[1] 11w Allyl sulfide counteracts 1-bromopropane-induced neurotoxicity by inhibiting neuroinflammation and oxidative stress

[1] 252w Chronic exposure to 1-bromopropane (1-BP), an alternative to ozone-depleting solvents, produces potential neurotoxicity in occupational populations. However, no therapeutic strategy is available currently. Accumulating evidence suggests that cytochrome P4502E1 (CYP2E1) is critical for the active metabolism of 1-BP. The purpose of this study is aimed to test whether inhibition of CYP2E1 by allyl sulfide, a specific inhibitor of CYP2E1, could be able to protect against 1-BP-induced neurotoxicity. Male Wistar rats were intoxicated with 1-BP for 9 continuous weeks with or without allyl sulfide pre-treatment. Results clearly demonstrated that 1-BP exposure induced decrease in NeuN + cells and increase in cleaved caspase-3 expression and TUNEL + cells in motor cortex of rats, which was significantly ameliorated by allyl sulfide. Allyl sulfide treatment also recovered the motor performance of rats treated with 1-BP. Mechanistically, allyl sulfide inhibited 1-BP-induced expression of CYP2E1 in microglia, which was associated with suppression of microglial activation and M1 polarization in motor cortex of rats. Reduced oxidative stress was also observed in rats treated with combined allyl sulfide and 1-BP compared with 1-BP alone group. Furthermore, we found that allyl sulfide abrogated 1-BP-induced activation of NF-κB and GSH/Thioredoxin/ASK1 pathways, the key factor for the maintenance of M1 microglial inflammatory response and oxidative stress-related neuronal apoptosis, respectively. Thus, our results showed that allyl sulfide exerted neuroprotective effects in combating 1-BPinduced neurotoxicity through inhibition of neuroinflammation and oxidative stress. Blocking CYP2E1 activity by allyl sulfide might be a promising avenue for the treatment of neurotoxicity elicited by 1-BP and other related neurotoxicants.

[1] 128w 1-Bromopropane (1-BP) is an alternative to ozone depleting solvent and has been used as detergent (metals, precise electronics and optical instruments), solvent (fats, waxes and resins), intermediate of synthesis products (pharmaceuticals, insecticides, flavors and fragrances) as well as dry cleaning agent. 1-BP has been proven to be neurotoxic in both occupational workers (Home, 2008;Ichihara et al., 2004;Ichihara et al., 2002;Ichihara et al., 2011;Majersik et al., 2007;Samukawa et al., 2012;Sclar, 1999) and experimental animals (Ichihara et al., 2000;Ohnishi et al., 1999;Sohn et al., 2002;Wang et al., 2002;Yu et al., 2006;Yu et al., 2001;Zhao et al., 2006;Zong et al., 2016). Human exposed to 1-BP display irreversible loss of vibration sense, anesthesia or dysesthesia in low extremities, ataxia, gait abnormality and even paralysis (Home, 2008;Ichihara, et al., 2011;Majersik, et al., 2007).

[2] 30w We recently reported that 1-BP exposure results in neuronal loss in the cortex of rats, which was associated with deficits of their learning and memory capacities (Chen et al., 2017).

[3] 224w Consistently, a dose-dependent decrease of neuron-specific enolase was also observed in the cerebrum of 1-BP-treated rats in Wang's study (Wang, et al., 2002). The widespread use of 1-BP significantly increases the exposure incidence in occupational populations and 1-BPrelated neurotoxicity gradually becomes a major health concern and occupational health hazard. However, no effective treatment is available at present. Accumulating evidence suggests that cytochrome P4502E1 (CYP2E1) is critical for the metabolism of low-molecular-weight chemicals, including 1-BP (Garner et al., 2014). The C2 oxidation of 1-BP catalyzed by CYP2E1 yields the toxic metabolites, 1-bromo-2-propanol and bromoacetone (Garner et al., 2015;Garner et al., 2006;Garner, et al., 2014), which has been shown to be involved in 1-BP's toxicity (Lee et al., 2010;Zong, et al., 2016). Elevation of CYP enzymes activities by phenobarbital significantly exaggerates 1-BP-induced hepatotoxicity in mice (Lee, et al., 2010). Consistently, inhibition of CYP2E1 by SKF-525A Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 or 1-aminobenzotriazole (1-ABT) protects against 1-BP-induced hepatic necrosis and hepatocytes degeneration in mice (Emoto et al., 2005;Zong, et al., 2016). One previous study performed by Zong et al showed that inhibition of CYP2E1 activity by 1-ABT also elevates the expression of hippocampal Ran protein and cerebral cortical GRP78 in response to 1-BP, indicating the involvement of CYP2E1 in neurotoxicity elicited by 1-BP (Zong, et al., 2016).

