Evaluation of exposure to trihalomethanes in tap water and semen quality: A prospective study in Wuhan,China

Trihalomethanes (THMs) have been demonstrated to adversely affect male reproductive health in animals, but the evidence in humans is limited. The study aimed to examine the association between THM exposure and semen quality in a Chinese population. We recruited 324 men from the same water supply district in Wuhan, China between April 2011 and May 2012. Exposure to THMs was evaluated based on their concentrations in tap water measured within 90 days preceding semen collection, the uptake factors of THMs and personal information on ingestion and showering/bathing. We found that TTHM [sum of chloroform (TCM) and brominated THMs (Br-THMs)], TCM and Br-THM uptakes via ingestion were associated with significant or suggestive decreasing trends in sperm concentration (P for trend = 0.01, 0.03 and 0.05, respectively) and sperm count (P for trend = 0.02, 0.05 and 0.09, respectively). Our results suggest that THM exposure via ingestion may adversely affect semen quality.     

Urinary bisphenol A and semen quality,the LIFE Study

Bisphenol A (BPA), a high-production volume industrial chemical found in several consumer products, has been negatively associated with sperm quality. This study aimed to estimate the association between BPA and 35 measures of semen quality among reproductive aged men recruited from 16 counties in Michigan and Texas, 2005–2009. Of 501 enrolled males, 418 (83.4%) provided a urine sample and at least one semen sample. Linear and logistic regression models assessed the association between urinary BPA levels and individual semen quality endpoints. Generalized estimating equations were used to account for repeated measures of semen quality and adjusted models accounted for 11 a priori covariates. Geometric mean total urinary BPA concentration among participants was 0.55 ng/mL (95% CI 0.49–0.63). A negative relation between BPA and DNA fragmentation was the sole significant finding in adjusted linear regression (β = −0.0544, p = 0.035) and suggestive of less sperm DNA damage.     

DNA methylation changes in the placenta are associated with fetal manganese exposure

Adequate micronutrient intake, including manganese (Mn), is important for fetal development. Both Mn deficiencies and excess exposures are associated with later-life disease, and Mn accumulates in the placenta. Placental functional alterations may alter fetal programming and lifelong health, and we hypothesized that prenatal exposures to Mn may alter placental function through epigenetic mechanisms. Using Illumina's HumanMethylation450 BeadArray, DNA methylation of >485,000 CpG loci genome-wide was interrogated in 61 placental samples and Mn associations assessed genome-wide via omnibus test (p = 0.045). 713 loci were associated with Mn exposure (p < 0.0001). Five significantly differentially-methylated (p < 1.3 × 10⁻⁷) loci reside in neurodevelopmental, fetal growth and cancer-related genes. cg22284422, within the uncharacterized LOC284276 gene, was associated with birth weight; for every 10% increase in methylation, lower birth weights were observed, with an average decrease of 293.44 g. Our observations suggest a link between prenatal micronutrient levels, placental epigenetic status and birth weight, although these preliminary results require validation.     

Associations between paternal urinary phthalate metabolite concentrations and reproductive outcomes among couples seeking fertility treatment

IntroductionLimited evidence suggests that male exposure to ubiquitous environmental phthalates may result in poor reproductive outcomes among female partners.MethodsThis analysis included male–female couples undergoing in vitro fertilization (IVF) and/or intrauterine insemination (IUI). We evaluated associations between the geometric mean of paternal specific gravity-adjusted urinary phthalate concentrations prior to the female partners’ cycle and fertilization, embryo quality, implantation, and live birth using generalized linear mixed models.ResultsTwo-hundred eighteen couples underwent 211 IVF and 195 IUI cycles. Trends were observed between paternal urinary mono-3-carboxypropyl phthalate (MCPP; P = 0.01) and mono(carboxyoctyl) phthalate (MCOP; P = 0.01) and decreased odds of implantation. MCPP and MCOP were also associated with decreased odds of live birth following IVF (P = 0.01 and P = 0.04, respectively), and monobutyl phthalate above the first quartile was significantly associated with decreased odds of live birth following IUI (P = 0.04). However, most urinary phthalate metabolites were not associated with these reproductive outcomes.ConclusionSelected phthalates were associated with decreased odds of implantation and live birth.     

