Mutation Research, 239 (1990) 133-142
133
Elsevier MUTREV 07283
Review of the genotoxicity and carcinogenicity of 4,4'-methylene-dianiline and 4,4'-methylene-bis-2-chloroaniline Charlene A. McQueen and Gary M. Williams American Health Foundation, One Dana Road, Valhalla, N Y 10595 (U.S.A.)
(Received4 December 1989) (Accepted 5 February 1990)
Keywords: 4,4'-Methylene-dianiline;4,4'-Methylene-bis-2-chloroaniline;Polycyclicaromatic amines
Summary 4,4'-Methylene-dianiline (MDA) and 4,4'-methylene-bis-2-chloroaniline (MOCA) are polycyclic aromatic amines that are currently used in industry. Both compounds have been found to be bacterial mutagens and to be positive in a number of assays for genotoxicity. In animal studies, MDA has induced thyroid and liver neoplasms while exposure to MOCA resulted in a variety of tumors including those of the liver, mammary gland and bladder. Epidemiologic proof of human carcinogenicity of both compounds is lacking; however, there is evidence that MOCA can be metabolized to mutagenic products by human tissue. In this paper, the major findings concerning the biotransformation, genotoxicity and carcinogenicity of MDA and MOCA are reviewed.
4,4'-Methylene-dianiline (MDA) and 4,4'methylene-bis-2-chloroaniline (MOCA) are structurally related polycyclic aromatic amines in which two aniline rings are linked by a methylene bridge (Fig. 1). MDA has been commercially produced for more than 50 years. It is in wide use mainly in the manufacture of polyurethane foams and resins and in the production of polyurethane for medical devices. MDA has been identified as the N-demethylation product of Michler's ketone, which is 4,4'-methylene-bis-(N, N'-dimethyl)aniline (McCarthy et al., 1982). The main source of exposure to MDA is occupational although it has been suggested that exposure could occur in patients
Correspondence: Dr. Charlene A. McQueen, American Health Foundation, One Dana Road, Valhalla, NY 10595 (U.S.A.).
due to the formation of MDA during sterilization of medical devices (Shintani and Nakamura, 1989). MOCA has been commercially available for more than 30 years, but is no longer produced in the United States. It is used as a curing agent for
4,4'-methylene dianiline (MDA)
4,4'-methylene bis-2-chloroaniline (MOCA) Fig. 1. Chemical structures.
0165-1110/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
134
Biotransformation
isocyanate-containing polymers such as polyurethane resins and in a number of other industrial processes. Exact usage of MOCA is difficult to determine because of the fact that many different curing agents are used by polyurethane manufacturers, but it has been estimated that up to 33000 workers are potentially exposed (Ward et al., 1987). Environmental contamination was reported in a Michigan town where MOCA was manufactured (Parris et al., 1980). Both compounds have structural similarities to benzidine and 4-aminobiphenyl, two compounds identified as human carcinogens (International Agency for Research on Cancer, 1971); hence the potential human effects of MDA and MOCA are of particular interest. The major findings from literature reports of the biotransformation, genotoxicity and carcinogenicity of MDA and MOCA are reviewed in this paper.
The biotransformation of polycyclic aromatic amines has been well documented (see review, Garner et al., 1984). In general, aromatic amines can undergo enzymatic ring and N-hydroxylation followed by the formation of sulfate and glucuronide conjugates. The extra-cyclic amino group of the parent aromatic amine can be acetylated and the acetylated product can be deacetylated. N-Hydroxylation is considered to be an activation process leading to the formation of reactive compounds. Liver is the major site of biotransformation although metabolism can also occur in tissues such as mammary gland and urinary bladder that are targets for development of aromatic amine induced tumors. Little has been specifically reported on the biotransformation of MDA. Clearly, acetylation of the free amino group occurs since N-acetyl-MDA
CI
.CI
H H H3COC-N-~CH--~N-COCH3 DiacetylMOCA(V)
?. 4,4'-Diamino-3,3'-dichlofobenzhydrol (VII Cl
.el
°' 5-Hydroxy-MOCA(111)
T
_@
Cl CI H2N--~CH2 --~H--cOCH3 AcetylMOCA(IV)
MOCA(I)
H2N-~CHz~HOH N-Hydrow-M0CA(11) Fig. 2. Some metabolitesof 4,4'-methylene-bis-2-chloroaniline.
