Cancer incidence and exposure to 4,4'-methylene-bis-ortho-chloroaniline (MbOCA).

AIMS: To monitor the occurrence of cancer in a recently defined cohort of UK workers engaged in the manufacture of polyurethane elastomers using 4,4'-methylene-bis-ortho-chloroaniline. METHODS: A cohort of 308 male production workers from seven factories have been enumerated. All employees had a minimum of 12 months employment and were first employed at one of the participating factories in the period 1973-2000. Mortality and cancer incidence data for the period 1979-2007 were compared with expected values based on national rates. RESULTS: Mortality from all cancers combined was below the expected value [observed (Obs) 5, standardized mortality ratio (SMR) 68]. There was a single death from bladder cancer (SMR 560). The incidence of all cancers combined was also below expectation [Obs 9, standardized registration ratio (SRR) 77]. Site-specific incidence was unexceptional except there was a non-significant excess of bladder cancer based on two cases (SRR 328). CONCLUSIONS: The findings for bladder cancer should be treated with caution as they relate to a relatively early period of follow-up and are based on very small numbers.

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.

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity.

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10 mM), hydrazine (HZ; 0.5-4 mM), cadmium sulfate (CD; 31.2 and 62.5 µM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125 µM), isobutyl nitrite (IBN; 7.8-31.2 µM) and tetranitromethane (TNM; 1.9-15.6 µM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.

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.

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens.

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using (32)P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had (32)P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.

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.

Mutagenicity of N-OH-MOCA (4-amino-4'-hydroxylamino-bis-3,3'-dichlorodiphenylmethane) and PBQ (2-phenyl-1,4-benzoquinone) in human lymphoblastoid cells.

The genotoxic potential of two occupationally significant chemicals, 4,4'-methylene-bis-2-chloroaniline (MOCA) and 2-phenyl-1,4-benzoquinone (PBQ), was explored by monitoring the induction of mutations at the HPRT locus of AHH-1 human lymphoblastoid cells. Exposure of AHH-1 cells to the putative carcinogenic metabolite of MOCA, N-OH-MOCA, induced a 6-fold increase in mutant frequency and resulted in base pair substitutions primarily at A:T base pairs. In contrast, exposure to PBQ did not result in an increased mutant frequency although this compound was significantly more cytotoxic than N-OH-MOCA at equimolar doses. The induction of mutations at A:T sites by N-OH-MOCA is consistent with the type of DNA damage known to be produced by MOCA and provides a specific marker of genotoxic damage for exposed populations.

Occupational applications of a human cancer research model.

Many bladder cancers are indolent, and since there are no biomarkers to predict progression, the prognosis is problematic. Utilizing an in vitro/in vivo human uroepithelial cell (SV-HUC.PC) transformation system, we investigated several molecular events occurring along the continuum of exposure to disease outcome as potential biomarkers for occupational carcinogenesis. The model also served to generate information on the occupational carcinogenicity of N-hydroxy-4,4'-methylene bis(2-chloroaniline) [N-OH-MOCA]. Two of 14 groups of SV-HUC.PC treated with various concentrations of N-OH-MOCA formed carcinomas in athymic nude mice. Each of the biomarkers investigated demonstrated potential for interventions/prevention applications of occupational bladder cancers but will require validation and further evaluation. Those investigated displaying potential occupational utility included the induction of ornithine decarboxylase (ODC), DNA adducts, and altered proteins, as detected on HUC two-dimensional polyacrylamide gel electrophoresis protein maps.

A critical evaluation of centromeric labeling to distinguish micronuclei induced by chromosomal loss and breakage in vitro.

