Effect of CYP3A4 genetic polymorphisms on the genotoxicity of 4,4'-methylene-bis(2-chloroaniline)-exposed workers.

OBJECTIVES: We investigated the relationship between 4,4'-methylene-bis(2-chloroaniline) (MBOCA) exposure and micronucleus (MN) frequency, and how this association was affected by genetic polymorphism of the cytochrome P450 enzyme (CYP3A4). METHODS: We divided the study population into an exposed group (n=44 with total urine MBOCA ≥20 µg/g creatinine) and a control group (n=47 with total urine MBOCA <20 µg/g creatinine). Lymphocyte MN frequency (MNF) and micronucleated cell (MNC) frequency were measured by the cytokinesis-block MN assay method. MNF reported as the number of micronuclei in binucleated cells per 1000 cells, and MNC reported as the number of binucleated cells with the presence of MN per 1000 cells. CYP3A4 alleles were measured by PCR-based restriction fragment length polymorphism (PCR-RFLP). RESULTS: The mean MNF (6.11 vs 4.46 MN/1000 cells, p<0.001) and MNC (5.75 vs 4.15 MN/1000 cells, p<0.001) in the exposed workers was significantly higher than that in the controls. The CYP3A4 polymorphism A/A+A/G influenced the difference in the mean MNF (5.97 vs 4.38 MN/1000 cells, p<0.001) and MNC (5.60 vs 4.15 MN/1000 cells, p<0.001) between the MBOCA-exposed and control groups. After adjusting risk factors, the MNF level in the MBOCA-exposed workers was 0.520 MN cells/1000 cells (p<0.001) higher than the control group among the CYP3A4 A/A+A/G genotype. Similarly, the MNC level in the MBOCA-exposed workers was 0.593 MN/1000 cells (p<0.001) higher than the control group among the CYP3A4 A/A+A/G genotype. However, the difference in adjusted MNF and MNC between the exposed and control groups was not significant for the CYP3A4 polymorphism with the G/G genotype. CONCLUSIONS: We recommend that lymphocytes MNF and MNC are good indicators to evaluate MBOCA genotoxicity. Individuals with the CYP3A4 polymorphism A/A and A/G genotypes appear to be more susceptible to MBOCA genotoxicity.

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.

New approaches to extraction techniques in determination of 4,4'-methylenebis(2-chloroaniline) in air and water solutions.

Extraction techniques for 4,4'-methylenebis(2-chloroaniline) (MOCA) in air samples and water solutions were developed and compared. Classic techniques for air sampling of MOCA were enhanced by incorporating a derivatization step (3,5-dinitrobenzoyl chloride solution in toluene), thus increasing the limit of detection and limit of quantification. Sampling of MOCA from water solution was performed using novel nanoporous polymeric (polypyrrole and polythiophene) fiber coatings and solid phase microextraction. Samples were analyzed by high-performance liquid chromatography coupled with a UV detector. Using the modified method for air sampling of MOCA, we found that the limit of detection was 7.90 ng m(-3) and the limit of quantification was 23.8 ng m(-3). In contrast, the limit of detection for MOCA in water samples was 11.26 ng mL(-1) (polypyrrole) and 84.62 ng mL(-1) (polythiophene) and the limit of quantification for MOCA was from 33.78 (polypyrrole) and 253.86 ng mL(-1) (polythiophene). Correlation coefficients were 0.9997 for air and 0.8790-0.9852 for water samples, respectively. The techniques presented provide alternative methods for the determination of MOCA in air samples and in water solutions that are more sensitive, quicker and less expensive than previously established procedures.

A follow up study of occupational exposure to 4,4'-methylene-bis(2-chloroaniline) (MbOCA) and isocyanates in polyurethane manufacture in the UK.