[4] 16w We therefore hypothesized that blocking CYP2E1 activity might be an effective approach for combating 1-BP-induced neurotoxicity.

[5] 180w Allyl sulfide, also named diallyl sulfide, is the major ingredient from garlic, which is proven to be an specific inhibitor of CYP2E1 (Brady et al., 1991). Previous studies revealed that allyl sulfide potently inhibited the oxidative metabolism of multiple environmental toxicants, such as 1,2-dichloroethane (Yang et al., 2016), paraquat and maneb (Ahmad et al., 2014), which was associated with attenuation of their toxicity. Diallyl sulfide has also been shown to be able to antagonize mercury-induced neurotoxicity by maintaining the balance of oxidant and antioxidant status in rats (Ansar, 2015). Despite of this, the protective efficacy of allyl sulfide against 1-BP-induced neurotoxicity is still unknown. In the current study, we aimed to investigate the protective efficacy of allyl sulfide and underlying mechanisms against 1-BP-induced neurotoxicity. Rats were intoxicated with 1-BP with or without allyl sulfide pre-treatment. We found that allyl sulfide potently ameliorated 1-BP-induced neurodegeneration in motor cortex and related motor deficits of rats. Suppression of microglia-mediated neuroinflammation and oxidative stress contributed to allyl sulfideafforded neuroprotection. Our findings suggest a promising strategy for combating 1-BPinduced neurotoxicity through inhibition of CYP2E1 activity.

[1] 186w To investigate the neuroprotective effects of allyl sulfide, an inhibitor of CYP2E1, against 1-BP-induced neurotoxicity, neurons in motor cortex of rats were initially stained with thionin dye. As seen in Fig. 1A, neurons in motor cortex of control rats filled with darkly stained nissl bodies. In contrast, the staining of nissl bodies of motor neurons in 1-BP-treated rats was very light, indicating damage of neurons. Interestingly, administration of allyl sulfide significantly reduced neuronal damage induced by 1-BP as shown by recovered thionin staining in motor cortex (Fig. 1A) Neurons in motor cortex among groups were further immunostained with antibody against NeuN, a neuron specific marker. Consistently, compared with vehicle controls, 1-BP intoxication decreased the number of NeuN + cells, which was also significantly attenuated by allyl sulfide (Fig. 1B). Quantitative analysis showed that 1-BP intoxication resulted in 47% loss of NeuN + cells in motor cortex of rats, which was reduced to 11% in combined allyl sulfide and 1-BP group (Fig 1C). In agreement with morphological observation, Western blot revealed that the reduced expression of NeuN in 1-BP-treated rats was recovered by allyl sulfide (Fig. 1D).

[2] 143w To further confirm the neuroprotective efficacy of allyl sulfide, the effects of allyl sulfide on 1-BP-induced apoptosis in neurons in motor cortex were investigated. The doubleimmunofluorescence staining by using antibodies against NeuN and cleaved caspase-3 was initially performed. 1-BP-treated rats displayed an elevated expression of cleaved caspase-3 in motor cortex of rats compared with vehicle controls. Co-staining with NeuN antibody revealed a high expression of cleaved caspase-3 in NeuN + cells in motor cortex of 1-BPtreated rats. Treatment with allyl sulfide markedly reduced 1-BP-induced expression of cleaved caspase-3 and number of cleaved caspase-3 + /NeuN + cells compared with 1-BP alone Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 group (Fig. 1E). Western blot analysis further confirmed the inhibitory effects of allyl sulfide on 1-BP-induced expression of cleaved caspase-3 in motor cortex of rats (Fig. 1F).

[3] 98w Since caspases also have crucial non-apoptotic-related roles in neurons (Mukherjee et al., 2017), TUNEL staining was employed to further confirm the motor neuronal apoptosis. 1-BP-treated rats displayed an increased number of TUNEL-positive cells in motor cortex compared with vehicle controls. Treatment with allyl sulfide markedly reduced 1-BP-induced apoptosis of neurons as shown by decreased number of TUNEL-positive cells compared with 1-BP alone group (Fig. 1G, H). No significant difference of thionin staining, NeuN + and TUNEL + cell number as well as expressions of NeuN and cleaved caspase-3 was observed between allyl sulfide alone and vehicle control group.

[4] 74w To determine whether allyl sulfide-afforded neuroprotection was functional, behaviors of rats including grip strength, gait and paralysis were measured. As shown in Fig. 2, rats intoxicated with 1-BP displayed significant behavioral deficits including progressive loss of hind-limb grip strength, gait abnormality, and high paralysis rates compared with vehicle controls. The decrease of grip strength with 1-BP-treated rats was observed after 5 weeks of 1-BP intoxication, while gait abnormality occurred after 8 weeks of intoxication.

