Evaluation of urinary cyclohexanediols and cyclohexanol as biomarkers of occupational exposure to cyclohexane.

OBJECTIVES: The aim of the present study was to comparatively evaluate the usefulness of urinary cyclohexanediols (CHdiols-U) and cyclohexanol (CHol-U) as biomarkers of occupational exposure to cyclohexane (CH). METHODS: Sixteen subjects (14 men and 2 women) were exposed to CH during proof-printing work. Personal exposure monitoring was conducted during the whole shift on the last working day of the week. The time-weighted average level of exposure to CH (CH-A) was measured using a diffusive sampler. Two urine samples were collected from each worker at different times during the same week: a baseline urine sample (before the first shift of the working week, after a 5-day holiday with no CH exposure) and an end-of-shift urine sample (after the last shift of the same working week, the same day personal exposure monitoring was conducted). CH-A, CHdiols-U and CHol-U were determined using a gas chromatograph-flame ionization detector. RESULTS: The CH-A concentrations ranged from 4.5 to 60.3 ppm, with a geometric mean (GM) of 18.1 ppm. The GMs and ranges (in parenthesis) of the creatinine (cr)-corrected end-of-shift 1,2-CHdiol-U, 1,4-CHdiol-U and CHol-U concentrations were 12.1 (4.1-36.6), 7.5 (2.4-20.1) and 0.4 (0.2-1.0) mg/g cr, respectively. Both CHdiols-U at the end of the shift were significantly correlated with CH-A (correlation coefficients for 1,2-CHdiol-U and 1,4-CHdiol-U of 0.852 and 0.847, respectively). No correlation was observed between CH-A and CHol-U. CONCLUSIONS: CHdiols-U at the end of the last shift of the working week are suitable biomarkers of occupational exposure to CH, but CHol-U is not suitable.

A method for routine analysis of urinary 4,4'-methylenebis (2-chloroaniline) by gas chromatography-electron capture detection.

OBJECTIVES: The purpose of this study was to develop a simple and cost-effective method for the determination of urinary 4,4'-methylenebis (2-chloroaniline) (MBOCA) by gas chromatography-electron capture detection (GC-ECD) for biological monitoring of occupational exposure to MBOCA. METHODS: MBOCA was prepared by liquid-liquid extraction after alkaline hydrolysis, derivatized with N-methyl-bis (trifluoroacetamide) and then analyzed using GC-ECD. The proposed method was validated in accordance with the US Food and Drug Administration guidance. RESULTS: The calibration curve showed linearity in the range 1-100 µg/l, with a correlation coefficient of >0.999. The limits of detection and quantification were 0.3 µg/l and 1 µg/l, respectively. The recovery was 94-99%. Intraday accuracy, expressed as the deviation from the nominal value, was 90.5-100.3%, and intraday precision, expressed as the relative standard deviation, was 0.3-2.4%. Interday accuracy and precision were 87.8-100.2% and 0.3-4.1%, respectively. CONCLUSIONS: The proposed method is a simple and cost-effective method suitable for routine analyses and could be useful for biological monitoring of occupational exposure to MBOCA.

Determination method for p-Phenylazoaniline and 2-methyl-4-(2-tolylazo)aniline in workplace air by high-performance liquid chromatography.

OBJECTIVES: The purpose of this research was to develop a method for the simultaneous determination of p-Phenylazoaniline (also called 4-aminoazobenzene, AAB) and 2-methyl-4-(2-tolylazo)aniline (also called o-aminoazotoluene, AAT) in workplace air for risk assessment. METHODS: The characteristics of the proposed method, such as recovery, limit of quantitation, reproducibility and storage stability of the samples were examined. RESULTS: An air sampling cassette containing two sulfuric acid-treated glass fiber filters was chosen as the sampler. The AAB and AAT were extracted from the sampler filters by methanol and then analyzed by a high-performance liquid chromatograph equipped with a photo-diode array detector. The overall recoveries from spiked samplers were 77-98 and 85-98% for AAB and AAT, respectively. The recovery after 5 days of storage in a refrigerator exceeded 96%. The overall limits of quantitation were 5.00 and 2.50 µg/sample for AAB and AAT, respectively. The relative standard deviations, which represent the overall reproducibility defined as precision, were 0.6-1.8 and 0.5-2.2% for AAB and AAT, respectively. CONCLUSIONS: The proposed method enables 4-h personal exposure monitoring of AAB and AAT at concentrations of 21 to 2,000 µg/m3 for AAB and 10 to 2,000 µg/m3 for AAT, respectively. The proposed method is useful for estimating worker exposure to AAB and AAT.

