Free and total 2,5-hexanedione in biological monitoring of workers exposed to n-hexane in the shoe industry

OBJECTIVES: To analyse the role of total 2,5-hexanedione (2,5-HD) compared with free 2,5-HD as a biological indicator of exposure to n-hexane at work. METHODS: One-hundred and thirty two workers in contact with this solvent during their occupation in the shoe industry in the province of Alicante (Spain) were studied. Environmental and biological tests were carried out analysing variations of the concentration of the metabolite in urine corresponding to different working conditions. Environmental exposure was evaluated in each work place using active personal monitors and measured by gas chromatography (GC). Dichloromethane extracts of the urine samples collected at the end of the working shifts were analysed, before (determining free 2,5-HD, the toxic metabolite) and after acid hydrolysis (pH 0.1) (yielding the total 2,5-HD) and also by GC. The concentration of conjugated metabolite 4,5-dihydroxy-2-hexanone was calculated from the difference between total and free 2,5-HD. RESULTS: Free 2,5-HD represented an average of 14.2% of the total 2,5-HD determined in urine, and this percentage increased significantly (P<0.01) with higher environmental levels of acetone. Other factors, such as absorption through the skin (depending on the use of gloves) and the day on which samples were taken also significantly affected the relation between the two indicators and their respective relationships with environmental concentrations of n-hexane. CONCLUSION: Although analyses of the relationship between the levels of atmospheric n-hexane and those of metabolites in urine show a greater correlation for total 2,5-HD than for free 2,5-HD, our results suggest that free 2,5-HD could be a better indicator in evaluating risk of exposure to n-hexane, since the concentration is directly related to the neurotoxic effect.     

Gas chromatographic determination of 2,5-hexanedione in urine as an indicator of exposure to n-hexane.

A sensitive and specific method for determination of 2,5-hexanedione in urine is described. Treatment of the urine specimen directly with n-butylamine yields n-butyl 2,5-dimethyl pyrrole. The latter is extracted into diisopropylether and analyzed by gas chromatography (GC) with flame ionization detection (FID), thermionic specific detection (TSD) (N mode), or mass spectrometric detection (MS). The minimum detectable quantities are 1 mg/L urine when employing FID with a coefficient of variation of less than 6%. Recovery of 2,5-hexanedione added to the urine at the level of 10 mg/L was 78.9%.     

Comparative neurotoxicity and pyrrole-forming potential of 2,5-hexanedione and perdeuterio-2,5-hexanedione in the rat

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, reacts with protein amines to form alkylpyrrole adducts. Pyrrolylation of neurofilament protein may be the initiating molecular event in 2,5-HD neuropathy. The present study compares the neurotoxic and pyrrole-forming potentials of 2,5-HD with those of perdeuterio-2,5-HD ([D10]-2,5-HD) in the rat. Due to a requirement for C-H bond breaking in the reaction mechanism, the latter derivative was expected to exhibit a primary isotope effect, thus forming the pyrrole at a slower rate. In vitro studies confirmed that [D10]-2,5-HD pyrrolylated protein at only one-third of the initial rate seen with native 2,5-HD. Prolonged incubation resulted in similar pyrrole concentrations with both derivatives. Adult, male Wistar rats were administered daily (5 days/week) ip doses of either 3.5 mmol 2,5-HD or [D10]-2,5-HD/kg/day for 17 days or 2.5 mmol/kg/day for 38 days. At termination, animals administered 2,5-HD and [D10]-2,5-HD exhibited 27 and 8% body weight loss, respectively. Moderate to severe hindlimb paralysis was present in the 2,5-HD groups while only mild effects were seen in [D10]-2,5-HD-dosed rats. Neuropathological changes were prominent in spinal cord sections from 2,5-HD-treated animals, while no effects were present in rats given the deuterated derivative. Pyrrole adduct concentrations in serum and axonal cytoskeletal proteins from 2,5-HD-treated animals were two- to threefold higher than in rats given equimolar doses of [D10]-2,5-HD. Levels of covalent crosslinking of axonal cytoskeletal proteins (assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) appeared to correlate with pyrrole concentrations. Tissue concentrations of each diketone isomer were not significantly different, indicating similar uptake of native and deuterated 2,5-HD. Mass spectrometry revealed rapid back exchange of the terminal (methyl) but not of the internal (methylene) deuteriums of [D10]-2,5-HD in vivo. These findings support an absolute requirement for pyrrole formation in gamma-diketone neurotoxicity.     