[5] 52w Administration of allyl sulfide significantly improved the behavioral performance of 1-BPexposed rats by showing recovered hind-limb grip strength and gait scores compared with 1-BP alone group. Consistently, 1-BP intoxication resulted in 30% paralysis of rats, which was reduced to 10% when allyl sulfide was co-treated with 1-BP (p < 0.01) (Fig. 2C).

[6] 105w Neuroinflammation mediated by glial cells including microglia and astroglia contributes to neurodegeneration in a variety of neurodegenerative disorders (Kerenshaul et al., 2017;Liddelow et al., 2017). To investigate whether allyl sulfide-afforded neuroprotection was related to suppression of neuroinflammation, we initially determined the effects of allyl sulfide on CYP2E1 expressions in microglia and astroglia. Double immunofluorescence staining by using antibodies against CYP2E1 and Iba-1 (a marker for microglia) or GFAP (marker for astroglia), revealed a co-localization of CYP2E1 and Iba-1 but not GFAP (Fig. 3A), suggesting that CYP2E1 expressed in microglia but not astroglia. Compared with vehicle controls, 1-BP intoxication elevated the expressions of CYP2E1 in microglia.

[7] 32w However, the elevated expressions of CYP2E1 in microglia induced by 1-BP were not observed in combined allyl sulfide and 1-BP-treated rats (Fig. 3B). Western blot analysis supported the immunofluorescence observations (Fig. 3C).

[8] 57w The effects of allyl sulfide on 1-BP-induced microglial activation were subsequently detected. Activation of microglia in motor cortex was morphologically observed by immunostaining with Iba-1. In 1-BP-treated rats, activated microglia, characterized by a hypertrophied morphology and intensified Iba-1, were observed in motor cortex (Fig. 4A).Analysis of Iba-1 + cell number supported these morphological observations (Fig. 4 B).

[9] 57w Compared with 1-BP group, allyl sulfide treatment markedly attenuated microglial activation, as shown by a ramified morphology and a reduced numbers of Iba-1 + cells in motor cortex (Fig. 4A and B). Western blot analysis also showed a reduced expression of Iba-1 in motor cortex in combined allyl sulfide and 1-BP-treated rats compared with 1-BP alone group.

[10] 27w Since activated microglia are capable of inducing neurotoxic reactive astrocyte, astroglial activation was further determined. Consistently, astroglial activation was also mitigated by allyl sulfide (Supplementary Fig. 1).

[11] 218w Activated microglia can be classified into two phenotypes, i.e., M1 and M2, to produce either detrimental or beneficial effects in the central nervous system (Jha et al., 2016;Loane et al., 2016;Rojo et al., 2014). To determine whether allyl sulfide-inhibited microglial Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 activation was associated with suppression of M1 polarization, the expressions of iNOS and Arginase-1, the maker for M1 and M2 microglia, respectively, were determined. As shown by double immunofluorescence staining, compared with vehicle control, a high expression of iNOS was manifested in motor cortex of 1-BP-treated rats (Fig. 5A). In contrast to iNOS, the expression of Arginase-1 was reduced by 1-BP (Fig. 5B), indicating an imbalanced ratio of M1/M2 microglia in 1-BP-treated rats. Interestingly, allyl sulfide treatment reversed the alterations of both iNOS and Arginase-1 induced by 1-BP as shown by decreased iNOS and increased Arginase-1 expressions in motor cortex of combined allyl sulfide and 1-BP-treated rats compared with 1-BP alone group (Fig. 5A and B). Western bolt and qRT-PCR analysis further supported these finds by showing reduced levels of iNOS and mRNA transcripts of iNOS, TNF-α, IL-1β, while elevated protein and mRNA levels of Arginase-1 in motor cortex of allyl sulfide and 1-BP-treated rats compared with 1-BP alone group (Fig. 5C, D).

[12] 147w Neuroinflammation and oxidative stress are usually involved in brain pathological event simultaneously. We therefore determined the effects of allyl sulfide on 1-BP-induced oxidative stress. As indicated in Fig. 7, compared with vehicle controls, 1-BP intoxication significantly decreased the levels of GSH in motor cortex of rats. Consistently, the contents of MDA in 1-BP-treated rats were increased. Administration of allyl sulfide significantly mitigated the alterations of GSH and MDA in 1-BP-treated rats, indicating allyl sulfide Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 attenuates 1-BP-induced oxidative stress. Double immunofluorescence staining by using antibodies against NeuN and 4-HNE, a product of protein oxidation, revealed a high degree of oxidative stress in NeuN + cells of 1-BP-treated rats, which was significantly reduced by allyl sulfide (Fig. 7C). These results suggested that inhibition of CYP2E1 by allyl sulfide ameliorates 1-BP-induced oxidative damage in neurons.

