RESUMEN
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.
Asunto(s)
Contaminantes Ocupacionales del Aire/orina , Metilenobis (cloroanilina)/análogos & derivados , Metilenobis (cloroanilina)/análisis , Extracción en Fase Sólida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Contaminantes Ocupacionales del Aire/química , Biomarcadores/química , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Humanos , Metilenobis (cloroanilina)/química , Metilenobis (cloroanilina)/metabolismo , Exposición Profesional/análisisRESUMEN
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.
Asunto(s)
Benzoquinonas/toxicidad , Carcinógenos/toxicidad , Linfocitos/patología , Metilenobis (cloroanilina)/análogos & derivados , Mutágenos/toxicidad , ADN/análisis , ADN/efectos de los fármacos , ADN/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Linfocitos/efectos de los fármacos , Metilenobis (cloroanilina)/toxicidad , Pruebas de Mutagenicidad , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Carcinógenos/toxicidad , Enfermedades Profesionales/inducido químicamente , Enfermedades Profesionales/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/metabolismo , Animales , Biomarcadores , Humanos , Metilenobis (cloroanilina)/análogos & derivados , Metilenobis (cloroanilina)/toxicidad , Ratones , Ratones Desnudos , Modelos BiológicosRESUMEN
Urine samples from workers exposed to 4,4'-methylenebis (2-chloroaniline) (MbOCA) contain a labile metabolite(s) that, on hydrolysis, yields the parent compound at concentrations two to three times those of free MbOCA. Evidence has now been obtained that the major labile metabolite is an N-glucuronide of MbOCA. The N-glucuronide of MbOCA was synthesised chemically, characterised by thermospray mass spectrometry, and found to have a pseudomolecular (M + 1) ion at m/z 443/445. MbOCA and [14C] uridine diphosphoglucuronic acid [( 14C]UDPGA) were incubated with liver microsomes from rats induced with polychlorinated biphenyls. The stoichiometry of the reaction product was about 1:1 (MbOCA:UDPGA). This product, the chemically synthesised glucuronide, and the labile urinary metabolite had identical chromatographic and hydrolytic (heat and beta-glucuronidase) properties. These studies show that the major labile conjugate of MbOCA in the urine of workers exposed to this compound is probably the mono N-glucuronide. In view of the lability of this compound and the fact that its concentration in urine is two to three times that of free MbOCA, it is essential that any strategy for the biological monitoring of exposed workers takes into account the N-glucuronide.
Asunto(s)
Compuestos de Bencidrilo/análisis , Compuestos de Bencidrilo/metabolismo , Metilenobis (cloroanilina)/análisis , Metilenobis (cloroanilina)/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Exposición a Riesgos Ambientales , Monitoreo del Ambiente/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Hidrólisis , Metilenobis (cloroanilina)/efectos adversos , Metilenobis (cloroanilina)/análogos & derivadosRESUMEN
A sensitive and specific gas chromatographic/mass spectrometric assay is described for the determination of N-acetyl 4,4'-methylene dianiline (N-acetyl MDA) and N-acetyl 4,4'-methylene-bis(2-chloroaniline) (N-acetyl MbOCA) in urine. The method is based on the solvent extraction of the compounds together with deuterium-labelled internal standards, the compounds being separated and detected by capillary gas chromatography/mass spectrometry as their pentafluoropropyl derivatives. The method has been applied to the detection of N-acetyl MbOCA and N-acetyl MDA in the urine of workers occupationally exposed to MbOCA and MDA. The results show that whilst N-acetyl MbOCA is a relatively minor urinary metabolite a significant proportion of MDA is excreted as the N-acetylated compound.
Asunto(s)
Acetanilidas/orina , Compuestos de Bencidrilo/orina , Cromatografía de Gases y Espectrometría de Masas , Metilenobis (cloroanilina)/orina , Exposición a Riesgos Ambientales , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Metilenobis (cloroanilina)/análogos & derivadosRESUMEN
The structure of the major urinary metabolite of 4,4'-methylenebis(2-chloroaniline) (MBOCA) in dogs was identified and the reactivity of the metabolite was characterized in vitro. Arylsulfatase but not beta-glucuronidase hydrolyzed the metabolite in a time- and enzyme concentration-dependent manner. Electron impact mass spectrometry following derivatization and transesterification indicated that the major metabolite was ring hydroxylated and fast atom bombardment mass spectrometry confirmed the molecular weight as a sulfate ester. Proton nuclear magnetic resonance studies indicated that the ring substitution was ortho to an amine. These analytical and enzymatic data supported the proposed structure of the major urinary metabolite of MBOCA in dogs as 5-hydroxy-3,3'-dichloro-4,4'diaminodiphenylmethane-5-sulfate. Protein and DNA binding in vitro and mutagenicity were investigated. During hydrolysis with arylsulfatase, time- and enzyme concentration-dependent protein binding and time-dependent DNA binding were observed. Mutagenicity during enzymatic hydrolysis in the presence of Salmonella typhimurium TA1538 with up to 20 micrograms metabolite/plate was negative and at 50 micrograms/plate the metabolite was cytotoxic. These results indicated that the metabolite was the sulfate conjugate of a reactive molecule. This study demonstrated that the major metabolite of MBOCA in canine urine is an orthohydroxysulfate and thus is similar to the major metabolites of benzidine, 2-naphthylamine, and 4-aminobiphenyl.