This is a follow up survey of exposure to 4,4'-methylene-bis(2-chloroaniline) (MbOCA) and isocyanates in the UK polyurethane industry. Urine samples (n=446) were collected from 90 different workers. MbOCA levels were below the limit of detection in 170 samples and 26 were above the UK Biological Monitoring Guidance Value (BMGV) of 15 µmol MbOCA/mol creatinine. Detailed advice and guidance was given to each workplace at the end of the survey in 2008 and the 90% value reduced from 10 to 3 µmol MbOCA/mol creatinine in samples collected since. There was a positive correlation between glove contamination and urinary MbOCA and levels were dependent upon individual working practices especially how gloves were used. Of the 446 samples analysed for urinary metabolites of toluene diisocyanate 280 were below the detection limit and 126 were above the BMGV (1 µmol/mol creatinine). Of the 326 urine samples that were analysed for metabolites of methylenediphenyl diisocyanate, 270 were below the detection limit and 13 were above the BMGV for isocyanates. There was no correlation between urinary levels of isocyanates and MbOCA suggesting different routes of absorption, most likely inhalation and dermal respectively.

A new method for the determination of 2,2'-dichloro-4,4'-methylenedianiline in workplace air samples by HPLC-DAD.

A new procedure has been developed for the assay of 2,2'-dichloro-4,4'-methylenedianiline (MOCA) using high-performance liquid chromatography with diode array detector. MOCA was sampled from workplace air and derivative before determination using 3,5-dinitrobenzoyl chloride. The determination was carried out in the reverse-phase system (mobile phase: acetonitrile: water) using an Ultra C(18) column. The measurement range was 2-40 µg/m(3) for a 100 dm(3) air sample. Limit of detection: 7.9 ng/m(3) and limit of quantification: 23.8 ng/m(3).

A survey of occupational exposure to 4,4'-methylene-bis (2-chloroaniline) (MbOCA) in the UK.

OBJECTIVES: The main objective of the study was to gather information about the current controls and levels of exposure to 4,4'-methylene-bis (2-chloroaniline) (MbOCA) in a representative cross section of workplaces that use it to manufacture polyurethane elastomers. The study also aimed to investigate whether controls and guidance could be improved and to investigate exposure to isocyanates in these workplaces using biological monitoring. METHODS: An occupational hygienist and a field scientist visited the two UK suppliers and 20 out of the 25 workplaces known to be using MbOCA in the UK during 2005 and 2006. They collected air samples, surface wipes, gloves, and urine samples and made observations to assess exposure and the adequacy of controls. All samples were analysed for MbOCA and urine samples were additionally analysed for isocyanate metabolites. A statistical analysis was made of the results. RESULTS: Only 2.5% of the 80 personal inhalation exposures to MbOCA exceeded the workplace exposure limit of 5 microg m(-3) 8-h time-weighted average and 84% were below the limit of detection (LOD). Surface samples (n = 334) were collected from MbOCA users and suppliers and 60% had detectable levels of MbOCA ranging from 0.019 to 400 microg cm(-2). The highest levels were around a hopper, ovens, and the weighing and pouring areas. MbOCA was also detected in 8 of the 75 samples collected from areas not likely to be in contact with MbOCA. At the two suppliers, samples (n = 28) were collected from the outside surfaces of recently imported kegs, pallets, and the floor around kegs. Six samples had detectable levels and four of these (0.2, 0.8, 1, and 6 microg cm(-2)) were from the floor and pallets in both suppliers. The other two positive results were found on the outside rim (18 microg cm(-2)) and side (23 microg cm(-2)) of a keg at one supplier indicating contamination by the manufacturer. Urine samples (n = 79) were collected and 49% were below the LOD for MbOCA and only three samples had levels of MbOCA that exceeded the biological monitoring guidance value (BMGV) of 15 micromol mol(-1) creatinine. The highest urinary MbOCA concentrations were in samples from workers casting and moulding. The 90th percentile of the urine MbOCA results was 8.6 micromol MbOCA per mol creatinine. Urine samples were also analysed for the diamine metabolites of toluene diisocyanate and hexamethylene diisocyanate and 33% had detectable levels with 22 and 13% of results, respectively, above the BMGV for isocyanates (1 micromol isocyanate-derived diamine per mol creatinine). The maximum urinary concentration of toluene diamine and hexane diamine were 15.6 and 10.1 micromol mol(-1) creatinine, respectively. CONCLUSIONS: The survey found that the measures used to control exposure to MbOCA could be improved. Although air levels of MbOCA were generally low, there was evidence of spread of surface contamination and poor maintenance of controls such as local exhaust ventilation. A BMGV based on the 90th percentile of data from workplaces with good control would be less than the 90% value of 8.6 micromol mol(-1) creatinine found in this study and suggests that the current BMGV of 15 micromol mol(-1) creatinine is no longer acting as a stimulus to reduce exposure. The metabolites of isocyanates found in urine samples in this study could arise from inhalation exposure to isocyanates or from dermal exposure to either isocyanates or their diamine breakdown product and need further investigation.