[13] 133w Thioredoxin is known to be an intracellular redox regulator and plays an important role in regulating oxidative stress and apoptosis through interaction with apoptosis signal-regulating kinase 1 (ASK1) (Matsuzawa, 2016;Sakauchi et al., 2016). The effects of allyl sulfide on the expression of thioredoxin and activation of ASK1 were further determined. As shown in Fig. 7D, 1-BP intoxication reduced the expressions of thioredoxin, which was significantly mitigated by allyl sulfide in motor cortex of rats. In agreement with elevated thioredoxin expression, allyl sulfide administration abrogated 1-BP-induced activation of ASK1 and p38 in motor cortex by showing reduced phosphorylation of ASK1 and p38 in combined allyl sulfide and 1-BP rats compared with 1-BP alone group. No significant difference of thioredoxin expression and ASK1, p38 activation was observed between allyl sulfide alone and vehicle control group.

[1] 68w The neurotoxicity of 1-BP is well-documented in both epidemiological and laboratory studies. In this study, we revealed a critical role of CYP2E1 in 1-BP-induced neurotoxicity and blocking CYP2E1 activity by allyl sulfide significantly protected against 1-BP-induced neuronal apoptosis in motor cortex and related motor deficits in rats. Mechanistic study strongly supported that the neuroprotective effects of allyl sulfide occurred by inhibiting microglial activation, M1 polarization and oxidative damage.

[2] 115w Strong evidence suggests that chronic neuroinflammation mediated by microglia, the innate immune cells in the brain, has been implicated in neuronal damage induced by a Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 variety of neurotoxicants, such as manganese (Chen et al., 2018), rotenone (Sharma et al., 2018), paraquat and maneb (Hou, et al., 2017;Srivastava et al., 2012). Microglia, as the first line of immune defense, continuously survey the microenvironment in CNS under physiological conditions, and are activated immediately in response to pathologically stimulation. Activated microglia can be polarized into M1 and M2 phenotypes to produce detrimental and beneficial effects, respectively (Kempuraj et al., 2016;Rojo, et al., 2014).

[3] 280w Sustained microglial M1 activation induces collateral damage of neurons through continually release of pro-inflammatory cytokines and reactive oxygen species (ROS) (Siddiqui et al., 2016). While, M2 activated microglia are anti-inflammatory and promote tissue reconstruction (Siddiqui, et al., 2016). Therefore, suppressing microglial M1 polarization and/or stimulating M2 polarization is considered to be a potential therapeutic approach for the treatment of neuroinflammation-related damage. CYP2E1 is highly expressed in microglia and plays a critical role in microglial activation. Caito et al. reported that microglial CYP2E1 contributes to acrylonitrile, a high volume vinyl monomer, -induced microglial activation and production of proinflammatory cytokines (Caito et al., 2014). Furthermore, increased CYP2E1 mRNA and protein levels were detected in LPS-treated human cholinergic neuroblastoma IMR-32 cells, which aggravate the neuroinflammatory damage to neuronal cells (Na et al., 2016). Although previous study demonstrated that exposed to 1-BP for 4 weeks results in morphological changes of microglia and oxidative damage in the cerebellum of rats (Subramanian et al., 2012), the role of CYP2E1 remains unclear. In this study, we extended previous finding and showed that 1-BP stimulated microglial activation and M1 polarization in motor cortex of rats. Allyl sulfide, a well-known inhibitor of CYP2E1, displayed potent inhibitory effects against 1-BP-induced activation of CYP2E1 by showing reduced content of 1-bromo-2-propanol, the metabolite of 1-BP catalyzed by CYP2E1, in both blood and brain samples of combined allyl sulfide and 1-BP-treated rats compared with 1-BP alone group (Supplementary Fig. 5). Moreover, allyl sulfide significantly suppressed 1-BP-induced microglial activation and release of proinflammatory cytokines, which was associated with reduction of neuronal apoptosis and recovery of motor functions of rats. Our results suggest that inhibition of microglia-mediated neuroinflammation via CYP2E1 may underlay allyl sulfide-afforded neuroprotection.

[4] 160w The inhibitory effects of allyl sulfide on microglial M1 polarization might relate to its inactivation of NF-κB pathway. NF-κB is a central regulator of innate and adaptive immune response, controlling the expression and secretion of the chemokines and cytokines under inflammatory conditions (Moynagh, 2005). Once activation, the NF-κB can amplify M1 inflammatory responses, resulting in a sustained inflammatory state in the brain (Rothe, 1999). Frakes et al reported that the activation of NF-κB contributes to microglial M1 polarization in a mouse models of amyotrophic lateral sclerosis (ALS) and selective inhibition of NF-κB in microglia greatly reduced neuroinflammation and related damage of motor neurons (Frakes et al., 2014). In the present study, 1-BP treatment significantly increased the phosphorylation of NF-κB, which was significantly reduced by allyl sulfide. In agreement with our findings, the potent inhibitory effects of raw garlic extract on NF-κB activation was also observed in BV2 microglial cells treated with LPS, a classic M1 stimulator (Ho et al., 2014).