Protective role of sodium para-amino salicylic acid against manganese-induced hippocampal neurons damage

Manganese (Mn) is an essential trace element of human. However, excessive Mn can cause manganism. Mn selectively accumulated in Mn-exposed workers’ hippocampus which is crucial for higher brain functions such as learning, memory, and motivation during our postnatal life. Studies suggested sodium para-aminosalicylic acid (PAS) appeared to be therapeutic for manganism. We aimed to explore whether PAS could block Mn-induced neuronal injury in hippocampus in vitro. Hippocampal neurons were exposed to 50 μM manganese chloride (MnCl₂) for 24 h, following by 50, 500, or 5000 μM PAS treatment for 24 h. Cell viability, apoptosis rate, mean fluorescence intensity of mitochondrial and DNA damage were respectively performed. MnCl₂ significantly decreased neurons’ viability and fluorescence intensity of comet head of DNA, while increasing the apoptosis rate, mean fluorescence intensity of mitochondrial, percentage of tail DNA, and Olive tail moment of DNA. PAS reduced the percentage of tail DNA and Olive tail moment of Mn-exposed neurons. These data suggested that Mn caused hippocampal neurons’ injury, and 50–5000 μM PAS could inhibit Mn-induced DNA damage.     

Toxic trace metals and human oocytes during in vitro fertilization (IVF)

Trace exposures to the toxic metals mercury (Hg), cadmium (Cd) and lead (Pb) may threaten human reproductive health. The aim of this study is to generate biologically-plausible hypotheses concerning associations between Hg, Cd, and Pb and in vitro fertilization (IVF) endpoints. For 15 female IVF patients, a multivariable log-binomial model suggests a 75% reduction in the probability for a retrieved oocyte to be in metaphase-II arrest for each μg/dL increase in blood Pb concentration (relative risk (RR) = 0.25, 95% confidence interval (CI) 0.03–2.50, P = 0.240). For 15 male IVF partners, each μg/L increase in urine Cd concentration is associated with an 81% decrease in the probability for oocyte fertilization (RR = 0.19, 95% CI 0.03–1.35, P = 0.097). Because of the magnitude of the effects, these results warrant a comprehensive study with sufficient statistical power to further evaluate these hypotheses.     

Evaluation of neurobehavioral and neuroinflammatory end-points in the post-exposure period in rats sub-acutely exposed to manganese

Manganese (Mn) can cause manganism, a neurological disorder similar to Parkinson’ Disease (PD). The neurobehavioral and neuroinflammatory end-points in the Mn post exposure period have not been studied yet. Rats were injected on alternate days with 8 doses of MnCl₂ (25 mg/kg) or saline, then euthanized 1, 10, 30 or 70 days following the last dose. Whole-blood (WB) (p < 0.05), urine (p < 0.05) and brain cortical (p < 0.0001) Mn levels were significantly increased 24 h after the last dose. Decreases in the rats’ ambulation were noted 1, 10 and 30 days after the last Mn dose (p < 0.001; p < 0.05; p < 0.001, respectively) and also in the rearing activity at the four time-points (p < 0.05). Cortical glial fibrillary acid protein immunoreactivity (GFAP-ir) was significantly increased at 1, 10, 30 (p < 0.0001) and 70 (p < 0.001) days after the last Mn dose, as well as tumor necrosis α (TNF-α) levels (p < 0.05) but just on day 1. Taken together, the results show that, during the 70-day clearance phase of Mn, the recovery is not immediate as behavioral alterations and neuroinflammation persist long after Mn is cleared from the cortical brain compartment.     

Heavy metals in agricultural soils and crops and their health risks in Swat District,northern Pakistan

This study assessed the concentrations of heavy metals such as cadmium (Cd), chromium (Cr), copper (Cu), manganese (Mn), nickel (Ni) and zinc (Zn) in agricultural soils and crops (fruits, grains and vegetable) and their possible human health risk in Swat District, northern Pakistan. Cd concentration was found higher than the limit (0.05 mg/kg) set by world health organization in 95% fruit and 100% vegetable samples. Moreover, the concentrations of Cr, Cu, Mn, Ni and Zn in the soils were shown significant correlations with those in the crops. The metal transfer factor (MTF) was found highest for Cd followed by Cr > Ni > Zn > Cu > Mn, while the health risk assessment revealed that there was no health risk for most of the heavy metals except Cd, which showed a high level of health risk index (HRI ⩾ 10E-1) that would pose a potential health risk to the consumers.     

