min. Following observation, a gait score was assigned from 1 to 4 where 1 ¼ a normal, unaffected gait; 2 ¼ a slightly abnormal gait (hindlimbs show uncoordinated placement, exaggerated or overcompensated movements, or are splayed slightly, walks on tiptoes); 3 ¼ moderately abnormal gait (obvious movement abnormalities characterized by markedly splaying hindlimbs, ataxia, swaying, rocking, lurching, stumbling); 4 ¼ severely abnormal gait (flat foot walk, hindlegs flat on surface, crawling, or unable to support weight).
2.5. Free 2,5-HD detection A 50 ml of 0.5 mg/ml of cyclohexanone (internal standard) and 0.2 g of NaCl were added to 100 ml plasma sample. After addition of 1.0 ml of dichloromethane, samples rotated vigorously and centrifuged at 3500 rpm for 10 min. The dichloromethane extraction solution was evaporated to about 0.1 ml with a nitrogen flow and then injected into a gas chromatography system [18]. The GC used was a Shimadzu GC-2010 (Kyoto, Japan) equipped with a flame ionization detector (FID) and an HP-5 column (30 m 0.32 mm 0.25 mm, Agilent Technologies). The injector temperature was set at 180 C and the detector temperature at 260 C. The oven was set at 80 C and the injection volume was 2 ml. The carrier gas was nitrogen at a flow rate of 1.5 ml/min and the split ratio was set at 3:1. 2.6. Pyrrole adducts detection The pyrrole adducts were measured spectrophotometrically after reaction of 0.1 ml of plasma with 0.1 ml guanidine hydrochloride (70%) and 0.1 ml of Ehrlich's reagent (3% 4dimethylaminobenzaldheyde in 40 ml of methanolic 14% boron trifluoride and 60 ml of ethanol) [19]. Absorption values were measured at 526 nm, using automatic microplate reader (Infinite® 200 PRO, TECAN Inc. Switzer). The calculations were based on standard curve prepared with different concentrations of 2,5dimethylpyrrole (5e80 nmol/ml). Liver, kidney, spinal cord and brain samples were homogenized with a homogenizer (10,000e15,000 rev./min, 10 s) at 4 C in physiological saline at 0.5 g wet weight/ml. A 0.5 ml of 0.7 g/ml of aqueous guanidine hydrochloride was added to 0.1 ml tissue homogenate. After digestion at room temperature for 30 min, the pyrrole adducts was determined by the above procedure [20]. Sciatic nerves were initially broken into powder with pestle in liquid nitrogen, and then homogenized in physiological saline at a concentration of 0.5 g wet weight/ml, followed by adding 0.3 ml of 2.5% trypsin. After digestion at 56 C for 1 h, the pyrrole adducts was determined by the above procedure. Values were expressed as nanomoles of 2,5-dimethylpyrrole per milliliter or gram of tissue. 2.7. Western blot analysis of CYP proteins in the liver Total protein extract was obtained as previously described [21]. Briefly, livers samples were homogenized in ice-cold RIPA lysis buffer (50 mM Tris-HCl PH7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 5 mM NaF, 50 mM TriseHCl) supplemented with 1 mM PMSF and 1% cocktail protein protease inhibitors. After a 20-min centrifugation at 12,000 g, the supernatant was collected and the protein concentration was determined using the BCA™ protein assay kit. Equal aliquots of samples were mixed with loading buffer, and then heated at 100 C
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for 10 min. The pre-treated protein samples (about 20e50 mg) were separated by 10% or 15% SDS/polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Blots were blocked with 5% skim milk in TBST for 1 h at room temperature. Then the membrane was incubated with the anti-CYP1A1, anti-CYP1A2, anti-CYP2B1/2, anti-CYP2E1 or anti-bactin antibodies overnight at 4 C. After washing off unbound primary antibodies, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated antibodies for 1 h at room temperature. The blots were then washed, visualized by enhanced chemiluminescence detection kit and exposed on x-ray film. The relative optical densities of the bands were quantified and normalized by b-actin, using Kodak Imaging Program and ImagePro Plus software (Eastman Kodak Company, New Haven, CT, USA).