Development of an analytical method for the determination of arsenic in urine by gas chromatography-mass spectrometry for biological monitoring of exposure to inorganic arsenic.

OBJECTIVES: The purpose of this study was to develop an analytical method for the simultaneous determination of inorganic arsenic [As(III) and As(V)] and monomethylarsonic acid (MMA) in urine by gas chromatography-mass spectrometry (GC-MS) for the biological monitoring of exposure to inorganic arsenic. METHODS: Arsenic compounds (after reduction of arsenic to the trivalent state) were derivatized with 2,3-dimercapto-1-propanol and then analyzed using a GC-MS. The proposed method was validated according to the US Food and Drug Administration guidelines. The accuracy of the proposed method was confirmed by analyzing Standard Reference Material (SRM) 2669 (National Institute of Standards and Technology). RESULTS: Calibration curves showed linearity in the range 1-100 µg/l for each of the arsenic species, with correlation coefficients of >0.999. For each of the arsenic species, the limits of detection and quantification were 0.2 µg/l and 1 µg/l, respectively. The recoveries were 96-100%, 99-102% and 99-112% for As(III), As(V) and MMA, respectively. Intraday accuracy and precision were 82.7-99.8% and 0.9-7.4%, respectively. Interday accuracy and precision were 81.3-100.0% and 0.8-9.9%, respectively. The analytical values of SRM 2669 obtained by the proposed method were sufficiently accurate. CONCLUSIONS: The proposed method overcame the disadvantages of high-performance liquid chromatography with inductively coupled plasma mass spectrometry. It was a robust, selective and cost-effective method suitable for routine analyses and could be useful for the biological monitoring of occupational exposure to inorganic arsenic.

Determination method for mono- and diethanolamine in workplace air by high-performance liquid chromatography.

OBJECTIVES: The purpose of this research was to develop a simultaneous determination method for monoethanol-amine (MEA) and diethanolamine (DEA) in workplace air for risk assessment. METHODS: The characteristics of the proposed method, such as recovery, quantitation limit, reproducibility and storage stability of the samples, were examined. RESULTS: An air sampling cassette containing two sulfuric acid-treated glass fiber filters was chosen as the sampler. The MEA and DEA were extracted from the sampler filters, derivatized with 9-fluorenylmethyloxycarbonyl chloride and then analyzed by a high-performance liquid Chromatograph equipped with a fluorescence detector or photo-diode array detector. The overall recoveries from spiked samplers were 86-99 and 88-99% for MEA and DEA, respectively. The recovery after 5 days of storage in a refrigerator exceeded 95%. The overall limits of quantitation were 0.750 and 0.100 jug/sample for MEA and DEA, respectively. The relative standard deviations, which represent the overall reproducibility defined as precision, were 0.3-1.6 and 0.4-5.7% for MEA and DEA, respectively. CONCLUSIONS: The proposed method enables 4-h personal exposure monitoring of MEA and DEA at concentrations equaling 1/3,000-2 times the threshold limit value-time-weighted average (TLV-TWA: 3 ppmfor MEA, 1 mg/m(3) for DEA) adopted by the American Conference of Governmental Industrial Hygienists and also by the Japan Society for Occupational Health. The method is useful for estimating worker exposure to MEA and DEA.

Determination method for xylidines in workplace air.

OBJECTIVES: The purpose of this research was to develop a determination method for xylidines (XLDs) in workplace air for risk assessment. METHODS: The characteristics of the proposed method, such as recovery, detection limit, reproducibility, and storage stability of the samples were examined. RESULTS: An air sampler cassette containing two sulfuric acid-treated glass fiber filters was chosen as the sampler. The XLDs were extracted from the sampler filters, derivatized with heptafluorobutyric anhydride, and then analyzed by a gas chromatograph equipped with a mass spectrometer. The average recoveries of XLDs from the spiked sampler were 83-101% for personal exposure monitoring. The recovery after 5 days of storage in a refrigerator exceeded 90%. The overall limit of quantitation (LOQ) was 0.600 g/sample. The relative standard deviation, which represents the overall reproducibility defined as precision, was 0.8-10.3%. CONCLUSIONS: The proposed method enables 4-hour personal exposure monitoring of XLDs at concentrations equaling 0.001-2 times the threshold limit value-time-weighted average (TLV-TWA: 0.5 ppm) adopted by the American Conference of Governmental Industrial Hygienists, and is useful for estimating worker exposure to XLDs.

Use of a holder-vacuum tube device to save on-site hands in preparing urine samples for head-space gas-chromatography, and its application to determine the time allowance for sample sealing.