Manganese acts centrally to activate reproductive hormone secretion and pubertal development in male rats

Manganese (Mn) is an important element for normal growth and reproduction. Because Mn accumulates in the hypothalamus and is capable of stimulating puberty-related hormones in female rats, we assessed whether this metal could cause similar effects in male rats. We have demonstrated that MnCl₂, when administered acutely into the third ventricle of the brain, acts dose dependently to stimulate luteinizing hormone (LH) release. Furthermore, there was a dose dependent stimulation in the secretion of LH-releasing hormone (LHRH) from the medial basal hypothalamus in vitro, and administration of an LHRH receptor antagonist in vivo blocks Mn-induced LH release. To assess potential chronic effects of the metal, male pups were supplemented with 10 or 25 mg MnCl₂ per kg by gastric gavage from day 15 until days 48 or 55, at which times developmental signs of spermatogenesis were assessed. Results demonstrate that while significant effects were not observed with the 10 mg/kg dose, the animals receiving the 25 mg/kg dose showed increased LH (p < 0.05), FSH (p < 0.01) and testosterone (p < 0.01) levels at 55 days of age. Furthermore, there was a concomitant increase in both daily sperm production (p < 0.05) and efficiency of spermatogenesis (p < 0.05), demonstrating a Mn-induced acceleration in spermatogenesis. Our results suggest Mn is a stimulator of prepubertal LHRH/LH secretion and may facilitate the normal onset of male puberty. These data also suggest that the metal may contribute to male precocious pubertal development should an individual be exposed to low but elevated levels of Mn too early in life.     

In vitro exposure of human spermatozoa to bisphenol A induces pro-oxidative/apoptotic mitochondrial dysfunction

As bisphenol A (BPA) exerts oxidative/pro-apoptotic effects in several cell types, we explored whether the in vitro exposure to BPA could affect human sperm integrity through the induction of pro-oxidative/apoptotic mitochondrial dysfunction. The exposure of motile sperm suspensions to scalar BPA concentrations for 4 h produced a decrease in the mitochondrial membrane potential, starting from 300 μM. It was associated with an increased mitochondrial generation of superoxide anion, caspase-9 and caspase-3 activation and motility decrement. Vitality decline was observed at BPA ≥ 400 μM. Twenty hours exposure to 300 μM BPA, but not to lower concentrations, produced a significant loss in sperm vitality associated with a complete sperm immobilization. Finally, 300 μM BPA also produced a significant DNA oxidative damage, as revealed by the formation of oxidized base adduct 8-hydroxy-2′-deoxyguanosine. In conclusion, BPA affected human sperm integrity by inducing pro-oxidative/apoptotic mitochondrial dysfunction.     

Influence of MRP1 G1666A and GSTP1 Ile105Val genetic variants on the urinary and blood arsenic levels of Turkish smelter workers

To understand the cellular mechanisms responsible for arsenic metabolism and transport pathways plays a fundamental role in order to prevent the arsenic-induced toxicity. The effect of MRP1 G1666A and GSTP1 Ile105Val polymorphisms on blood and urinary arsenic levels were determined in 95 Turkish smelter workers. Blood and urinary arsenic concentrations were measured by GF-AAS with Zeeman correction and gene polymorphisms were investigated by PCR-RFLP method. The mean blood and urinary arsenic levels were 21.60 ± 12.28 μg/L and 5.58 ± 4.37 μg/L, respectively. A significant association between MRP1 1666A allele and urinary arsenic levels was found (p = 0.001). GSTP1 Ile105Val polymorphism was detected not to be associated with either blood or urinary arsenic levels (p = 0.384, p = 0.440, respectively). Significant association was also detected between MRP1A^-/GSTP1Val⁻ genotypes and urinary arsenic levels (p = 0.001). This study suggested that MRP1 G1666A alone and, also, combined with GSTP1 Ile105Val were associated with inter-individual variations in urinary arsenic levels, but not with blood arsenic levels.     