3
3.2. DATS co-treatment led to the decrease of plasma concentration of 2,5-HD and pyrrole adducts The mean plasma concentration-time profiles of 2,5-HD and pyrrole adducts after chronic oral exposure of n-hexane (3 g/kg) in the absence and presence of DATS (10, 20, 30 mg/kg) are depicted in Fig. 2. The toxicokinetic parameters are summarized in Table 1. Compared with the rats in n-hexane group, the AUC0-t value of 2,5HD in rats of DATS (10, 20 and 30 mg/kg) groups were decreased by 3.3%, 17.1% and 37.4% (P < 0.05), respectively, while the AUC0-t of pyrrole adducts were reduced by 6.9%, 7.0% and 26.5% (P < 0.05), respectively. The Cmax and Cmin of 2,5-HD and pyrrole adducts were
2.8. Isolation of liver microsomes The liver microsomal fractions were prepared by homogenization and differential centrifugation as previously described [22]. Briefly, liver tissue was homogenized in four volume ice-cold TMS buffer (50 mM Tris-HCL, 6.4 mM MgCl2, 0.2 M saccharose, pH ¼ 7.5). The resulting homogenate was centrifuged at 12,000 g for 15 min and the supernatants were further centrifuged at 105,000 g for 60 min. The final pellet was reconstituted in the TMS buffer. The protein concentrations were quantified using BCA™ protein assay kits. All the procedure was conducted at 4 C. 2.9. Determination of the activities of CYP1A1, CYP1A2, CYP2B1/2 and CYP2E1 The activity of CYP2E1 was measured with aniline as the substrate as previously described [22]. CYP1A1, CYP1A2 and CYP2B1/2 activities were detected by measurement of the dealkylation of the ethoxyresorufin, methoxyresorufin, and pentoxyresorufin using Hitachi fluorescence spectrophotometer (Hitachi HighTechnologies Corporation) [23]. 2.10. Statistical analysis Values are presented as the mean ± SD and analyzed statistically using the SPSS 13.0 software. For Forelimb and hind limb grip strength parameters, the data was analyzed by two-way repeatedmeasures analysis of variance, followed by one-way ANOVA coupled with LSD post hoc test for each two weeks' grip strength. For all other data, one-way ANOVA was adopted, followed with LSD for the multiple comparisons. The level of statistical significance was set at P < 0.05. The maximum concentration (Cmax) and the time to maximum concentration (Tmax) were obtained from the observed data. The area under the serum concentrationetime curve (AUC(0-t)) and the half-life time (t1/2) were calculated using DAS 3.0 software. 3. Results 3.1. Effect of DATS on n-hexane induced peripheral neuropathy n-Hexane exposure led to significant peripheral neuropathy, shown by the decrease of grip strength and increase of gait scores. Before n-hexane exposure, all rats exhibited normal, unaffected grip strength. By the end of 8 weeks' exposure, rats exposed to nhexane exhibited a significantly grip strength lost compared with the control group, which was significantly suppressed by DATS (20 and 30 mg/kg) co-treatment. Parallelly, 20 and 30 mg/kg DATS also inhibited the increase of gait scores induced by n-hexane. (Fig. 1).
Fig. 1. The changes of grip strength and gait score after DATS co-treatment on nhexane induced peripheral neuropathy. (A) Every two weeks limb grip strength of the n-hexane experiment; (B) Abnormalities of gait during n-hexane experiment. The data were expressed as mean ± S.D. (n ¼ 12 per group). Significant statistical difference was indicated by *P < 0.05, **P < 0.01, with respect to the control rats; #P < 0.05, ##P < 0.01, with respect to the model rats.