[5] 334w Oxidative stress usually occurs simultaneously with neuroinflammation, which works in concert to damage neurons. It's well-known that M1 activated microglia not only produce pro-inflammatory cytokines but also generate ROS and reactive nitrogen species (RNS) (Rojo, et al., 2014). In turn, these toxic factors further signal microglia and exaggerate microglial M1 inflammatory response, fuelling a self-renewing cycle of microglial activation followed by further neuron damage (Z et al., 2012). Previous studies demonstrated that 1-BP intoxication increases the production of ROS and nitric oxide (NO) in experimental animals (Subramanian, et al., 2012;Xu et al., 2016). Consistently, in the current study, we found that beyond neuroinflammation, 1-BP intoxication also elevated the levels of MDA and 4-HNE, two products of oxidative damage in motor cortex of rats. Interestingly, 1-BP-induced expressions of MDA and 4-HNE were accompanied with reduction of anti-oxidant GSH. It's well-known that GSH is thought to work with thioredoxin in parallel in many cases (Ren et al., 2017). Thioredoxin is widely expressed in the mammalian CNS, and has been proved to be a negative regulator of ASK1, a member of mitogen-activated protein kinase kinase kinase (MAP3K) (Liu et al., 2002;Saitoh et al., 1998). Activated ASK1 led to a sustained activation of JNK and p38 signaling cascades, resulting in neuronal apoptosis (Liu, et al., 2002;Myers et al., 2011). Under pathological conditions, thioredoxin is readily oxidative and nitrosative modified, leading to dissociation of thioredoxin from thioredoxin-ASK1 complex and occurrence of ASK1-dependent apoptosis. In this study, allyl sulfide significantly abrogated 1-BP-induced expressions of MDA and 4-HNE, which was associated with recovery of GSH contents in motor cortex of rats. Moreover, 1-BP-induced reduction of thioredoxin and phosphorylation of ASK1 and p38, a down-stream signal of ASK1 (Kazuki et al., 2009), were also abrogated by allyl sulfide. These results suggest that blocking oxidative stress and subsequent GSH/Thioredoxin/ASK1 apoptosis pathway also contributes to allyl sulfideafforded neuroprotection. In agreement with our finding, the potent anti-apoptotic effects of allyl sulfide was also observed in transient focal cerebral ischemia in rat (Lin et al., 2012).

[6] 534w Altogether, our results provided strong evidence that blocking CYP2E1 activity by allyl sulfide significantly protected against 1-BP induced neurotoxicity in rats. Our findings may provide a promising strategy for combating 1-BP-induced neurotoxicity and allyl sulfide might be considered as a potential drug candidate for future clinical studies. Fig. 1 Allyl sulfide protects against 1-BP-induced neurotoxicity in motor cortex of rats. A) The nissl bodies were stained with thionin staining, B) neurons in motor cortex were immunostained with anti-NeuN antibody and the representative images were shown (Scale bar = 200 µm). C) The numbers of NeuN + cells were quantified. D) The levels of NeuN were determined using western blot. E) Double immunofluorescence staining with antibodies against NeuN and Cleaved caspase-3 was performed in motor cortex of rats and the representative images were shown (Scale bar = 20 µm). F) The expression of cleaved caspase-3 was determined using western blot. G) The neuronal apoptosis was confirmed by TUNEL staining (Scale bar = 200 µm). H) The numbers of TUNEL positive cells were quantified. ** p < 0.01, ## p < 0.01; n = 3-4 in each group. Hindlimb grip strength (analyzed as hindlimb grip strength per body weight (g/kg.bw)). B) Gait score. C) Paralysis rate. * p < 0.05, ** p < 0.01, # p < 005, ## p < 0.01; n = 10 in each group. Double immunofluorescence staining with antibodies against CYP2E1 and Iba-1 or GFAP was performed in motor cortex of rats and the representative images were shown. B) Doubleimmunofluorescence staining with antibodies against CYP2E1 and Iba-1 was performed in motor cortex of rats intoxicated with 1-BP with or without allyl sulfide pre-treatment and the representative images were shown. C) The expression of CYP2E1 was determined using western blot. ** p < 0.01, ## p < 0.01; n = 3-4 in each group; Scale bar = 20 µm. immunofluorescence staining with antibodies against iNOS (M1 marker) and Iba-1 in the motor cortex of rat was performed and the representative images were shown. B) Double immunofluorescence staining with antibodies against Arginase-1 (M2 marker) and Iba-1 in the motor cortex of rats was performed and the representative images were shown. C) The expressions of iNOS and Arginase-1 were determined using western blot. D) The relative mRNA expression of M1 (iNOS, TNF-α, IL-1β, IL-6) and M2 (Arginase-1, IL-10, IL-4, Tgfb1) marker were determined using qRT-PCR analysis. ** p < 0.01, ## p < 0.01; n = 3-4 in each group; Scale bar = 20 µm. and non-phosphorylated NF-κB in the motor cortex of rats were determined by western blot and the density of blots was quantified. ** p < 0.01, ## p < 0.01; n = 3-4 in each group. BP. A) The activities of GSH and levels of MDA. B) were determined in the motor cortex of rats. C) Double immunofluorescence staining with antibodies against NeuN and 4-HNE in the motor cortex of rats was performed and the representative images were shown. D) The expressions of Thioredoxin 1, phosphorylated and non-phosphorylated ASK1 and p38 in the motor cortex of rats were determined by western blot. ** p < 0.01, # p < 0.05; n = 3-4 in each group; Scale bar = 20 µm.