Protective effect of nimesulide against hepatic ischemia/reperfusion injury in rats: Effects on oxidant/antioxidants,DNA mutation and COX-1/COX-2 levels

BackgroundNimesulide is a pharmacological agent and selective COX-2 inhibitor. It has anti-inflammatory, analgesic and antipyretic properties. The purpose of this study was to investigate the effect of nimesulide on oxidant/antioxidant, DNA mutation and COX-1/COX-2 activities in rat liver tissue with induced ischemia/reperfusion (I/R).MethodsBefore the experiment, rats were divided into four groups; liver ischemia/reperfusion (LIR), 50 mg/kg nimesulide + liver ischemia/reperfusion (NLIR50), 100 mg/kg nimesulide + liver ischemia/reperfusion (NLIR100) and a control group to be given a sham operation (SG). Malondialdehyde (MDA), total glutathione (GSH) levels and myeloperoxidase (MPO), COX-1/COX-2 enzyme activities and DNA damage product level results from liver tissues and serum AST and ALT levels were determined. The data obtained were compared with the results from the liver ischemia/reperfusion and sham operation groups.ResultsMDA levels, MPO and COX-2 activities and products of DNA injury were significantly lower in the groups given nimesulide, and particularly the NLIR100 group, compared to the LIR group (p < 0.05), while tGSH levels were significantly higher (p < 0.05). There was no significant difference between the NLIR50 and NLIR100 groups and the LIR group in terms of COX-1 levels (p > 0.05). AST and ALT levels were significantly lower in the other groups compared to the LIR group (p < 0.05).ConclusionsNimesulide at 100 mg/kg prevented oxidative liver damage induced with I/R significantly better than at a dose of 50 mg/kg. These experimental findings indicate that nimesulide may be useful in the treatment of hepatic I/R damage.     

Assessment of methyl thiophanate–Cu (II) induced DNA damage in human lymphocytes

Dimethyl 4,4′-(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a category-III acute toxicant and suspected carcinogen to humans. Hence, the ability of this benzimidazole class of fungicide to engender DNA strand breaks was investigated using alkaline single cell gel electrophoresis (SCGE), alkaline unwinding and cytokinesis-blocked micronucleus (CBMN) assays. The SCGE of human lymphocytes treated with 1 mM MT for 3 h at 37 °C showed much higher Olive tail moment (OTM) value of 40.3 ± 2.6 (p < 0.001) vis-à-vis 3.3 ± 0.09 in DMSO control. Treatment of cultured lymphocytes for 24 h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). Stoichiometric data revealed the intrinsic property of MT to bind with Cu (II) and its reduction to Cu (I), which is known to form reactive oxygen species (ROS). We have detected the intracellular ROS generation in MT treated lymphocytes and observed an elevated level of MT-induced strand breaks per unit of calf thymus DNA in presence of Cu (II). Overall the data suggested that the formation of MT–Cu (II)–DNA ternary complex and consequent ROS generation, owing to Cu (II)/Cu (I) redox cycling in DNA proximity, is responsible for MT-induced DNA damage.     

Pyrethroid insecticide metabolites are associated with serum hormone levels in adult men

Experimental studies have reported that pyrethroid insecticides affect male endocrine and reproductive function, but human data are limited. We recruited 161 men from an infertility clinic between years 2000–2003 and measured serum reproductive and thyroid hormone levels, as well as the pyrethroid metabolites 3-phenoxybenzoic acid (3PBA) and cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA and trans-DCCA) in spot urine samples. When adjusting for potential confounders, categories for all three metabolites, as well as their summed values, were positively associated with FSH (all p-values for trend <0.05). Statistically significant or suggestive positive relationships with LH were also found. In addition, cis-DCCA and trans-DCCA were inversely associated with inhibin B (p for trend = 0.03 and 0.02, respectively). Finally, there was evidence that trans-DCCA was inversely associated with testosterone and free androgen index (the ratio of testosterone to sex hormone binding globulin; p for trend = 0.09 and 0.05, respectively). The observed relationships were consistent with previous findings, but further research is needed for a better understanding of the potential association between pyrethroid insecticides and male reproduction.     