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Fig. 2. Mean plasma concentrations-time curves of 2,5-HD (A) and pyrrole adducts (B) in rats following oral administration of 3 g/kg of hexane alone (C) or coadministered with 10 mg/kg (B), 20 mg/kg (-) and 30 mg/kg (,) of DATS. Bars represent standard error (n ¼ 6).
Table 1 Toxicokinetic parameters of plasma 2,5-HD and pyrrole adducts following n-hexane administration after coadministration of DATS (n ¼ 6). Group 2,5-HD Hexane Hexane þ DATS(10 Hexane þ DATS(20 Hexane þ DATS(30 Pyrrole Adducts Hexane Hexane þ DATS(10 Hexane þ DATS(20 Hexane þ DATS(30
AUC0-t (nmol/ml$h)
mg/kg) mg/kg) mg/kg)
8231.2 7956.4 6824.2 5150.9
mg/kg) mg/kg) mg/kg)
770.4 717.4 716.1 566.5
± ± ± ±
± ± ± ±
2811.4 2049.5 553.4 1648.6*
79.3 69.8 91.6 180.8*
Tmax (h) 10.3 10.3 10.0 10.3
Cmax (nmol/ml) ± ± ± ±
0.8 0.8 0.0 1.5
10.9 ± 0.1 11.7 ± 2.3 11.0 ± 0.1 9.7 ± 2.9
727.5 642.6 601.1 507.2 41.5 37.1 37.6 30.8
± ± ± ±
± ± ± ±
248.5 196.4 85.5 153.8
3.6 5.1 7.1 9.5*
Cmin (nmol/ml) 138.6 ± 73.9 173.7 ± 77.3 123.0 ± 18.5 65.7 ± 22.7 31.6 29.9 29.7 23.1
± ± ± ±
4.7 3.0 1.8 7.2*
Values are presented as mean ± S.D., AUC0-t area under the curve, Tmax the time to peak plasma concentration, Cmax the peak plasma concentration, Cmin the minimum plasma concentration. * P < 0.05, **P < 0.01 with respect to rats in the n-hexane group.
also suppressed by the DATS co-treatment. 3.3. Tissue distribution of pyrrole adducts Tissue distribution of pyrrole adducts after repeated n-hexane and DATS administration were presented. As shown in Fig. 3, the nervous system had higher adducts levels after adjusting by protein concentration, compared with the pyrrole adducts levels in the liver and the kidney. DATS (20 and 30 mg/kg) co-administrate led to a significant decline of the pyrrole adducts levels in the brain, spinal cord and sciatic nerves.
64%, 274% and 79%, respectively (P < 0.05). In DATS group, the activities of CYP2E1 were decrease by 47%, while the activities of CYP1A1 and CYP2B1 were increased by 44% and 114%. The changes of protein levels of the above four phase I enzymes were paralleled with those of the activities (Fig. 4). The protein level of CYP2E1 showed a dose-dependent decrease in rats treated with 10, 20 and 30 mg/kg DATS. The expression changes of CYP2B1 induced by nhexane were suppressed by mega dose DATS co-treatment. The expression of CYP1A1 was upregulated after DATS co-treatment but CYP1A2 was not changed significantly. Meanwhile, DATS cotreatment also significantly inhibited the increases of the activities of CYP2E1 and CYP2B1 and increased the activity of CYP1A1.
3.4. Effects of DATS and n-hexane on the protein levels of hepatic cytochrome P450s in rats
4. Discussion
As shown in Table 2, n-hexane exposure resulted in the dramatic increase of the activities of CYP1A1, CYP1A2, CYP2B1 and CYP2E1. Compared with the control group, the activities of CYP1A1, CYP1A2, CYP2B1 and CYP2E1 in n-hexane group were increased by 113%,
The current study demonstrated that DATS, a garlic-derived organosulfur compound, could attenuate chronic n-hexane induced peripheral neurotoxicity. Treatment with DATS restrained the lesions of n-hexane on peripheral neuropathy. Through
S. Wang et al. / Chemico-Biological Interactions 265 (2017) 1e7
Fig. 3. Tissue distribution of pyrrole adducts of n-hexane group or coadministration group (10 mg/kg, 20 mg/kg or 30 mg/kg) 24 h later after the last administration. Data were shown as mean ± S.D (n ¼ 12). Compared with the n-hexane group, *P < 0.05, **P < 0.01.