Simultaneous analysis of urinary 4,4'-methylenebis(2-chloroaniline) and N-acetyl 4,4'-methylenebis(2-chloroaniline) using solid-phase extraction and liquid chromatography/tandem mass spectrometry.

Analysis of 4,4'-methylenebis(2-cholroaniline) (MOCA) or its metabolites in urine has been considered as the appropriate method to assess MOCA exposures through inhalation and skin absorption. MOCA and its metabolite, N-acetyl 4,4'-methylenebis(2-chloroaniline) (acetyl-MOCA), are analyzed using methods either limited by sensitivity or sample preparation. Therefore, a solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to simultaneously analyze MOCA and acetyl-MOCA in urine to serve as biomarkers for MOCA exposure. Protein was precipitated by using acetonitrile, and SPE were applied to clean up samples to eliminate the matrix effect and to improve the recovery. The limit of quantitation of this method was at 1.0 ng/mL for MOCA and 0.03 ng/mL for acetyl-MOCA (signal-to-noise (S/N) ratio = 10). Urinary MOCA and acetyl-MOCA levels in MOCA-exposed workers were analyzed and quantitated to be 191.9 +/- 373.2 (mean +/- standard deviation (SD)) and 11.79 +/- 23.8 ng/mL (N = 54) with the median values 38.6 and 1.8 ng/mL, respectively. MOCA concentrations are significantly correlated with their corresponding acetyl-MOCA levels in urine (Spearman correlation coefficient r = 0.916, p < 0.001). These results show that this method has been successfully developed and provides high-throughput potential to analyze MOCA and acetyl-MOCA to serve as exposure biomarkers for future study of the potential health effects associated with MOCA exposures.

Oxidative DNA damage estimated by plasma 8-hydroxydeoxyguanosine (8-OHdG): influence of 4, 4'-methylenebis (2-chloroaniline) exposure and smoking.

Oxidative DNA damage may play an important role in the human carcinogenic process. Recently, we reported a case of bladder cancer among 4, 4'-methylenebis (2-chloroaniline) (MBOCA)-exposed workers. By measuring the plasma level of 8-hydroxydeoxyguanosine (8-OHdG), we investigated the association between oxidative DNA damage and MBOCA exposure. In addition, we examined the effects of different confounders on the plasma level of 8-OHdG. We undertook a cross-sectional survey at four MBOCA-producing factories in Taiwan (158 subjects). Plasma 8-OHdG levels and urinary MBOCA concentrations were measured by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). Personal characteristics were collected by questionnaire. The workers were classified according to their job titles as exposed (n=57) or unexposed (n=101) groups as well as classified according to urinary MBOCA levels as high urinary MBOCA (>20 microg/g creatinine) (n=45) or low urinary MBOCA (n=108) groups. Neither the MBOCA-exposed workers nor the high urinary MBOCA workers had a significant increase in the mean plasma 8-OHdG level, even after adjustment for potential confounders. Age and gender were significantly positively correlated with plasma 8-OHdG levels. Smokers among high urinary MBOCA workers also had significantly higher 8-OHdG levels than non-smokers among high urinary MBOCA workers. Our study provides evidence that smoking rather than MBOCA exposure induces elevation of plasma 8-OHdG levels among workers exposed to MBOCA, indicating that oxidative DNA damage does not play an important role in the carcinogenic processes of MBOCA.