Activation of soluble guanylate cyclase by nitric oxide is increased in lymphocytes from both rats chronically exposed to 2,5-hexanedione and workers chronically exposed to n-hexane

Although occupational exposure to n-hexane induces neurotoxic effects in the central and peripheral nervous systems, the mechanisms of its neurotoxicity remain unclear. n-Hexane is metabolized to 2,5-hexanedione (2,5-HD), which is the neurotoxic agent and the indicator chosen for the biological monitoring of exposed workers. It has been previously reported that chronic exposure to 2,5-HD impairs the glutamate-nitric oxide-cyclic GMP pathway at the level of activation of soluble guanylate cyclase (sGC) enzyme by nitric oxide (NO), both in cultured neurons and in the cerebellum of rats in vivo. The aim of this study was to assess whether the activation of sGC by NO is also altered in lymphocytes from rats treated with 2,5-HD and/or workers chronically exposed to n-hexane. Lymphocytes were isolated from male Wistar rats treated with 2,5-HD in drinking water, and from blood samples from shoe-factory workers environmentally and chronically exposed to n-hexane. Urine samples were also collected from workers at the end of the shift in order to measure the urinary levels of 2,5-HD. Activation of sGC by NO was significantly higher (p<0.05) in lymphocytes from rats treated with 2,5-HD than in control rats. In isolated lymphocytes from exposed workers the activation of sGC by NO also increases (p<0.05) in contrast to the controls. The results presented here indicate that the activation of lymphocytes could be an indicator of the toxicity produced by being exposed to n-hexane, since the effects observed in workers chronically exposed to n-hexane are similar to those found in rats chronically treated with 2,5-HD in drinking water.     

The relative neurotoxicities of n-hexane,methyl n-butyl ketone, 2,5-hexanediol,and 2,5-hexanedione following oral or intraperitoneal administration in hens

The sensitivity of the hen to neurotoxicity produced by po and ip administration of n-hexane, methyl n-butyl ketone (MnBK), 2,5-hexanediol (2,5-HDOH), and 2,5-hexanedione (2,5-HD) was investigated. While po administration of one or two doses of these chemicals at a 21-day interval caused acute effects, it did not induce neuropathy in treated hens. Subchronic po or ip administration of n-hexane caused only weakness, which subsided after cessation of administration. By contrast, subchronic administration of the other three related compounds caused neurotoxicity characterized by ataxia, which progressed to paralysis in some hens. Severity of the neurotoxic effect was dependent on both the test compound and its route of administration of a similar dosage. Generally, ip injection caused more severe effects than po administration. Pathological examination of nervous system tissues of hens treated with the 2,5-HD, 2,5-HDOH, and MnBK showed giant paranodal axonal swelling followed by Wallerian degeneration of axons and myelin in peripheral nerve and spinal cord. Wallerian degeneration in the spinal cord was observed almost exclusively in the ventral columns of the lower spinal cord. n-Hexane failed to produce the characteristic pathological features produced by related compounds. The neurotoxic potency of these chemicals which considers onset and magnitude of clinical signs and severity of histopathologic changes was in descending order: 2,5-HD > 2,5-HDOH > MnBK > n-hexane when given by either method.     

