Biological monitoring of n-hexane exposure in shoe workers

To analyse working conditions and to provide information about the degree to which shoe workers are exposed to n-hexane, the urinary excretion of total 2,5-hexanedione (2,5-HD) was determined in 81 employees in 12 shoe factories. Twenty-five individuals who had experienced no exposure to solvents were used as controls. 2,5-HD was measured in spot urine samples collected from workers at the end of shift. In the urine of shoe workers, the 2,5-HD presented a mean value of 2.33 mg g^-1 creatinine, a median of 1.96 mg g^-1 creatinine. The mean 2,5-HD concentration in the urine samples from non-exposed subjects was 0.28 mg g^-1 creatinine, the median value was 0.18 mg g^-1 creatinine. The mean time-weighted average (TWA) concentration of n-hexane in 12 shoe workshops was 126.1 ppm, ranging from 23 to 215 ppm. We found a significant, but low, correlation (r = 0.40; p < 0.001) between TWA intensity of environmental exposure to n-hexane and the concentration of 2,5-HD in urine. The probable effect of toluene on the concentration of 2,5-HD was also discussed in the present study.     

Determination of 2,5-hexanedione,a metabolite of n-hexane,in urine: evaluation and application of three analytical methods

Three methods for the determination of 2,5-hexanedione (2,5-HD) in urine were compared in order to assess their applicability for toxicokinetic studies and biological monitoring of occupational exposure to n-hexane. Two of them were based on derivatization, followed by gas chromatography and electron-capture detection. of these two, one is a modification of the other, already published, method. The third one involves direct extraction of 2,5-HD followed by gas chromatography and flame-ionization detection. To determine 2,5-HD in urine of workers occupationally exposed to n-hexane, the most straightforward method, direct extraction of 2,5-HD from urine, has been proven to be the most suitable. However, in case of very low concentrations of 2,5-HD in urine, or analysis of small samples of blood, e.g. in kinetic studies, it is necessary to use a more sensitive procedure. The sensitivity of the methods based on the derivation of 2,5-HD followed by electron-capture detection, was, as expected, much higher in terms of analytical reliability. By using these methods, however, precautions are necessary to avoid a matrix effect.     

Microwave-assisted derivatization of 2,5-hexanedione in urine: evaluation using GC-MS and GC-ECD

2,5-Hexanedione, the main metabolite of n-hexane, can be responsible for axonal degeneration symptoms via formation of pyrrol-adducts with several amino acids. In order to make it amenable to gas chromatographic analysis, a protocol including microwave assisted derivatization is presented and compared to state-of-the-art technique of urine analysis. The applied methodology includes derivatization with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine, extraction of the oximes and final analysis using either GC-MS or GC-muECD. Furthermore, the mass spectra of derivatized 2,5-hexanedione and 5-hydroxy-2-hexanone as well as preliminary excretion kinetics are provided. Orthogonal regression methodology demonstrated superior sensitivity for the microwave heating. Limits of detection were calculated to be approximately 20 ng mL(-1) with both MS and electron capture detection, the decompositon of excess derivatizing agent using sulfuric acid, following the reaction is beneficial. A matrix effect caused by urine was not observed, a calibration in aqueous matrix ensures accurate results therefore. Microwave heating yields excellent results regarding recovery, sensitivity and the time needed for sample preparation, furthermore, it is demonstrated that both mass selective as well as electron capture detection are of comparable suitability for this task.     

Evaluation of DNA damage by the Comet assay in shoe workers exposed to toluene and other organic solvents

The alkaline single-cell gel electrophoresis (or Comet) assay was applied to evaluate DNA damage in cryopreserved peripheral blood mononuclear leukocytes from 34 female shoe workers exposed to organic solvents and a group of 19 non-exposed women. We also investigated whether the polymorphisms of glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes affect individual level of DNA damage possibly induced by the solvent exposure. Chemical measurements of workplace air in the two factories studied showed that the workers were exposed to acetone, gasoline, and toluene in both factories and to ethylacetate and diisocyanate in one factory. In the exposed workers, the average level of blood hemoglobin was lower and that of urinary hippuric acid higher than in the non-exposed individuals. However, the occupational exposure to organic solvents did not affect the Comet values. Neither did age, smoking, or the GSTM1 genotype have any effect on the outcome of this assay. The low prevalence of the GSTT1-null genotype precluded conclusions on the influence of GSTT1 polymorphism.     