Levels of dialkylphosphate metabolites in urine among general U.S. population

Data from National Health and Nutrition Examination Survey for years 2003–2008 were used to study the factors that affect urinary levels of dialkylphosphate (DAP) metabolites in urine. Separate regression models were fitted for children aged 6–11 years, adolescents aged 12–19 years, and adults aged ≥20 years. Specifically, DAP metabolites that were analyzed were: dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP). Males had statistically significantly lower adjusted levels than females for DMP for adolescents, for DEP for adults, for DMTP for both adolescents and adults, and for DETP for both children and adults. Nonsmokers had statistically significantly higher adjusted levels than smokers for DMTP for adolescents and for DMP and DMTP for adults. Exposure to second hand smoke at home was associated with relatively higher levels of DMP among children (p = 0.01) but the reverse was found to be true for DMTP (p < 0.01) among adolescents as well as adults (p = 0.02). Children had higher levels of DMTP than both adolescents and adults (p < 0.01) and higher levels of DETP than adolescents (p = 0.02). Age was found to be negatively associated with the levels of DMTP (p = 0.01) among children and positively associated (p < 0.01) with the levels of all four metabolites among adults.     

Cell damage induced by copper: An explant model to study anemone cells

Sea anemones are benthic organisms, of low mobility and can be directly affected by water pollution. This work studied the defense mechanisms and DNA damage caused by copper toxicity in cells from the anemone Bunodosoma cangicum. For this, exposure of anemones cells were held, kept in primary culture through explant of podal disk to copper (7.8 and 15.6 μg/L), and the control group, for 6 and 24 h. Cytotoxicity was seen through the viability and cell number, MXR phenotype through the accumulation of rhodamine-B, ROS generation by H₂DCF-DA and DNA damage by comet assay. The results obtained show that there is a drop in viability and number of cells, especially after exposure of 24 h in 15.6 μg/L. There is an induction of the MXR activity only at 7.8 μg/L for 24 h. As for ROS, there is an increase in the generation of reactive species in greatest concentration of copper for 6 h, and in both for 24 h, which leads to oxidative stress, which culminates with a DNA damage. What was evidenced by the increase of the tail size, % DNA presented and moment of tail. Therefore, the copper represents an adversity to the anemones cells, being cytotoxic and genotoxic.     

Safety evaluation of metal exposure from commonly used moisturizing and skin-lightening creams in Nigeria

The concentrations of ten metals (Cd, Pb, Ni, Cr, Cu, Co, Fe, Mn, Zn and Al) were measured in some commonly used moisturizing and skin-lightening creams in Nigeria with a view to providing information on the risk of exposure to metals from the use of these products. The metal concentrations in these products were measured by atomic absorption spectrometry after acid digestion of the samples. The measured concentrations of metals in the skin moisturizing creams ranged from <0.15 to 6.3 μg/g Cd, <0.02 to 17.5 μg/g Cu, 2.25 to 6.25 μg/g Cr, <0.25 to 124.3 μg/g Al, 0.2 to 7.3 μg/g Pb, <0.03 to 10.7 μg/g Ni, 17.3 to 372.0 μg/g Zn, <0.02 to 1.0 μg/g Co, 17.75 to 28.8 μg/g Mn, <0.1 to 89.8 μg/g Fe while the concentrations of metals in the skin-lightening products ranged from <0.15 to 16.5 μg/g Cd, <0.02 to 10.0 μg/g Cu, 4.25 to 8.0 μg/g Cr, <0.25 to 128.0 μg/g Al, 0.5 to 4.5 μg/g Pb, <0.03 to 1.65 μg/g Ni, 24.7 to 267.5 μg/g Zn, <0.02 to 2.5 μg/g for Co, 19.3 to 31.8 μg/g Mn, 9.5 to 211.63 μg/g Fe. In a significant number (>93%) of the samples investigated the concentrations of Pb, Cd, Ni and Co were below the specified limit, or the maximal limit for impurities in colour additives in cosmetics for external use. However, Cr was found at concentrations above the allergenic limit of 1 μg/g. The results also showed that skin-lightening creams contained higher concentrations of the studied metals than the moisturizing creams, except for Ni, which indicates that persons who uses skin-lightening creams in preference to moisturizing ones, are exposed to higher concentrations of metals.     