Biomonitoring at the UK Health and Safety Laboratory.

The UK Health and Safety Laboratory (HSL) provides research and analytical support to the Health and Safety Executive, other Government Departments and employers. In the area of biomonitoring HSL conducts research studies and provides an analytical service for regular surveillance of worker exposure to hazardous substances. This paper gives brief examples of how data from such studies can be used to develop biological monitoring guidance values for isocyanates, polycyclic aromatic hydrocarbons and hexavalent chromium. In addition, a study of occupational exposure to copper chrome arsenic wood preservatives is briefly described to show how biological monitoring can be used for post-approval surveillance of a biocide.

Occupational bladder cancer in a 4,4 -methylenebis(2-chloroaniline) (MBOCA)-exposed worker.

A 52-year-old male chemical worker was admitted to the hospital with a history of paroxysmal microscopic hematuria for about 2 years and nocturia with gross hematuria about five times per night for 2 months. He was a nonsmoker and denied a history of any other bladder carcinogen exposure except for occasional pesticide application during agricultural work. Intravenous urogram imaging showed a mass occupying half of the bladder capacity. Cystoscopy revealed a mass over the left dome of the bladder. Cystoscopic biopsy revealed a grade 3 invasive transitional cell carcinoma with marked necrosis. From 1987 until hospital admission in 2001, the patient had worked in a company that produced the 4,4 -methylenebis(2-chloroaniline) (MBOCA) curing agent. He did not wear any personal protective equipment during work. Ambient air MBOCA levels in the purification process area (0.23-0.41 mg/m3) exceeded the U.S. Occupational Safety and Health Administration's permissible exposure level. Urinary MBOCA levels (267.9-15701.1 microg/g creatinine) far exceeded the California Occupational Safety and Health Administration's reference value of 100 microg/L. This patient worked in the purification process with occupational exposure to MBOCA for 14 years. According to the environmental and biologic monitoring data and latency period, and excluding other potential bladder carcinogen exposure, this worker was diagnosed as having occupational bladder cancer due to high exposure to MBOCA through inhalation or dermal absorption in the purification area. This case finding supports that MBOCA is a potential human carcinogen. Safe use of skin-protective equipment and respirators is required to prevent workers from MBOCA exposure.

Biological monitoring of workers exposed to 4,4'-methylene-bis-(2-orthochloroaniline) (MOCA). I. A new and easy determination of "free" and "total" MOCA in urine.

OBJECTIVE: The objective of the study was to validate a new and simple method to determine MOCA in the urine of exposed workers. METHODS: The separation, identification and quantification of urinary MOCA were performed in spiked urines by a sensitive and practical high-performance liquid chromatography (HPLC) method and applied to urine samples of 11 workers occupationally exposed to MOCA; the postshift urinary levels of MOCA in their urine samples with and without hydrolysis, "total" and "free" MOCA respectively, were determined. In addition, we investigated the use of citric or sulfamic acid as preservatives of urine samples. RESULTS: The "total" and "free" MOCA were extracted with isooctane from hydrolysed and nonhydrolysed 20-ml urine samples respectively. After evaporation, the residue was dissolved in 4 ml of 2.10(-2) M aqueous hydrochloric acid and analysed by an isocratic HPLC system using both ultraviolet (UV) detection at 244 nm and electrochemical detection working in oxidation mode (0.9 V) with an Ag/AgCl reference electrode. Mobile phase (50% acetonitrile in water containing 0.4% acetate buffer solution pH = 4.6) was used to complete the 20-min analysis. "Free" and "total" MOCA were chromatographed on a reversed-phase C8 column (5 microns, 250 mm x 4 mm). The standard curve of MOCA was linear over the range 5-500 micrograms/l in human urine. The detection limit was 1 microgram/l for a 20-microliter injection volume; the repeatability ranged from 5.6 to 1.3% (n = 6) for spiked urines at 5 and 500 micrograms/l, with a percentage recovery of 94 +/- 3%. The reproducibility of the method was 7.3% (n = 4) for spiked urine at 10 micrograms/l. The use of sulfamic acid as a preservative of urine samples is important to improve the precision and accuracy of the analysis. CONCLUSION: The results indicate that these analytical procedures using conventional apparatus may be used routinely and reliably with large numbers of urine samples for biological monitoring of the exposure to MOCA. The occupational exposure to MOCA in some factories in France is studied in the second part of this work.