The in vitro micronucleus assay in conjunction with CREST-staining and fluorescence in situ hybridization (FISH) with centromere-specific DNA probes is being increasingly utilized for the detection of clastogenic and aneuploidy-inducing agents. Although potentially powerful techniques, both methods have unique characteristics that can influence sample processing and the interpretation of results. In this article, the use of the CREST and the FISH modifications of the in vitro micronucleus assay have been used to characterize the origin of the micronuclei induced by cyclophosphamide, 4,4'-methylene-bis(2-chloroaniline), 4-nitroquinoline N-oxide and ionizing radiation in metabolically competent MCL-5 cells or a derived cell line lacking metabolic activation. Using these results and our previous experiences with these techniques, a detailed comparison including the strengths and limitations of each technique as well as potential problems in performing each assay and in analyzing the data is discussed. In spite of their limitations, our results to date indicate that CREST-staining as well as FISH with centromere-specific DNA probes can be used to accurately distinguish micronuclei formed from chromosome loss from those originating from chromosome breakage and that these techniques can be valuable complements to the in vitro micronucleus assay.

Characterization of 1-hydroxypyrene as a novel marker substrate of 3-methylcholanthrene-inducible phenol UDP-glucuronosyltransferase(s).

Rats were treated with acetone, pyrazole, phenobarbital, 4,4'-methylenebis-(2-chloroaniline) (MOCA), 3-methylcholanthrene, creosote oil, or a mixture of polychlorinated biphenyls (Aroclor 1254) to study the inducibility and enzyme kinetics of UDP-glucuronosyltransferases towards 1-hydroxypyrene, which is a human metabolite and a urinary biomarker of exposure to pyrene. The rate of 1-hydroxypyrene glucuronidation was analyzed in rat liver microsomes by a fluorometric HPLC assay of the formed glucuronide. The apparent K(m) and Vmax values in untreated controls (K(m) = 0.27 mM; Vmax = 31 nmol/min./mg protein) did not differ markedly from those in rats treated with acetone, pyrazole or phenobarbital, whereas the significantly decreased K(m) and increased Vmax values of the rats treated with the carcinogenic chemicals, MOCA (0.11; 51), creosote (0.06; 137), 3-methylcholanthrene (0.07; 141) or the Aroclor-1254 polychlorinated biphenyl (PCB) mixture (0.08; 226), implicated major changes in the hepatic expression of UDP-glucuronosyltransferases. 1-Hydroxypyrene proved to be a high affinity substrate and a sensitive marker of the polycyclic aromatic hydrocarbon (PAH) metabolizing UDP-glucuronosyltransferase(s). Catalytically, the most efficient isoforms were induced in creosote, 3-methylcholanthrene and PCB-treated rats showing Vmax/K(m) ratios which were 22-27 times greater than in untreated controls. Our findings suggest the existence of a 3-methylcholanthrene type inducible and a functionally efficient low-K(m)/ high-Vmax form(s) of UDP-glucuronosyltransferase(s) that detoxify 1-hydroxypyrene and probably other polycyclic aromatic hydrocarbons as well.

In vitro effects of MOCA and dapsone on rat hepatic and splenic immune cells.

The industrial curing agent 4,4'-methylene-bis(2-chloroaniline) (MOCA) and the structurally related medicinal agent 4,4'-sulphonyldianiline (dapsone), are two commonly used aromatic amine compounds and documented animal carcinogens. In this study the effects of in vitro exposure to MOCA and dapsone (over a dose range of 1-200 microM on spleen and liver immune cell functions were investigated. MOCA exposure caused a dose-dependent inhibition of natural immune activities (i.e. natural killer, NK and natural P815 killer, NPK) and mitogen-stimulated proliferation of T- and B-lymphocytes, with 50% inhibition (IC50) of splenic-cell activities occurring at 145, 85, 21 and 31 microM, respectively. Liver NK and NPK activities were less sensitive to MOCA exposure and exhibited higher IC50's of 165 and 160 microM, respectively. Dapsone exposure slightly enhanced both T- and B-cell mitogenesis at low doses (1 microM) but decreased B-cell mitogenesis at high doses (IC50 = 130 microM). Natural tumouricidal activities were generally unaffected by dapsone, whilst natural cytotoxic (NC) activity was unaffected by both compounds. These results indicate that MOCA is generally immunotoxic in vitro, and may have the potential to enhance the carcinogenic effects of its genotoxic metabolite by inhibiting the tumour surveillance activities of the immune system. The relationship between immune effects and the carcinogenicity of dapsone remains unclear.

Neoplastic transformation and DNA-binding of 4,4'-methylenebis(2-chloroaniline) in SV40-immortalized human uroepithelial cell lines.