toxicokinetic studies and tests of related P450 enzymes, the possible mechanism may be connected to the modulatory effects of DATS on hepatic cytochrome P450s which disturbed the bioactivation of n-hexane. The exact mechanism of toxicity of n-hexane neurotoxicity remains obscure. However, previous studies have shed some light on this issue. Generation of 2,5-HD, the ultimate toxicant of n-hexane, is thought to play an important role in n-hexane toxicity [24]. It is formed via sequential hydroxylation of n-hexane at the 2- and 5carbon atoms by CYP2E1 and CYP2B1/2, followed by oxidation of the hydroxyl functions by alcohol dehydrogenase [25,26]. The critical roles of CYP2E1 in the bioactivation of n-hexane and the consequent neurotoxicity have been highlighted. One previous study showed that continuous daily n-hexane exposure resulted in increased urinary level of 2,5-HD in wild-type mice but not in CYP2e1(/) mice [2]. CYP2E1 inducer phenobarbital and competitive inhibitors methyl ethyl ketone could significantly affect the 2,5-HD formation in rats administered n-hexane [27,28]. The current study demonstrated that DATS administration inhibited the expression and activity of CYP2E1, which might at least partially account for the protection of DATS against n-hexaneinduced neuropathy. In addition, result of the current study also suggested that co-treatment with DATS could decrease the protein level and activity of CYP2B1/2. DATS was known as an activator of CYP2B1/2 [29], and was manifested by the present study in DATS group. However, the antagonistic actions of CYP2B1 in coadministrate group might be a subsidiary effect of CYP2E1 inhibition. As the total amount of bioactive n-hexane decrease, activation
5
related-enzymes became less compared with the n-hexane group. n-Hexane could also be hydroxylated by other P450 isozymes including CYP1A1 and CYP1A2. However, this is a detoxification way, which produced 1-hexanol, 3-hexanol, 4,5-dihydroxy-2hexanone and other compounds [27,29]. In the present study, the protein level and activity of detoxification way related-enzymes CYP1A1 was upregulated. This might accelerate n-hexane to metabolize by the detoxification way. Accompanied with the P450 enzymes changes, the toxicokinetic parameters of 2,5-HD and pyrrole adducts in serum was also changed, manifested as a decreased Cmax and AUC0-t. Pyrrole adducts in the tissue, which reflected the accumulative total of toxic effect, were also parallel to neurobehavioral manifestation and enzymes changes. Particularly, the amount of pyrrole adducts in nerve system seemed to be the highest following the adjustment by the protein concentration. The results were seemed to be different from a previous study when pyrrole adducts were adjusted by the weight of tissues, which showed that liver and kidney contained more pyrrole adducts [18]. This might be because of the variations of tissues protein concentration. The reason for the nerve system to be more vulnerable to form pyrrole adducts might be longevity of the axonal proteins; neurofilaments are extremely stable and have an extremely long lifetime, facilitating continued pyrrole adduct formation [30]. This can also be the underlying reason why the nerve system is the target organ of n-hexane. Previous study has found that free 2,5-HD and pyrrole adducts in serum were related to exposures of n-hexane [31,32]. They might be served as biomarkers in prevention of n-hexane neurotoxicity. However, in present study the amount of free 2,5-HD was periodical variated, but serum pyrrole adducts was relative stable with continuous exposure. If using the pyrrole adducts reduced in DATS group as effect indicators, the point of departure (POD) of the protective effects of DATS is 9.14 mg/kg calculated by Benchmark Dose