Biological monitoring of workers exposed to 4,4'-methylene-bis-(2-orthochloroaniline) (MOCA). II. Comparative interest of "free" and "total" MOCA in the urine of exposed workers.

OBJECTIVES: The objectives of the study were to investigate the best urinary marker of exposure to MOCA in urine samples of exposed workers and to study its applicability in exposure evaluation in polyurethane resin production plants. In addition, our intention was to contribute to the question of biological target value in order to reduce exposure levels. METHODS: Urinary MOCA markers were measured in urine samples collected at the end of the workshifts. 40 workers from four factories were observed for three consecutive days in the same week. "Free" MOCA in non-acid-stabilized urines, "total" MOCA in urines after alkaline hydrolysis, "acid-labile" MOCA in sulfamic or citric acid-protected urines were measured in all urine samples. MOCA liberated by heating the non-acid-stabilized urines ("heat-labile" MOCA) was also measured in 17 urine samples of exposed workers. RESULTS: The median value of "free" MOCA was lower than that of "sulfamic acid-labile" MOCA: 6.0 and 12.5 micrograms/l respectively. The difference between "free" and "total" MOCA was statistically significant (Mann-Whitney test, P = 0.023) for the urine samples collected without acid. Correlations between urinary MOCA concentrations in hydrolysed and non-hydrolysed urine samples were high (r = 0.984, 0.966 and 0.950; P < 0.001) for urine samples with sulfamic acid, with citric acid and without acid respectively. For all the factories, the postshift urinary MOCA concentrations ranged between 1 microgram/l (detection limit) and 570 micrograms/l; 25% of "sulfamic acid-labile" MOCA concentrations exceeded 50 micrograms/l. Workers handling crystallized MOCA excreted the highest amounts of MOCA in urine. The urinary MOCA concentrations (median) were: 84.0 micrograms/l (mixer), 15.5 micrograms/l (moulder). 59.0 micrograms/l (maintenance) and 3.0 micrograms/l (others). CONCLUSIONS: MOCA measured in sulfamic acid-protected urine samples without hydrolysis provides a more practical and reliable biomarker than "total" MOCA (after hydrolysis) or "free" MOCA. A biological guiding value of 20 micrograms/l expressed as "sulfamic acid-labile" MOCA is proposed.

Micronuclei in peripheral lymphocytes and exfoliated urothelial cells of workers exposed to 4,4'-methylenebis-(2-chloroaniline) (MOCA).

4,4'-Methylenebis-(2-chloroaniline) (MOCA) is used in the manufacture of polyurethane. The IARC classifies MOCA as a probable human carcinogen. Suggested changes to guidelines for health surveillance of MOCA-exposed workers in Australia include a reduction in acceptable levels of urinary MOCA to below 15 mumol/mol creatinine. Twelve male workers aged 24 and 42 years were recruited into this study from four work locations where MOCA is used. Exfoliated urothelial cells from prework urine samples on a midweek work day were assessed for micronucleus (MN) frequencies. Postwork urine samples were analysed for total MOCA. Blood samples collected on the same day were cultured for 96 h and cytochalasin-B-blocked cells were scored for MN. Eighteen male control subjects (23-59 years) provided corresponding urine and blood samples. Median urinary MOCA concentrations were 6.5 mumol/mol creatinine (range 0.4-48.6 mumol/mol creatinine) in postwork samples of MOCA-exposed workers. MOCA was not detected in urine of control workers. Mean MN frequencies were higher in urothelial cells and lymphocytes of MOCA workers (14.27 +/- 0.56 and 13.25 +/- 0.48 MN/1000 cells) than in controls (6.90 +/- 0.18 and 9.24 +/- 0.29 MN/1000 cells). The mean number of micronucleate cells was also higher in both tissues of exposed workers (9.69 +/- 0.32 and 8.54 +/- 0.14 MN cells/1000 cells) than in controls (5.18 +/- 0.11 and 5.93 +/- 0.13 MN cells/1000). There was no correlation between postwork urinary MOCA concentrations and MN frequencies in either tissue. This study suggests that exposures to MOCA in South Australia are similar to those of a decade ago and are at levels similar to those currently acceptable in Australia. These are associated with genotoxic effects in urothelial cells and peripheral blood lymphocytes. It may be prudent to reduce MOCA exposures in line with proposed guidance values.