Amelioration of ethanol-induced oxidative stress and alcoholic liver disease by in vivo RNAi targeting Cyp2e1

Alcoholic liver disease(ALD) results from continuous and heavy alcohol consumption. The current treatment strategy for ALD is based on alcohol withdrawal coupled with antioxidant drug intervention, which is a long process with poor efficacy and low patient compliance. Alcohol-induced CYP2E1 upregulation has been demonstrated as a key regulator of ALD, but CYP2E1 knockdown in humans was impractical, and pharmacological inhibition of CYP2E1 by a clinically relevant approach for treating ALD was no...     

Alternative biomarkers of n-hexane exposure: Characterization of aminoderived pyrroles and thiol-pyrrole conjugates in urine of rats exposed to 2,5-hexanedione

The identification of pyrrole derivatives in urine of rats exposed to 2,5-hexanedione (2,5-HD), was performed to select an adequate peripheral biomarker predictive of 2,5-HD neurotoxicity. Studies on molecular mechanism of 2,5-HD neurotoxicity have revealed that 2,5-hexanedione reacts with free amino groups of lysine in proteins forming primary pyrrole adducts, which may autoxidize and form pyrrole dimers, responsible for protein crosslinking in neurofilaments, or react with sulfhydryl groups of cysteine in peptides and proteins, forming secondary pyrrole adducts, which probably may inhibit the process responsible by 2,5-HD neurotoxicity. In this work, the analysis of excreted 2,5-HD and pyrrole derivatives in urine of rats i.p. treated with 3 doses of 2,5-HD (400 mg/kg bw/48 h) was performed using ESI-LC–MS/MS. Several pyrrole compounds were identified, namely dimethylpyrrole norleucine (DMPN), cysteine-pyrrole conjugate (DMPN NAC), glutathione-pyrrole conjugate (DMPN GSH) and 2,5-dimethylpyrrole (2,5-DMP). Additionally, free and total 2,5-HD, DMPN and DMPN NAC were quantified. The observed results suggest that DMPN is a sensitive and specific indicator of repeated exposure to 2,5-HD.     

Pattern of neurotoxicity of n-hexane, methyl n-butyl ketone, 2,5-hexanediol, and 2,5-hexanedione alone and in combination with O-ethyl O-4-nitrophenyl phenylphosphonothioate in hens

This investigation was designed to study the neurotoxicity produced in hens by the aliphatic hexacarbons n-hexane, methyl n-butyl ketone (MnBK), 2,5-hexanediol (2,5-HDOH), and 2,5-hexanedione (2,5-HD) following daily dermal application of each chemical alone and in combination with O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). Dermal application was carried out on the unprotected back of the neck. To assess whether the joint neurotoxic action of various chemicals is caused by the enhancement of absorption through the skin or by interaction at the molecular level, two additional experiments were performed. In the first experiment, EPN was dissolved in each of the aliphatic hydrocarbons prior to their topical application. In the second experiment, EPN was dissolved in acetone and applied at a different location from that of the aliphatic hexacarbons. Dermal application was carried out for 90 d followed by a 30-d observation period. The results show that hens treated with EPN developed severe ataxia followed by improvement during the observation period; n-hexane produced leg weakness with subsequent recovery, whereas the same dose of MnBK, 2,5-HDOH, or 2,5-HD produced clinical signs of neurotoxicity characterized by gross ataxia; concurrent dermal application of EPN with n-hexane or 2,5-HDOH at the same site or at different sites produced an additive neurotoxic action; simultaneous dermal application of EPN and MnBK at different sites resulted in an additive effect, whereas it caused potentiation when applied at the same site; and concurrent topical application of EPN and 2,5-HD produced a potentiating neurotoxic effect. While no histopathologic lesion was produced at the end of the observation period when any test chemical was applied alone, binary treatments of EPN and aliphatic hexacarbons resulted in histopathologic changes in some hens, with morphology and distribution characteristic of EPN neurotoxicity. The joint potentiating or additive action of aliphatic hexacarbons on EPN neurotoxicity was: 2,5-HD greater than MnBK greater than 2,5-HDOH greater than n-hexane. The mechanism of this joint action seems to be related both to enhancing skin absorption of EPN and/or its metabolic activation by n-hexane and its related chemicals.     