Clastogenic activity in urine of workers occupationally exposed to pesticides

Genotoxicity in the urine of orchardists occupationally exposed to pesticides was investigated. Urine samples were obtained during pre-spraying and spraying periods from 22 non-smoking orchardists who spray large amounts of pesticides during the fruit growing season. For comparison purposes, urine was collected from 11 non-smoking personnel at an agricultural research station located near the application site and from 21 non-smoking individuals (reference controls) in a non-agricultural area. Organic material was isolated from urine by preparative reversed-phase high-pressure liquid chromatography, and assayed for clastogenic activity using Chinese hamster ovary cells. Urine samples collected during the pre-spraying period showed no significant differences in clastogenic activity compared to that found for the reference control group. However, clastogenic activity of urine specimens collected during the spraying period was significantly elevated (p less than 0.001) for the highly-exposed orchardists, but not for the research station personnel. Clastogenicity of orchardists' urine was observed within 8 h of pesticide application.     

Effect of 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione on retrograde axonal transport in sciatic nerve

The effects of systemically introduced neurotoxic solvents 2,5-hexanedione (2,5-HD) and 3,4-dimethyl-2,5-hexanedione (DMHD) on retrograde axonal transport (RT) of¹²⁵I-labeled tetanus toxin (TT) was studied in rat and mouse sciatic nerves. The rate of retrograde transport of TT in control rat sciatic nerves was slightly higher (6.8±0.4 mm/h) than in mouse sciatic nerves (5.4±0.5 mm/h). A single high dose of 2,5-HD (1,000 mg/kg, i.p.) produced a time-dependent effect on RT in mouse sciatic nerves. 2,5-HD caused a gradual decrease in the velocity of RT (approximately 65% inhibition between 2.0–2.5 h) with a reversal to normal rate 3–5 h after the toxin administration. The effect of DMHD on RT was examined following semi-chronic treatment in rats. DMHD caused a significant decrease (approximately 50%) in the rate of TT transport, in addition, it produced weight loss and hind-limb paralysis.I had the good opportunity of being a member of Professor Alan N. Davison' research team during 1971–1977. This research paper is dedicated to his retirement.     

Analysis of n-hexane, 2-hexanone, 2,5-hexanedione, and related chemicals by capillary gas chromatography and high-performance liquid chromatography

Analytical methods, using capillary gas chromatography and normal-phase high-performance liquid chromatography, were developed for the analysis of the neurotoxic chemicals n-hexane, 2-hexanone, and 2,5-hexanedione and their suspected metabolites. Two gas chromatographic methods, using a 50-m glass capillary OV 101 column and cyclohexane as an internal standard, were employed. In both methods, the injector and detector temperatures were 220 and 280 degrees C, respectively. In method I the following temperature program was used: isothermic at 50 degrees C for 30 min, followed by a temperature increase of 10 degrees C/min to a final temperature of 180 degrees C, which was then maintained for 7 min. This method was used to analyze the following compounds: n-hexane, 2,5-dimethylfuran, 2-hexanone, 3-hexanone, hexanal, 1-hexanol, 2-hexanol, 3-hexanol, 5-hydroxy-2-hexanone, gamma-valerolactone, 2,5-hexanedione, and 2,5-hexanediol. Method II, which was developed for n-hexane and eight of its more common metabolites, used the following temperature program: isothermic at 70 degrees C for 15 min, followed by a temperature increase of 40 degrees C/min to a final temperature of 220 degrees C, which was maintained for 5 min. A linear relationship between peak area and amount injected was observed over a 100-fold range. The minimum detectable amounts ranged from 0.05 to 1 microgram, depending on the compound. Normal-phase HPLC, using a 5-micron silica cartridge fitted into an RCM-100 radial-compression separation system, was utilized to analyze 2-hexanone and its metabolites 2,5-dimethylfuran, gamma-valerolactone, 5-hydroxy-2-hexanone, and 2,5-hexanedione.(ABSTRACT TRUNCATED AT 250 WORDS)     

A retrospective mortality study of workers exposed to radon in a Brazilian underground coal mine

Recently a high radon concentration was detected in the underground coal mine of Figueira, located in the south of Brazil. This coal mine has been operating since 1942 without taking cognizance of the high radon environment. In order to assess possible radon-related health effects on the workers, a retrospective (1979-2002) mortality study of 2,856 Brazilian coal miners was conducted, with 2,024 underground workers potentially exposed to radon daughters. Standard mortality ratio (SMR) analysis hints at lower mortality from all causes for both underground (SMR = 88, 95% CI = 78-98) and surface workers (SMR = 96, 95% CI = 80-114). A high statistically significant SMR for lung cancer mortality was observed only in the underground miners (SMR = 173, 95% CI = 102-292), with a statistically significant trend reflecting the duration of underground work. High statistically significant SMRs were observed for pneumonia as a cause of death between both surface (SMR = 304, 95% CI = 126-730) and underground miners (SMR = 253, 95% CI = 140-457). Because mortality from smoking-related cancers other than lung cancer was not found elevated in underground workers and because diesel equipments were not used in this mine, it can be concluded that the enhanced lung cancer mortality observed for underground miners is associated with exposure to radon and radon daughters, rather than other confounding risk factors.     