Monitoring for occupational exposure to 4,4'-methylenebis(2-chloroaniline) by gas chromatographic-mass spectrometric analysis of haemoglobin adducts, blood, plasma and urine.

The feasibility of using plasma, blood and haemoglobin adducts for monitoring occupational exposure to the suspected human carcinogen 4,4'-methylenebis(2-chloroaniline) (MOCA) was investigated. A method utilising capillary gas chromatography-negative-ion chemical-ionisation mass spectrometry (GC-MS) for the determination of pentafluoropropionyl (PFP) derivatives of MOCA, released by alkaline hydrolysis from protein adducts and conjugates, was both sensitive and selective. When selected ion monitoring was used, sub-femtomole amounts of PFP-MOCA could be measured. The detection limit for haemoglobin adducts of MOCA was below 10 fmol/g Hb, well below the levels found for occupationally exposed individuals. Capillary GC with electron-capture detection also had the required sensitivity for the determination of MOCA in blood samples, however, the presence of interfering compounds in some samples limited its use. The levels of MOCA in the blood and urine of five individuals who were exposed to MOCA during the manufacture of polyurethane elastomers were determined by the GC-MS method. The MOCA concentrations for the various blood fractions and urine were within the following ranges: haemoglobin adducts, 0.73-43.3 pmol MOCA/g Hb; plasma alkaline hydrolysate, 0.05-22.0 nmol/l; whole blood, 0.13-17.4 nmol/l; urine, 4.5-2390 nmol/l. Because the products of MOCA in the blood reflect metabolic activation of MOCA and integrate exposure over a period of weeks, the use of blood samples for monitoring exposure to MOCA offers advantages over the currently used urinary MOCA measurements.

32P-postlabelling analysis of DNA adducts of 4,4'-methylenebis(2-chloroaniline) in target and nontarget tissues in the dog and their implications for human risk assessment.

4,4'-Methylenebis(2-chloroaniline) (MOCA) has considerable human occupational exposure and it induces urinary bladder tumors in the dog, a species that has been often used as a model for aromatic amine-induced urinary bladder carcinogenesis in humans. Metabolic activation and formation of DNA adducts are considered to be critical steps in this process; and two major C8-adenine adducts have been shown to be formed in vitro by reaction with the proximate carcinogenic metabolite N-hydroxy-MOCA. MOCA-DNA adducts have also been detected in vivo in treated rats and in exfoliated urothelium of a worker accidentally exposed to MOCA. Thus, the aim of this study was to detect and quantify DNA adducts in the urinary bladder of dogs exposed to MOCA and thereby provide data that could be useful for risk assessment after human exposure to MOCA. Beagle dogs were treated with single and multiple doses of MOCA and DNA adduct levels were determined in liver and bladder epithelium. After a single dose, adduct levels in the liver were 1.5-fold higher than that in the bladder epithelium. Adduct levels in these two organs increased 3- to 5-fold after 10 doses and adducts in the liver were then 2.8-fold higher than that in the bladder epithelium. The levels found in these two organs after single exposures were compared, per unit exposure dose, with that reported for other carcinogenic aromatic amines. The comparison showed that MOCA was as effective in DNA adduct formation as most other potent urinary bladder carcinogens. These results suggest that MOCA may have high carcinogenic potential in humans and are consistent with the recent classification of MOCA as a probable human carcinogen.