[7] 419w Primer sequence (5'-3') Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 Fig. 2 Allyl sulfide improves the neurobehavioral performances of 1-BP treated rats. A) Hindlimb grip strength (analyzed as hindlimb grip strength per body weight (g/kg.bw)). B) Gait score. C) Paralysis rate. *p < 0.05, **p < 0.01, #p < 005, ##p < 0.01; n = 10 in each group. 306x82mm (72 x 72 DPI) Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 Fig. 3 Allyl sulfide decreases 1-BP induced CYP2E1 expression in activated microglia. A) Double immunofluorescence staining with antibodies against CYP2E1 and Iba-1 or GFAP was performed in motor cortex of rats and the representative images were shown. B) Double-immunofluorescence staining with antibodies against CYP2E1 and Iba-1 was performed in motor cortex of rats intoxicated with 1-BP with or without allyl sulfide pre-treatment and the representative images were shown. C) The expression of CYP2E1 was determined using western blot. **p < 0.01, ##p < 0.01; n = 3-4 in each group; Scale bar = 20 µm. 422x346mm (72 x 72 DPI) Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 Fig. 5 Allyl sulfide inhibits microglial M1 polarization in response to 1-BP. A) Double immunofluorescence staining with antibodies against iNOS (M1 marker) and Iba-1 in the motor cortex of rat was performed and the representative images were shown. B) Double immunofluorescence staining with antibodies against Arginase-1 (M2 marker) and Iba-1 in the motor cortex of rats was performed and the representative images were shown. C) The expressions of iNOS and Arginase-1 were determined using western blot. D) The relative mRNA expression of M1 (iNOS, TNF-α, IL-1β, IL-6) and M2 (Arginase-1, IL-10, IL-4, Tgfb1) marker were determined using qRT-PCR analysis. **p < 0.01, ##p < 0.01; n = 3-4 in each group; Scale bar = 20 µm. 517x382mm (72 x 72 DPI) Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 Fig. 6 Allyl sulfide inhibits 1-BP induced NF-κB activation. The levels of phosphorylated and nonphosphorylated NF-κB in the motor cortex of rats were determined by western blot and the density of blots was quantified. **p < 0.01, ##p < 0.01; n = 3-4 in each group. 203x146mm (72 x 72 DPI) Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018

[1] 23w Chemicals. 1-BP (99.999% purity) was provided by Sinopharm Chemical Reagent Co. and Ltd. (Shanghai, China). Allyl sulfide (≧97% purity) was purchased from Sigma-Aldrich,

[2] 15w Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018

[3] 23w St. Louis, MO, USA. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) detection kit for apoptosis was obtained from Beyotime (Shanghai, China).

[4] 51w Anti-NeuN (ab177487), anti-CYP2E1 (ab28146) and anti-4-HNE (ab46545) were obtained from Abcam (Cambridge, UK). Anti-Iba-1 (CTP1721) was obtained from wako pure chemical industries Ltd. Anti-GFAP (12389), anti-Arginase-1 (9819), anti-Thioredoxin 1 (2429), anti-ASK1 (8662), anti-ASK1 (Thr 845) (3765), anti-phospho-P38 (4511), anti-P38 (8690), anti-Cleaved Caspase-3 (9664), anti-p-NF-κB ( 13346) were all purchased from CST.

[5] 20w Anti-NF-κB (YT3107) was obtained from ImmunoWay Biotechnology Company. Anti-iNOS (C-11) (sc-7271) and anti-GAPDH (sc-32233) were obtained from Santa Cruz Biotechnology.

[6] 29w GSH and MDA commercial kits were purchased form Nanjing Jiancheng Biological Medical Engineering Institute (Jiangsu, China). Other chemicals used in this study were of the highest grade commercially available.