To facilitate urine sample preparation prior to head-space gas-chromatographic (HS-GC) analysis. Urine samples containing one of the five solvents (acetone, methanol, methyl ethyl ketone, methyl isobutyl ketone and toluene) at the levels of biological exposure limits were aspirated into a vacuum tube via holder, a device commercially available for venous blood collection (the vacuum tube method). The urine sample, 5 ml, was quantitatively transferred to a 20-ml head-space vial prior to HS-GC analysis. The loaded tubes were stored at +4 ℃ in dark for up to 3 d. The vacuum tube method facilitated on-site procedures of urine sample preparation for HS-GC with no significant loss of solvents in the sample and no need of skilled hands, whereas on-site sample preparation time was significantly reduced. Furthermore, no loss of solvents was detected during the 3-d storage, irrespective of hydrophilic (acetone) or lipophilic solvent (toluene). In a pilot application, high performance of the vacuum tube method in sealing a sample in an air-tight space succeeded to confirm that no solvent will be lost when sealing is completed within 5 min after urine voiding, and that the allowance time is as long as 30 min in case of toluene in urine. The use of the holder-vacuum tube device not only saves hands for transfer of the sample to air-tight space, but facilitates sample storage prior to HS-GC analysis.

Determination method for nitromethane in workplace air.

OBJECTIVES: The purpose of this research was to develop a determination method for nitromethane (NM) in workplace air for risk assessment. METHODS: A suitable sampler and appropriate desorption condition were selected by a recovery test in which a spiked sampler was used. The characteristics of the proposed method, such as recovery, detection limit, and reproducibility, and the storage stability of the sample were examined. RESULTS: A sampling tube containing bead-shaped activated carbon was chosen as the sampler. NM in the sampler was desorbed with acetone and analyzed by a gas chromatograph equipped with a flame ionization detector. The recoveries of NM from the spiked sampler were 81-97% and 80-98% for personal exposure monitoring and working environment measurement, respectively. On the first day of storage in a refrigerator, the recovery from the spiked samplers exceeded 90%; however, it decreased dramatically with increasing storage time. In particular, the decrease was more remarkable for the smaller spiked amounts. The overall LOQ was 2 microg/sample. The relative standard deviation, which represents the overall reproducibility, was 1.1-4.0%. CONCLUSIONS: The proposed method enables 4-hour personal exposure monitoring of NM at concentrations equaling 0.001-2 times the threshold limit value-time-weighted average (TLV-TWA: 20 ppm) proposed by the American Conference of Governmental Industrial Hygienists, as well as 10-minute working environment measurement at concentrations equaling 0.02-2 times TLV-TWA. Thus, the proposed method will be useful for estimating worker exposure to NM.

[Dietary fatty acid intake and concentrations in serum and erythrocyte membranes of students with allergic disease].

PURPOSE: An attempt was made to utilize accumulated scientific evidence for nutritional guidance concerning dietary fatty acids (FAs) for students with allergic diseases METHODS: A questionnaire survey on dietary fatty acid intake was conducted with 128 women students aged 19-20. In addition to hematological and physical examinations, fatty acid analyses were performed with serum and erythrocyte membrane samples using gas chromatography. Based on the answers to questions about allergic diseases, the subjects were divided into three groups (59 healthy students, 45 with previous experience of allergies, and 24 with allergies). We then investigated the influences of dietary fatty acids and other nutritional components on fatty acid compositions of serum and erythrocyte membranes, and statistically analyzed the results by comparing the three groups. RESULTS: 1) Dietary n-3 (g) intake by all students was lower than the tentative dietary goal in the Dietary Reference Intakes. However, there were no effects of allergic diseases on physical measurements and blood test data. Dietary n-3 (g) was negatively correlated with erythrocyte membrane n-3 (%), and dietary n-3 (%) was positively correlated with the number of acidophils (%). In addition, a positive correlation was found between serum n-3 (%) and dietary S (Saturated fatty acid) (%). 2) For the allergic group, the ratio of erythrocyte membrane M (Monounsaturated fatty asid) (%) to dietary M (%) was high. For the allergic predisposition group (allergic subjects and subjects with past history of allergic disease), a negative correlation was found between erythrocyte membrane M (%) and dietary S (%). CONCLUSION: Dietary n-3 (g) was insufficient in all subjects enrolled in this study, but erythrocyte membrane n-3 (Y%) decreased with increase of dietary n-3 (g). There was a tendency for acidophil number (%) to increase with the dietary n-3 (%). Therefore, it was suggested that if nutritional guidance is to made with attention to increasing dietary S (%), it should be stressed that serum n-3 (%) might become elevated and erythrocyte membrane M (%) depressed, especially in those with a predisposition to allergies.