Neurotoxicity and protein binding of 2,5-hexanedione in the hen

Previous studies in this laboratory have demonstrated 2,5-dimethylpyrrole adduct formation during in vitro exposure of protein amino groups to the neurotoxic n-hexane metabolite 2,5-hexanedione (2,5-HD). The present investigation reports in vivo pyrrole adduct formation in neural and nonneural protein from 2,5-HD-treated animals. Adult, White-Leghorn hens were given daily doses of either 200 or 70 mg 2,5-HD/kg, po, for up to 55 or 135 days, respectively. Additional animals were given 70 mg/kg for 63 days and then allowed to recover for 72 more days. Protein separation by gel electrophoresis followed by staining with a pyrrole-specific reagent yielded evidence of widespread adduct formation in protein from serum, liver, kidney, brain, and purified myelin. Binding was particularly strong in serum albumin nd myelin basic protein. Quantitation of the adduct in these tissues revealed that its formation reached peak levels at 20 days in high dose and 30 days in low-dose animals. Levels subsequently declined, suggesting the presence of a clearance mechanism capable of removing altered protein during continuing 2,5-HD exposure. Protein from animals on the recovery regimen contained no detectable pyrrole adduct. Pyrrole adduct formation was also detected in neurofilament protein preparations, although protein yields were too low to allow assessment of clearance. Hens at both dosages displayed clinical signs indicative of CNS and PNS neuropathy. Histologic findings included axonal swelling and degeneration in peripheral nerve and some spinal cord nerve tracts. A hypothesis is proposed involving differential clearance of pyrrole adduct from neural vs nonneural tissue to explain the mechanism of action and target organ specificity of 2,5-hexanedione.     

Role of mouse CYP2E1 in the O-hydroxylation of p-nitrophenol: comparison of activities in hepatic microsomes from Cyp2e1(-/-) and wild-type mice.

Enzymatic activities are routinely used to identify the contribution of individual forms of cytochrome P450 in a particular biotransformation. p-Nitrophenol O-hydroxylation (PNPH) has been widely used as a measure of CYP2E1 catalytic activity. However, rat and human forms of CYP3A have also been shown to catalyze this activity. In mice, the contributions of CYP3A and CYP2E1 to PNPH activity are not known. Here we used hepatic microsomes from Cyp2e1(-/-) and wild-type mice to investigate the contributions of constitutively expressed and alcohol-induced murine CYP2E1 and CYP3A to PNPH activity. In liver microsomes from untreated mice, PNPH activity was much greater in wild-type mice compared with Cyp2e1(-/-) mice, suggesting a major role for CYP2E1 in catalyzing PNPH activity. Hepatic PNPH activities were not significantly different in microsomes from male and female mice, although the microsomes from females have dramatically higher levels of CYP3A. Treatment with a combination of ethanol and isopentanol resulted in induction of CYP3A proteins in wild-type and Cyp2e1(-/-) mice, as well as CYP2E1 protein in wild-type mice. The alcohol treatment increased PNPH activities in hepatic microsomes from wild-type mice but not from Cyp2e1(-/-) mice. Our findings suggest that in untreated and alcohol-treated mice, PNPH activity may be used as a specific probe for CYP2E1 and that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in catalyzing PNPH activity.     