The tumorigenic transformation of certain occupationally significant chemicals, such as N-hydroxy-4-4'-methylenebis[2-chloroaniline] (N-OH-MOCA), N-hydroxy-ortho-toluidine (N-OH-OT), 2-phenyl-1,4-benzoquinone (PBQ) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) were tested in vitro using the well established SV40-immortalized human uroepithelial cell line SV-HUC.PC. SV-HUC cells were exposed in vitro to varying concentrations of N-OH-MOCA, N-OH-OT, N-OH-ABP and PBQ that caused approximately 25% and 75% cytotoxicity. The carcinogen treated cells were propagated in culture for about six weeks and subsequently injected subcutaneously into athymic nude mice. Two of the fourteen different groups of SV-HUC.PC treated with different concentrations of N-OH-MOCA, and one of the three groups exposed to N-OH-ABP, formed carcinomas in athymic nude mice. 32P-postlabeling analyses of DNA isolated from SV-HUC.PC after exposure to N-OH-MOCA revealed one major and one minor adduct. The major adduct has been identified as the N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorob enz yl alcohol (pdAp-ACBA) and the minor adduct as N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-amino-3-chlorot oluene (pdApACT). Furthermore, SV-HUC.PC cytosols catalyzed the binding of N-OH-MOCA to DNA, in the presence of acetyl-CoA, to yield similar adducts. The same adducts were also formed by chemical interaction of N-OH-MOCA with calf thymus DNA, suggesting that the aryl nitrenium ion may be the ultimate reactive species responsible for DNA binding. The tumorigenic activity of N-OH-MOCA in this highly relevant in vitro transformation model, coupled with the findings that SV-HUC.PC cells formed DNA-adducts in vitro and contained enzyme systems that activated N-OH-MOCA to reactive electrophilic species that bound to DNA, strongly suggest that MOCA could be a human bladder carcinogen. These findings are consistent with the International Agency for Research on Cancer's classification of MOCA as a probable human carcinogen.

Alterations of histone phosphorylation in rat spleen cells after treatment with the aromatic amine, 4,4'-methylene-bis(2-chloroaniline).

Alterations of the phosphorylation pattern of histones by the carcinogen, 4,4'-methylene-bis(2-chloroaniline) (MOCA) were investigated using rodent spleen cells. Spleen cells were isolated from Sprague-Dawley rats and treated with either 5, 10, 25, or 50 microM MOCA or acetone vehicle controls for 1, 2, 4, or 8 hours. Cells were incubated with 32P-phosphoric acid, and histones from these cells were fractionated utilizing two-dimensional polyacrylamide gel electrophoresis. Marked stimulation of histone phosphorylation was observed with the 10 microM MOCA treatment. A transient decrease in histone phosphorylation was observed at the 1 and 2 hour time points followed by a marked stimulation at 4 hours.

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.

Characterization of 4,4'-methylenebis(2-chloroaniline)--DNA adducts formed in vivo and in vitro.

4,4'-Methylenebis(2-chloroaniline) (MOCA) is a genotoxic and carcinogenic industrial chemical to which there is considerable potential human exposure. Since metabolic activation and formation of DNA adducts are believed to be important for the induction of these effects, DNA was treated in vitro with radiolabeled N-hydroxy-MOCA, the presumed proximate carcinogenic metabolite formed in vivo. Two major radioactive peaks were observed after HPLC separation of enzymatic hydrolysates. The two products were analyzed by MS and characterized as N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosin-8-yl)-4-amino-3-chlorotoluene. The same adducts were also the major adducts formed in DNA of tissues from rats treated with radiolabeled MOCA. They were eliminated from rat liver with non-linear kinetics, in agreement with observations made for other carcinogens. The selective reaction of N-hydroxy-MOCA with DNA-adenine and the formation of single arylamine ring adducts suggest a substitution mechanism involving an intermediate with strong SN1 character, aided by the negative inductive effect of the ortho-chlorine. Due to tautomer formation, the initial adduct may be inherently unstable and undergo cleavage at the 1'-carbon-methylene bond to yield the observed adducts.