[7] 246w Animals and experimental design. The animal studies were approved by the Animal Experimentation Committee of Shandong University, and were in compliance with the Nation Institute of Health (NIH) guidelines for the Care and Use of Laboratory Animals. A total of 48 specific pathogen free (SPF) male Wistar rats (200 ± 20 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). During the study, animal were kept under 24 ± 2℃ with a relative humidity of 50-60% and a 12-hour light/dark cycle. The standard chow and tap water were ad libitum. After acclimation for 7 days, all rats were randomly divided into four groups (n=12): 1-BP group, 1-BP+ allyl sulfide group, allyl sulfide group and control group. 1-BP (800 mg/kg.bw) and allyl sulfide (100 mg/kg.bw) were orally treated to the rats in corresponding groups once daily for 9 continuous weeks, control rats were received equivalent volume of corn oil which used as dosing vehicle. Allyl sulfide administration was 4-hour in advance of 1-BP. The human occupational exposure route in workplace was inhalation; however, the neurotoxicity induced by 1-BP in experimental Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 animals intoxicated by this way was unstable. Although Ichihara et al reported that rats exposed to 400 or 800 ppm of 1-BP 8 h per day for 12 weeks display decreased grip strength, motor nerve conduction velocity and distal latency of the tail nerve (Ichihara, et al., 2000).

[8] 202w On the contrary, in Sohn' study, neither neurobehavioral disorder nor morphological changes was observed in rats treated with 200, 500, and 1250 ppm 1-BP for consecutive 13 weeks (6 h/d and 5 d/w) (Sohn, et al., 2002). In present study, rats exposed to 1-BP by gavage exhibited similar neurotoxic signs as observed in human. It was reported that human with severe 1-BP poisoning displayed paresthesia of the lower limbs, difficulty to walk and even paralysis (Home, 2008;Ichihara, et al., 2004;Ichihara, et al., 2002;Ichihara, et al., 2011;Majersik, et al., 2007;Samukawa, et al., 2012;Sclar, 1999). Similarly, 1-BP intoxication in rats by gavage resulted in gait abnormality, dragging and paralysis of hindlimbs, although the forelimbs were not affected. In this sense, similar toxic targets might be useful for pathogenesis study of neurologic deficits induced by 1-BP. The dosages of 1-BP (Wang et al., 2012) and allyl sulfide (Sheen et al., 1999) used in this study were employed based on the previous studies. The body weight and neurobehavioral performances of all rats were measured once a week. Eight rats per group were subjected to the biochemical index and western blot examination, the remaining 4 rats were for morphological evaluation of cerebral cortex. Animals suffering were minimized.

[9] 114w Hind limb grip strength test and evaluation of gait score. YLS.13A rats and mice grip tester (Jinan Yi Yan Technology Development Co. Ltd.) was used to determine the hind limb grip strength. A smooth film was placed under rats' forelimbs, allowing the hind limbs of rats to grasp the metal rods of the meter. Put rat on the pedal made of the metal rods, as rat held the rod tightly with its hind limbs, gently pull its tail back. When the force exceeded its grip strength, the hind limbs of rats released the metal rods, the tester automatically recorded the force value as the grip strength. Test was duplicated and average values were taken.

[10] 94w The gait score of ten rats from each group were evaluated once a week. Let each rat walked freely for 3 min and a score of 1 to 4 were assigned following the observation. A normal gait without any affect was scored 1, a slightly abnormal gait (limb slightly weakness, instability standing, lying low during walking) was scored 2, a moderately abnormal gait (hind limb weakness and unable to support its body, foot splay and slide) was scored 3, a serious abnormal gait (an absolutely dragging hind limb with foot upturning) was scored 4.

[11] 14w This test was performed by blinded observer, who knew nothing about this experiment design.

[12] 89w Histopathological examination. At the end of experiment, rats were deeply anesthetized with pentobarbital sodium and transcardially perfused with 4% paraformaldehyde. Then, the brains of rat were harvested and post-fixed for 48-hour with 4% paraformaldehyde. After dehydration in 30% sucrose, the brains were sliced continuous coronal sections of 40 µm thickness with freezing microtome and store in PBS with azide. The interesting regions including primary motor cortex (M1) and secondary motor cortex (M2) were selected according to the stereotaxic coordinates in the The Rat Brain Atlas (Paxinos et al., 2007).

[13] 135w Nissl Staining. The sections were mounted onto the superfrost plus and dried for 30 min in 37°C incubator. After rinsing the slides with double-distilled water, they were stained with thionin staining solution for 20 min. Then, the slices were dehydrated with serial grades of 70%, 70%, 95%, absolute ethanol and cleared with xylene. Sections were observed under light microscope (Olympus Corp., Japan). TUNEL staining. In brief, the sections were fixed with 4% paraformaldehyde for 1-hour and rinsed twice with PBS. After incubation with 0.5% Triton X-100 in PBS for 5 min at room temperature, the section was reacted with 50 µl TUNEL reaction mix (5µl enzyme solution and 45µl label solution) at 37℃ for 1-hour in dark and humidified thermostat, then rinsed three times with PBS. Fluorescence microscope was used to observe and take photos.