神经生长因子对2,5-己二酮中毒大鼠周围神经损伤的治疗作用

目的　观察鼠源性神经生长因子 (mNGF)对2 ,5 己二酮中毒大鼠周围神经病的治疗效果。方法采用 2 ,5 己二酮 4 5 0mg·kg- 1·d- 1ig染毒 ,通过对姿势、步态、前后肢肌力的体征评分、坐骨神经运动神经传导速度 (MNCV)测定和神经病理检查 ,证实制成 2 ,5 己二酮中毒大鼠周围神经病模型 ,而后将相同周围神经体征评分的中毒大鼠 2 6只随机分为mNGF治疗组与染毒未治组进行疗效比较。结果染毒组大鼠于染毒 2 5d体征评分显示 ,中度与重度周围神经损伤者约占总数的 92 % ;坐骨神经MNCV比对照组明显减慢 ;神经病理检查发现 ,胫神经部分神经纤维轴索变性。经mNGF 30 μg·kg- 1·d- 1im治疗 2 1d后 ,治疗组大鼠的姿势、步态与前后肢肌力的恢复情况显著优于染毒未治组。结论　mNGF能显著加速 2 ,5 己二酮中毒性周围神经病的恢复 ,为临床应用mNGF治疗正己烷中毒性周围神经病提供了动物实验依据。     

Further observations on in vitro and in vivo effects of 2,5-hexanedione on glyceraldehyde-3-phosphate dehydrogenase

The in vitro and in vivo effect of aliphatic diketones has been studied on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) d,l-glyceraldehyde-3-phosphate: NAD oxidoreductase (phosphorylating EC 1.2.1.12 activity). Neurotoxic diketone, 2,5-hexanedione (2,5-HD), but not 2,4-hexanedione (2,4-HD), a non-neurotoxic diketone, inhibited GAPDH in rat brain homogenate preincubated with 25 mM diketones for 20 min. If the preincubation period was increased to 2 h, approximately 25% and 55% inhibition of GAPDH activity was observed with 1 mM and 5 mM 2,5-HD respectively. The inhibiton of GAPDH activity was also seen in sciatic nerves but not in the brain or liver homogenates of rats chronically intoxicated with 2,5-HD for 12 weeks. The inhibition of GAPDH by 2,5-HD appears to be selective, and thus confirms earlier data from this laboratory.     

Response force titration for the assessment of the neuromuscular toxicity of 2,5-hexanedione in rats

A silent, non-moving glass lever combined with strain gauges and mounted in an operant chamber measured the vertical isometric force exerted by the forepaw of rats. Every weekday the rats were subjected to a force titration schedule consisting of a sequence of discrete trials signalled by a lever light. The required force, above which a water reinforcement is delivered, was regulated by a generalized bisection algorithm. A stable force level which the rat was able to attain at 50% of the trials was quickly reached in each session by this algorithm. This technique was used to measure the neuromuscular performance decrement due to repeated 2,5-hexanedione treatment (250 and 500 mg/kg/day per os). The results were compared with fore- and hindlimb grip strength measurements on the same animals. This experiment showed that the force titration technique is able to distinguish between motivational and true force decrements, a distinction which cannot be made by the grip strength technique.     

三种茶饮料对小鼠肠道首过效应及肝脏Cyp3a、Cyp2e1的影响

目的:研究市售罐装绿茶、冰红茶及茉莉清茶饮料对小鼠肠道首过效应和肝脏细胞色素氧化酶(cytochrome P450,CYP)Cyp3a、Cyp2e1的影响。方法:SPF级小鼠随机分为6组,其中绿茶组、冰红茶组、茉莉清茶组小鼠分别自由饮用绿茶、冰红茶、茉莉清茶7 d。用血中扑热息痛(ac-etaminophen,APAP)的浓度推断肠道首过效应。采用差速离心法分离肝/肠微粒体,以Bradford法定量蛋白,紫外分光光度法定量Cyp3a、Cyp2e1及血中APAP浓度。结果:绿茶组血中APAP浓度较空白组明显升高(P<0.01),而冰红茶组与空白组相比则显著降低(P<0.01);绿茶组和冰红茶组小肠Cyp3a酶活性较空白组显著升高(P<0.05或P<0.01)。各供试物组肝脏Cyp3a酶活性较空白组明显升高(P<0.01),各供试物组肝脏Cyp2e1酶活性较空白组显著降低(P<0.01)。结论:绿茶、冰红茶能显著改变肠道首过效应(绿茶抑制肠道P-gp,诱导肠道Cyp3a;冰红茶则诱导肠道P-gp及肠道Cyp3a;而茉莉清茶对肠道首过效应无影响)。各供试物均诱导肝脏Cyp3a的酶活性,抑制肝脏Cyp2e1的酶活性。故在服用经CYP3A、CYP2E1和P-gp代谢/转运的药物期间,大量或经常饮用上述茶可能影响这些药物的临床疗效和/或不良反应。     