Monitoring exposure to 4,4'-methylene-bis(2-chloroaniline) through the gas chromatography-mass spectrometry measurement of adducts to hemoglobin.

4,4'-Methylene-bis(2-chloroaniline) (MOCA) is widely used as a curing agent in the plastics industry. The determination of the covalently bound reaction products to hemoglobin (Hb) has been investigated as a biomonitoring method for occupational exposure to this potential human carcinogen. Initial studies using the 14C-ring-labeled MOCA showed that 24 hr after a single IP dosage to rats (3.74 mumole/kg), 0.08% of the administered dose was adducted to the Hb, and base hydrolysis liberated 38% of the bound radioactivity. The only product released on hydrolysis was the parent diamine. A specific and sensitive assay procedure using capillary gas chromatography-mass spectrometry has been developed for determining the base-released MOCA adduct down to levels of 20 pmole/g Hb. This method has been used to establish a linear dose-response relationship in IP dosed rats between production of the adduct and dose of MOCA (3.74-44.94 mumole/kg). It is proposed to use analysis of the Hb adduct as a dosimeter for industrial workers exposed to MOCA.

4,4'-Methylene-bis(2-chloroaniline)-DNA adduct analysis in human exfoliated urothelial cells by 32P-postlabeling.

4,4'-Methylene-bis(2-chloroaniline) (MOCA) is an aromatic amine used widely in industry, and human exposure to this compound is well documented. MOCA induces lung and liver tumors in rodents and urinary bladder tumors in dogs, and it is regarded as a suspect urinary bladder carcinogen in humans. In this pilot study, we investigated the occurrence of MOCA-DNA adducts in exfoliated urothelial cells of a MOCA-exposed worker by 32P-postlabeling analysis. Urine samples were collected from the worker at various times after accidental acute exposure to MOCA. DNA isolated from exfoliated urothelial cells collected from each urine sample was enzymatically digested and postlabeled with excess [32P]ATP. Thin-layer chromatographic analysis of the labeled digests revealed the presence of a single, major DNA adduct that cochromatographed with the major N-hydroxy-MOCA-DNA adduct, N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, formed in vitro. The MOCA-DNA adduct was detected in samples obtained between 4 and 98 h after initial exposure but not in samples collected at later times. The level of DNA adducts 4 h after exposure was determined to be 516 adducts/10(8) nucleotides. A 5-fold decrease in adduct level was observed 14 h later, followed by a gradual decrease over subsequent days. The results indicate that MOCA is potentially genotoxic to human urinary bladder in vivo and that 32P-postlabeling analysis of exfoliated urothelial cells provides a noninvasive means for biomonitoring the formation of MOCA-DNA adducts resulting from occupational exposure.

Determination of 4,4'-methylenebis(2-chloroaniline) in urine using capillary gas chromatography and negative ion chemical ionization mass spectrometry.

A highly sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay for the determination of 4,4'-methylenebis(2-chloroaniline) (MBOCA) in urine is reported. It is based on the solvent extraction of the hydrolysed MBOCA conjugates, together with deuterium-labelled benzidine-d8 added as an internal standard, and a two-phase derivatization procedure involving use of pentafluoropropionic anhydride in the presence of ammonia as the phase-transfer catalyst. The reaction is complete within 2 min at room temperature. The pentafluoropropyl derivatives are determined by use of capillary column GC-MS with selected-ion monitoring in the negative ion chemical ionization mode. The lower limit of detection for MBOCA was 1 microgram dm-3 and the calibration graph showed linearity between 10 and 250 micrograms dm-3. The recovery of the analyte added to pooled urine was above 86%. Thirty urine specimens from workers employed in a polyurethane-producing plant were analysed for MBOCA by this method.