Different cytoprotective effect of antioxidants and change in the iron regulatory system in rodent cells exposed to paraquat or formaldehyde.

To study the mechanism of toxicity of paraquat and formaldehyde, the response of oxidant-exposed cultured NIH3T3 cells to antioxidants or an iron chelator was investigated. Paraquat-induced cell death was reduced by treatment with 10 microM pyrrolidine dithiocarbamate (PDTC) and 10 microM desferrioxamine (DFO), but not with N-acetyl-L-cysteine (NAC). Cells were protected from formaldehyde-induced cytotoxicity by 1 mM NAC, but not by PDTC or DFO. Moreover, paraquat modulated the cellular iron regulatory system. Paraquat induced a time-dependent increase in the binding of iron regulatory protein 1 (IRP1) to iron-responsive element (IRE), and the enhanced IRP1 activity continued over 24 h. On the other hand, no induction of increased IRP1 binding to IRE was observed in rodent cells exposed to formaldehyde. Previously, we observed stimulation of EpRE-mediated ferritin mRNA expression in the cells exposed to hydrogen peroxide. However, paraquat did not induce any transcriptional activation of ferritin genes. These results suggest that intracellular iron may be involved in paraquat-mediated cytotoxicity and the influence of paraquat on iron metabolism differs from that of hydrogen peroxide.

No relationship exists between itai-itai disease and TA repeat polymorphisms of the estrogen receptor alpha gene.

Itai-itai (ouch-ouch) disease is a syndrome accompanied by bone mineral disorders that may be related to oral cadmium exposure. Itai-itai predominantly affects postmenopausal women with a history of multiple childbirth. In a previous study we have examined the genotype distributions of PvuII and XbaI restriction fragment length polymorphisms of the estrogen receptor alpha (ER alpha) gene in patients with itai-itai disease and compared them with those of controls. However, no significant differences were shown between the genotype distributions of the patients and controls. In the present study, we determined the TA repeat polymorphisms of the patients and controls. The distributions of the patients were: HH 25.0%, HL 50.0%, and LL 25.0%; where HH includes two alleles with a high number of TA repeats (TA> or =16), HL includes one high number allele and one low number allele (TA< or =15), and LL includes two alleles with a low number of TA repeats. These patients' distributions were not significantly different from those of the controls. Although our sample number was limited, we concluded that a polymorphism variant of the ER alpha gene is not a predisposing factor for itai-itai disease.

Upregulation of stress response mRNAs in COS-7 cells exposed to cadmium.

Exposure of cells to cadmium (Cd) is known to stimulate the expression of various types of genes. These changes in gene expression are presumed to be related to the cellular response to Cd toxicity. To better understand the mechanisms related to Cd toxicity, suppression subtractive hybridization was carried out on COS-7 cells (African green monkey kidney cells) and the gene expression induced by Cd exposure was investigated. Heat shock protein (hsp) 10, 40, 60, 70, 89alpha and metallothionein II (MTII) mRNAs were found to be induced by Cd. This is the first report to describe the Cd-inducibility of hsp10, 40 and 89alpha mRNAs. Semi-quantitative reverse-transcription polymerase chain reaction showed the diverse expression patterns of these genes, depending on Cd concentration and exposure time. A marked elevation of hsp70 mRNA and induction of mRNA for the co-chaperone, hsp40, were detected. A relatively low level of hsp10 and hsp60 mRNAs was induced, with only a 2-fold increase within 24 h. Hsp89alpha mRNA was induced shortly after Cd exposure. These various induction patterns suggest that hsps play different roles in the cell against Cd toxicity.

Molecular genetics of spinal muscular atrophy: contribution of the NAIP gene to clinical severity.

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders characterized by degeneration of anterior horn cells in the spinal cord, and leads to progressive muscular weakness and atrophy. At least three SMA-related genes have been identified: SMN1, NAIP and p44t. We analyzed these genes in 32 SMA patients and found that the SMN1 gene was deleted in 30 of 32 patients (94 %), irrespective of clinical type. The NAIP gene was deleted in 6 patients and its deletion rate was higher in type I patients than that in type U or V. Further, in type I patients lacking the NAIP gene, deterioration in their respiratory function is more rapid than in those type I patients retaining the NAIP gene. Since complete p44t deletion was observed in only 3 patients, the correlation between the p44t deletion and severity of SMA remained ambiguous. We concluded that the NAIP deletion was closely related to the clinical severity of SMA and was a predictive marker of SMA prognosis, while the SMN1 deletion did not correlate with clinical severity.