Phenotype of the Cyp1a1/1a2/1b1-/- triple-knockout mouse

Crossing the Cyp1a1/1a2(-/-) double-knockout mouse with the Cyp1b1(-/-) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(-/-) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(-/-) produced the same degree of marked immunosuppression as seen in the Cyp1a1(-/-) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(-/-) mice showed the same "rescued" response as that seen in the Cyp1a1/1b1(-/-) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value < or = 0.00086). Gene Ontology "classes of genes" most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and activities.     

Comparative metabolism and disposition of trichloroethylene in Cyp2e1-/-and wild-type mice.

Trichloroethylene (TCE)1 is an important environmental contaminant, a well established rodent carcinogen, and a "probable human carcinogen". Metabolism of TCE occurs primarily via cytochrome P450 (P450)-dependent oxidation. In vitro studies suggested that CYP2E1 is the principal high-affinity enzyme responsible for TCE metabolism. The objective of the present work is to more directly assess the role of CYP2E1 in the metabolism and disposition of 1,2-14C-TCE administered at 250 or 1000 mg/kg (gavage) using Cyp2e1-/-[knockout (KO)] versus wild-type (WT) mice. After dosing, animals were individually placed in glass metabolism cages that allowed the collection of expired air, urine, and feces. Exhalation of TCE-derived 14CO2 increased in a dose-dependent manner in mice of both genotypes and was significantly higher in WT versus KO mice. A significantly greater percentage of the dose was exhaled in KO versus WT mice as organic volatiles (mainly as TCE). Urinary excretion was the major route of TCE metabolism in WT mice, and the percentage of dose eliminated in urine was significantly higher at the 250 versus 1000 mg/kg dose. Furthermore, urinary excretion and CO2 exhalation significantly decreased in KO versus WT mice. Pretreatment with 1-aminobenzotriazole clearly inhibited TCE metabolism as evident from increased exhalation of parent TCE, and decreased urinary excretion and CO2 exhalation in mice of both genotypes. In conclusion, these data showed that whereas CYP2E1 plays an important role in TCE metabolism and disposition, other P450s also play a significant role and may explain earlier results showing that TCE causes lung damage in KO and WT mice.     

2，5-己二酮对大鼠神经组织神经丝含量的影响

目的初步探讨正己烷中毒性周围神经病变中2,5己二酮(HD)对大鼠大脑、脊髓、坐骨神经中神经丝(NF)亚单位含量的影响。方法Wistar雄性大鼠分3组,2个染毒组,1个对照组,每组9只,经腹腔染毒HD,剂量分别为200和400mg/kg,连续8周(每周5d),分离大脑、脊髓、坐骨神经组织,匀浆,28000g、30min离心制备各组织上清和沉淀,采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)和WesternBlotting检测低分子量NF(NFL)、中分子量NF(NFM)、高分子量NF(NFH)的相对含量。结果大脑、坐骨神经上清中未检测到NFL、大脑上清中未能检测到NFM,其他NF亚单位的含量在沉淀和上清中均明显降低,下降为对照组的5%～77%,其中以NFM和坐骨神经组织中NF含量的降低更为明显。结论HD导致大鼠各神经组织中NF含量的降低,NF含量改变与正己烷中毒性周围神经病变的发生有关。