Software (EPA). DATS could also restrain the nerve system damage of n-hexane induced neurotoxicity by other mechanisms. As an organosulfur compound structurally containing three sulfur atoms, DATS is an exogenous donor of hydrogen sulfide (H2S) in vivo [33]. They might combine with the active 2,5-HD. Besides, DATS was also an effective antioxidant that might inhibit the oxidation of pyrrole adducts to reduce the extent of protein aggregation. Previous study with NAC, another organosulfur compound, had achieved good effects [15]. However, as the bioactive amount of n-hexane was changed in the present study, the mechanisms of GSH/cysteine conjugation and antioxidation in the present study are difficult to elucidate. Neuropathy built with 2,5-HD is a best choice and will be explored in the future study. In addition to modulated P450 enzymes, there are many other factors that can influence the bioavailability of n-hexane, for example, drug solubility, the gastrointestinal pH and
Table 2 Effect of n-hexane and DATS on hepatic CYP1A1, CYP1A2, CYP2B1 and CYP2E1 activities. Groups
CYP1A1 (mmol/min/mg microsomal proteins)
Control DATS n-Hexane Hexane þ DATS (10 mg/ kg) Hexane þ DATS (20 mg/ kg) Hexane þ DATS (30 mg/ kg)
0.62 0.89 1.32 1.43
± ± ± ±
0.13 0.17* 0.21** 0.25**
CYP1A2 (mmol/min/mg microsomal proteins) 2.39 2.27 3.93 3.96
± ± ± ±
CYP2B1 (mmol/min/mg microsomal proteins)
0.52 0.49 0.77** 0.67**
2.31 4.96 8.64 7.98
± ± ± ±
0.27 1.08** 1.53** 1.44**
CYP2E1 (nmol/min/mg microsomal proteins) 53.26 28.15 95.11 70.76
± ± ± ±
16.28 7.33** 29.30** 13.84#
1.63 ± 0.24**#
3.64 ± 0.68**
7.69 ± 1.76**
66.19 ± 12.70##
1.71 ± 0.29**##
2.79 ± 0.72**
6.64 ± 0.97**##
57.14 ± 16.05##
Compared with control group, *P < 0.05,
**
P < 0.01; compared with hexane group, #P < 0.05,
##
P < 0.01.
6
S. Wang et al. / Chemico-Biological Interactions 265 (2017) 1e7
Acknowledgments This research was supported by grants from National Natural Science Foundation of China (81373044, to K.X.). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.cbi.2017.01.013. References
Fig. 4. DATS effects on n-hexane induced activation of CYP1A1, CYP1A2, CYP2B1/2 and CYP2E1. Data were shown as mean ± SD (n ¼ 6). Representative blots (A) and the quantitative data (B) are shown; *P < 0.05, **P < 0.01, vs. control group; #P < 0.05, ## P < 0.01, vs. n-hexane group.
gastrointestinal transit [34]. To reduce the effects of these, the administration of DATS and n-hexane was done at an interval for at least 2 h. We even had tried to establish an n-hexane inhalation neurotoxicity model (1000 ppm, 8 h/day) which lasted for two years but observed no signs of paralysis. And until now there was no reported n-hexane inhalation paralysis model of rats. P450 enzymes are easily affected in daily life and have polymorphisms among population. Take the CYP2E1, the main CYP450 isoform responsible for the metabolism of n-hexane, as an example. Alcohol is an inducer well known to enhance its activity [35]. Other factors such as gene, gender, age and smoking also change its status [36,37]. People with high CYP2E1 activities had greater risks of peripheral nerve damage. So this must be borne in mind of the occupational prevention of n-hexane. In summary, our current study showed that DATS protected the rat from n-hexane induced neurotoxicity. These effects could be associated with the modulation of P450 enzymes that decreased the internal 2,5-hexanedione and pyrrole adducts. These data collectively suggest that DATS as a measure of control n-hexane occupational disease and strengthened the role of garlic as a beneficial substance when included in the diet for prevention of toxication.
Conflict of interest The authors declare that they have no conflict of interest.
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