Maternal exposure to environmental tobacco smoke,GSTM1/T1 polymorphisms and oxidative stress

Environmental tobacco smoking (ETS) is known to be associated with adverse pregnancy outcomes. The purpose of this study was to investigate the relationship between maternal exposure to ETS and oxidative stress for neonates, as well as the effect of maternal genetic polymorphisms, glutathione-S-transferase M1 (GSTM1) and GSTT1, on this relationship.We used the radioimmunoassay to measure the urinary concentration of cotinine in 266 pregnant women who denied smoking cigarettes during pregnancy and in their singleton babies. In addition, the urinary concentration of malondialdehyde (MDA) and 8-hydroxy-2-deoxyguanosine (8-OH-dG) were assessed using high-performance liquid chromatography and enzyme-linked immunosorbent assay, respectively. We also extracted DNA from whole blood obtained from the mothers and then conducted polymerase chain reaction on the samples to determine the GSTM1 and GSTT1 genotypes.The maternal cotinine concentration was found to be significantly associated with the fetal cotinine concentration, particularly for mothers whose urine cotinine concentrations were above 120 μg/g cr (p < 0.01). The fetal urine cotinine concentration was also found to be significantly associated with the fetal urine MDA concentration (p < 0.01). When the null type maternal GSTM1 or the wild type GSTT1 was present, the maternal oxidative stress level increased significantly as the maternal continine concentration increased (MDA: p < 0.01; 8-OH-dG: p < 0.01). No significant relationships were found between maternal cotinine and fetal oxidative stress markers, however, the fetal MDA levels increased significantly as fetal cotinine levels increased.These results suggest that the maternal exposure to ETS affects the fetal urine cotinine concentration and induces production of maternal oxidative stress. In addition, maternal genetic polymorphisms of GSTM1 and GSTT1 may modify the oxidative stress by maternal exposure to ETS.     

Arterial stiffness and sedentary lifestyle: Role of oxidative stress

Sedentary lifestyle is a risk factor for the development of cardiovascular disease, and leads to a quantifiable impairment in vascular function and arterial wall stiffening. We tested the hypothesis of oxidative stress as a determinant of arterial stiffness (AS) in physically inactive subjects, and challenged the reversibility of these processes after the completion of an eight-week, high-intensity exercise training (ET).AS was assessed before and after ET, measuring carotid to femoral pulse wave velocity (PWV) with a Vicorder device. At baseline and after ET, participants performed urine collection and underwent fasting blood sampling. Urinary 8-iso-PGF_2α, an in vivo marker of lipid peroxidation, total, HDL and LDL cholesterol, and triglyceride concentrations were measured.ET was associated with significantly reduced urinary 8-iso-PGF_2α(p < 0.0001) levels. PWV was significantly reduced after ET completion (p < 0.0001), and was directly related to urinary 8-iso-PGF_2α(Rho = 0.383, p = 0.021). After ET, cardiovascular fitness improved [peak oxygen consumption (p < 0.0001), peak heart rate (p < 0.0001)]. However, no improvement in lipid profile was observed, apart from a significant reduction of triglycerides (p = 0.022). PWV and triglycerides were significantly related (Rho = 0.466, p = 0.005) throughout the study period. PWV levels were also related to urinary 8-iso-PGF_2α in our previously sedentary subjects.We conclude that regular physical exercise may be a natural antioxidant strategy, lowering oxidant stress and thereby the AS degree.     

Role of N-acetylcysteine in protecting against 2,5-hexanedione neurotoxicity in a rat model: Changes in urinary pyrroles levels and motor activity performance

The interference of N-acetylcysteine (NAC) on 2,5-hexanedione (2,5-HD) neurotoxicity was evaluated through behavioral assays and the analysis of urinary 2,5-HD, dimethylpyrrole norleucine (DMPN), and cysteine-pyrrole conjugate (DMPN NAC), by ESI-LC–MS/MS, in rats exposed to 2,5-HD and co-exposed to 2,5-HD and NAC.Wistar rats were treated with 4 doses of: 400 mg 2,5-HD/kg bw (group I), 400 mg 2,5-HD/kg bw + 200 mg NAC/kg bw (group II), 200 mg NAC/kg bw (group III) and with saline (group IV). The results show a significant decrease (p < 0.01) in urinary DMPN and free 2,5-HD, a significant increase (p < 0.01) in DMPN NAC excretion, and a significant recovery (p < 0.01) on motor activity in rats co-exposed to 2,5-HD + NAC, as compared with rats exposed to 2,5-HD alone. Taken together, our findings suggest that at the studied conditions NAC protects against 2,5-HD neurotoxicity and DMPN may be proposed as a new sensitive and specific biomarker of 2,5-HD neurotoxicity in animals treated with a toxic amount of 2,5-hexanedione.