Occupational exposure to n-hexane produces a neuropathy characterized as a central-peripheral distal axonopathy, which is mediated by 2,5-hexanedione (HD). To investigate the mechanisms of the neuropathy induced by HD, the contents and activities of cyclin-dependent kinase 5 (CDK5) and activators (p35 precursor, p35 and p25) in rats’ cerebrum cortex (CC), spinal cord (SC) and sciatic nerve (SN) were determined. The results showed that the levels and activities of CDK5 in CC of 200 or 400 mg/kg HD-treated rats were significantly decreased in both the cytosolic and membrane fractions and negatively correlated with gait abnormality in the cytosolic fraction. However, CDK5 contents and activities in SN of rats treated with 200 or 400 mg/kg HD were significantly increased and positively correlated with gait abnormality in both the cytosolic and membrane fractions. Although increases of CDK5 contents in both the cytosolic and membrane fractions of SC in 200 and 400 mg/kg HD-treated rats were also observed, CDK5 activities were significantly decreased in the cytosolic fraction and negatively correlated with gait abnormality. The changes of p35 precursor, p35 and p25 contents in CC, SC and SN showed the same pattern with that of CDK5 activities. Thus, HD intoxication was associated with deregulation of CDK5 and its activator p35 or p25 in nerve tissues. The inconsistent changes of CDK5 activities in CNS and PNS might delegate the different mechanisms of HD-induced peripheral neuropathy.

Calcium-dependent mechanisms, particularly those mediated by Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC and SN were significantly increased, which indicated elevation of Ca2+ concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs.

The protective effects of single dose of garlic oil (GO) on acute ethanol-induced fatty liver were investigated. Mice were treated with ethanol (4.8 g/kg bw) to induce acute fatty liver. The liver index, the serum and hepatic triglyceride (TG) levels and the histological changeswere examined to evaluate the protective effects. Hepatic malondialdehyde (MDA), glutathione (GSH) levels and superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) activities were determined for the antioxidant capacity assay. Acute ethanol exposure resulted in the enlargement of the liver index and the increase of the serum and hepatic TG levels (P<0.01), which were dramatically attenuated by GO pretreatment in a dose-dependent manner (P<0.01). GO treatment (simultaneously with ethanol exposure) exhibited similar effects to those of pretreatment, while no obviously protective effects were displayed when it was used at 2h after ethanol intake. Histological changes were paralleled to these indices. Beside this, GO dramatically prolonged the drunken time and shortened thewaking time, and these effects were superior to those of silymarin and tea polyphenol. In addition, GO dose-dependently suppressed the elevation of MDA levels, restored the GSH levels and enhanced the SOD, GR and GST activities. Compared with the ethanol group, the MDA levels decreased by 14.2% (P<0.05), 29.9% and 32.8% (P<0.01) in GO groups 50, 100 and 200 mg/kg, respectively. The GST activity increased by 9.97%, 19.94% (P<0.05) and 42.12% (P<0.01) of the ethanol group in GO groups 50, 100 and 200mg/kg, espectively, while the GR activity increased by 28.57% (P<0.05), 37.97% (P<0.01), 50.45% (P<0.01) of the ethanol group in GO groups 50, 100 and 200mg/kg, respectively. These data indicated that single dose ofGOpossessed ability to prevent acute ethanol-induced fatty liver, but may lose its capacity when used after ethanol exposure. The protective effects should be associated with its antioxidative activities.

To investigate the mechanisms and biomarker of the neuropathy induced by 2,5-hexanedione (HD), male Wistar rats were administrated HD at dosage of 200 or 400 mg/kg for 8 weeks (five-times per week). All rats were sacrificed after 8 weeks of treatment and the cerebrum cortex (CC), spinal cord (SC) and sciatic nerves (SN) were dissected, homogenized and used for the determination of cytoskeletal proteins by western blotting. The levels of neurofilaments (NFs) subunits (NF-L, NF-M and NF-H) in nerve tissues of 200 and 400 mg/kg HD rats significantly decreased in both the supernatant and pellet fractions. Furthermore, significant negative correlations between NFs levels and gait abnormality were observed. As for microtubule (MT) and microfilament (MF) proteins, the levels of-tubulin-tubulin and-actin in the supernatant and pellet fraction of SN significantly decreased in 200 and 400 mg/kg HD rats and correlated negatively with gait abnormality. However, the contents of MT and MF proteins in CC and SC were inconsistently affected and had no significant correlation with gait abnormality. The levels of NF-L and NF-H in serum significantly increased, while NF-M-tubulin-tubulin and-actin contents remain unchanged. A significant positive correlation (R=0.9427, P<0.01) was observed between gait abnormality and NF-H level in serum as the intoxication went on. These findings suggested that HD intoxication resulted in a progressive decline of cytoskeletal protein contents, which might be relevant to the mechanisms of HD-induced neuropathy. NF-H was the most sensitive index, which may serve as a good indicator for neurotoxicity of n-hexane or HD.

To investigate the reversibility of the neuropathy induced by 2,5-HD, adult male rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week) for 2, 4, and 8 weeks, respectively. After stopping HD exposure, half of 8-week treated animals were allowed to naturally recover for 16 weeks. The relative levels of NF-H, NF-M, and NF-L in spinal cords and sciatic nerves of rats were determined by immunoblotting during the HD neuropathy. The results showed that NFs content in nerve tissues demonstrated a progressive decline as the intoxication continued. Furthermore, after a recovery of 16 weeks, the levels of three NF subunits in spinal cords of treated rats returned to normal while those in sciatic nerves displayed an inconsistent reversal. Among them, the level of NF-H in sciatic nerves returned to normal completely, and NF-L also showed a significant improvement, whereas NF-Mdid not demonstrate an obvious reversal. These findings suggest that HD-induced NFs decline is at least partially irreversible within the time frame of this study, which might be associated with the incomplete recovery of neurological dysfunctions of HD-treated rats.