[14] 11w The number of TUNEL-positive cells was counted with image J software.

[15] 181w Immunohistochemistry. Immunohistochemistry analysis was determined using the method of Y. Guo et al. (Guo et al., 2015). In brief, the sections were incubated with 1% H 2 O 2 for 15, and were blocked with 4% normal goat serum for 20 min. Sections were probed with specific primary antibody overnight at 4°C, followed by incubation with biotinylated secondary antibody for 2-hour at room temperature. Subsequently, the sections were reacted with avidin biotin complex for 1-hour and visualized with DAB. The motor cortex were observed under microscope and photographed with reference to the The Rat Brain Atlas (Paxinos, et al., 2007). The Image J software was employed to count the number of neurons, microglia and astrocytes (Hou et al., 2017). Double-immunofluorescence Staining. Sections were blocked with 4% normal goat or horse serum for 1-hour, and then incubated at 4℃ overnight with primary antibodies. After incubation with Alexa Fluor 594 and Alexa Fluor 488-conjugated IgG for 2-hour, the sections were mounted on slides. Digital images were captured using a confocal laser-scanning microscope (LSM800 Airy scan or LSM780 Airy scan; Carl Zeiss, Oberkochen, Germany).

[16] 116w Quantitative RT-PCR analysis. Total RNA was extracted from frozen motor cortex using TRIzol reagent (Roche Diagnostics, Barcelona, Spain) accroding to the manufacturer's instructions. Isolated mRNA was quantified using the NanoDrop ND-2000 spectrophotometer (NadroDrop Technologies, Wilmington, DE, USA). Then, single-stranded cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraserand oligo (dT) (Takara Bio INC., Shiga, Japan). Finally, qRT-PCR analysis was performed using specific primers and SYBR ® Premix Ex Taq TM (Tli RNaseH Plus) (Takara Bio INC., Shiga, Japan) on a LightCycler® 480 II (Roche Applied Science, Indianapolis, IN, USA). Results were normalized to the housekeeping gene GAPDH, and the ∆∆Ct method was employed for all qRT-PCR analysis. Primer sequences are listed in Table 1.

[17] 195w Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 performed as previously described (Guo, et al., 2015). Briefly, the cerebral cortex regions of brain tissue were homogenized in chilled RIPA lysis buffer (pH 7.5, 10 mM Tris-HCl containing 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM glycerophosphate, 5 mM sodium pyrophosphate, 1% protease inhibitor cocktail) using the bioprep-24 bead-based tissue homogenizer (Hangzhou allsheng instrument CO.; Ltd., Hangzhou, China), then centrifuged at 4℃ (12000 g × 10 min), the supernatants were collected and protein contents were determined by BCA TM protein assay kits (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of the protein lysates were loaded onto SDS-polyacrylamide gel electrophoresis to be separated, and transferred to PVDF membrane (Millipore, Bedford, MA). After blocking with 5% skim milk for 30 min, the membranes were incubated with specific primary antibodies at 4℃ overnight. Then, the membranes were reacted with secondary antibodies labeled horseradish peroxidase (HRP) for 1-hour and visualized by enhanced chemiluminescence system (ECL) (Biological Industries, Israel). Images were scanned and quantified by Jetta gel imaging analysis system (Jiangsu, China).

[18] 58w The GSH and MDA levels. 10% homogenate of cerebral cortex were prepared as previously described (Guo, et al., 2015), and protein concentration of the supernatants were determined by BCA TM protein assay kits following the kit instruction. The MDA and GSH levels were determined spectrophotometrically respectively with commercial kits (Nanjing Jiancheng Institute, China) according to the manufacturer's instruction.

[19] 87w All data are expressed as mean ± SEM. Hind limb grip strength was analyzed by Two-way analysis of variance (ANOVA) with Bonferroni test. For each training week in hind limb grip strength test and other data, One-way ANOVA was used, followed by post hoc analysis with Tukey's test. A value of p < 0.05 was considered statistically significant. GraphPad Prism 7.0 Version Software (San Diego, CA, USA) was used for Downloaded from https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfy240/5106016 by Serials Processing Library University of Canberra user on 26 September 2018 statistical analysis.

[1] 55w To further characterize the possible mechanisms involved in allyl sulfide-inhibited microglial M1 polarization, we analyzed the activation of NF-κB signaling pathway. As shown in Fig. 6, 1-BP significantly increased the levels of phosphorylated NF-κB. Whereas, rats co-treated with 1-BP and allyl sulfide displayed reduced phosphorylation of NF-κB, suggesting that allyl sulfide inhibits 1-BP-induced NF-κB activation.