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR = 5.58, 95% CI = 1.32-23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.     

Association between metabolic gene polymorphisms and susceptibility to peripheral nerve damage in workers exposed to n-hexane: A preliminary study

Abstract

Chronic exposure to n-hexane may result in peripheral neuropathy. 2,5-Hexanedione (2,5-HD) has been identified as a toxic metabolite of n-hexane. The CYP2E1, CYP1A1 and GST genes are involved in the formation of 2,5-hexanedione from n-hexane as well as the elimination of 2,5-HD-formed electrophile, and these genes are highly polymorphic in the general population. A nested case-control study in an industrial cohort was conducted to evaluate the associations between polymorphisms in these metabolic genes and n-hexane-induced peripheral nerve damage. The study subjects included 22 cases, who worked in a printing factory with symptoms of peripheral nerve damage, and 163 controls, who came from the same factory of cases. DNA was extracted from blood samples and genotyping was conducted for CYP2E1 Pst, CYP2E1 Dra, CYP2E1 Ins96, CYP1A1 Msp, GSTT1 null, GSTM1 null and GSTP1 105V. Unconditional logistic regression was applied to estimate the odds ratio and 95% confidence intervals. There were no significant differences between the two groups regarding age, sex, smoking and alcohol status. A significant association between Dra polymorphism and peripheral nerve damage was found. The frequency of CYP2E1 Dra homozygous mutation in the case group (18.2%) was higher than that in the control group (3.7%, p=0.015). Individuals with homozygote genotype (CC) of CYP2E1 Dra had a significantly higher risk of peripheral nerve damage compared with those with DD genotype (adjusted OR?=?5.58, 95% CI?=?1.32–23.65) after n-hexane exposure duration, sex, age, smoking and alcohol status were adjusted. No significant association was found that CYP2E1 Pst, CYP2E1 Ins96, CYP1A1 Msp, GSTT1, GSTM1, GSTP gene polymorphisms associated with the susceptibility of peripheral nerve damage. These findings suggested that CYP2E1 gene might increase the susceptibility to n-hexane-induced peripheral damage.     

Chromosomal aberrations, SCE and urine mutagenicity in workers occupationally exposed to cytostatic drugs

The genetic risk run by workers occupationally exposed to chemicals, including the newly developed cytostatics, was assessed in large chemical laboratories. The exposed group comprising 38 people consisted of chemists, laboratory assistants and pilot plant workers. The average rate of aberrant cells in peripheral blood lymphocytes (AB.C). was 3.9% and the value of break/cell ratio was 0.046. The group of matched controls (N = 18) was found to have 1.5% of AB.C. and 0.020 breaks/cell. In the exposed group, there were, on average, 8.94 SCEs/cell, and in controls, 5.81. Urine mutagenicity tests on Salmonella typhimurium tester strain TA98 revealed a significant increase of activity in 23 (64%) out of 36 urine samples tested as compared to the control group (N = 19) with 4 (21%) positive urine samples. Strain TA100 showed only a weak response to urine mutagens.     

Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and -valerolactone were used, 2,5-hexanedione at 75 g/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of flourescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.Abbreviations 2,5-Hxdn 2,5-hexanedione - 2-Hxl 2-hexanol - 2-Hxn 2-hexanone - PMN polymorphonuclear leukocyte - TMA DPH trimethylammonium diphenylhexatriene - -V1 -valerolactone     

Purge-and-trap gas chromatographic determination of styrene in urine and blood: Application to exposed workers

A simple purge-and-trap gas chromatographic method with flame ionization detection was developed for the determination of styrene in urine and blood. Styrene present in a 5 ml sample at room temperature was swept by helium at 40 ml/min for 11 min, trapped on a Tenax trap, desorbed by heating, cryofocused, and injected by flash heating into a DB-5 capillary GC column. The oven temperature program was from 80°C, held for 8 min, to 120°C at 5°C/min, and then held for 2 min. The detector temperature was 250°C. The calibration curves were linear in the range of 2.5–15 ppb styrene in urine and 25–150 ppb in blood. The detection limits calculated were 0.4 μg/l in urine and 0.6 μg/l in blood. The coefficients of variations within the day and day-to-day were 3 and 3.1%, respectively, for 2.5 ppb of styrene in urine, and 1 and 1.6% for 25 ppb of styrene in blood. The results obtained from samples taken from workers exposed to styrene were reported.     

Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.     

Redox status of the testes and sperm of rats following exposure to 2,5-hexanedione

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied.

Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days.

Results: Administration of 0.5% 2,5-HD significantly (P?<?0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats.

Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean ±SD = 15.24 ±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients ( ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean±SD = 15.24±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients (ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.