135
and N, N'-diacetyl-MDA were identified as urinary excretion products in rats exposed to MDA (Tanaka et al., 1985) and N-acetyl-MDA has been detected following occupational exposure to MDA (Cocker et al., 1986b). Other products have not been reported. The biotransformation pathways for MOCA have been elucidated through in vivo and in vitro studies in a number of species. In rats exposed to MOCA, less than 1% of the parent compound was recovered unchanged in urine (Farmer et al., 1981; Groth et al., 1984; Morton et al., 1988). Both sulfate and glucuronide conjugates were present. 9 metabolites were separated by HPLC but not subsequently identified (Farmer et al., 1981). Biotransformation products of MOCA have been identified using rat-liver microsomes (Fig. 2). N-Hydroxy-MOCA (II), N-nitroso-MOCA, 5-hydroxy-MOCA (III) and oxidation products of the methylene carbon were identified (Morton et al., 1988; Chen et al., 1989). In rat, N-hydroxylation of MOCA was catalyzed by the phenobarbital inducible forms of cytochrome P450, P-450ps_ B and P-450pB.D (Butler et al., 1989). C-Hydroxylation of MOCA was catalyzed by 10 cytochromes P-450 (Butler et al., 1989). The major urinary metabolite in dogs given M O C A was 5-hydroxy-3,3'-dichloro-4,4'-diaminodiphenylmethane-5-sulfate, the sulfate conjugate of 5-hydroxy-MOCA (III) (Manis and Braselton, 1984). Incubation of MOCA with dogliver slices yielded the sulfate as well as a glucose conjugate and O- and N-glucuronides (Manis and Braselton, 1986). Recently, N-hydroxy and nitroso metabolites of MOCA have been identified using dog-liver microsomes (Chen et al., 1989). Exposure of workers to MOCA has been monitored by its urinary excretion (Linch et al., 1971; Ducos et al., 1985). The detection of acetylated metabolites (IV, V) in urine has been reported although N-acetyl-MOCA (IV) appears to be a minor metabolite (Ducos et al., 1985; Cocker et al., 1988). Human-fiver microsomes catalyzed the formation of N-hydroxy-MOCA (II), 5-hydroxyMOCA (III) and 4,4'-diamino-3,3'-dichiorobenzhydrol (VI), which is produced by hydroxylation of the methylene carbon (Morton et al., 1988; Butler et al., 1989). Homogenates of human liver also have been shown to N-acetylate MOCA
(Glowinski et al., 1978). For this reaction, catalyzed by a polymorphic N-acetyltransferase, individuals are classified as being rapid or slow acetylators (see review, Weber and Hein, 1985). An association between acetylator phenotype and susceptibility to the carcinogenicity of benzidine and 2-naphthylamine has been described; among exposed workers, more cases of urinary bladder cancer were found in slow acetylators than in rapid acetylators (Cartwright et al., 1982). Genotoxicity The genotoxicity of MDA has been investigated both in vitro and in vivo. MDA was mutagenic to Salmonella typhimurium, strains TA98 and TA100, in the presence of an $9 activating system (Table 1). Two metabolites of MDA, Nacetyl-MDA and N,N'-diacetyl-MDA were not mutagenic in these strains (Tanaka et al., 1985; Cocker et al., 1986a). DNA-strand breaks were induced in V79 cells by MDA with exogenous activation (Swenburg, 1981) and in rat liver following in vivo exposure (Parodi et al., 1981). A small but significant increase in sister-chromatid exchanges in mouse bone marrow following exposure to MDA also has been reported (Parodi et al., 1983). DNA repair in rat hepatocytes was induced by MDA (Moil et al., 1988) and Michler's ketone, the N,N'-dimethyl derivative of MDA
TABLE 1 SALMONELLA/MICROSOME 4,4'-METHYLENE DIANILINE Strain a (TA) 98
100
+ +/-
+ +
+
+
+ +b
+
+
1537
+ +
OF
Reference 1535
+ -
MUTAGENICITY
+
a With rat-liver $9. b With mouse-liver $9.
1538
+/-
Darby et al., 1978 LaVoie et al., 1979 Andersen et al., 1980 Parodi et al., 1981 Rao et al., 1982 McCarthy et al., 1982 Tanaka et al., 1985 Cocker et al., 1986a Messerly et al., 1987
136
(Williams et al., 1982) while the isocyanate of MDA, methylene diphenylisocyanate was mutagenic in TA98 and TA100 (Andersen et al., 1980). The potential of MOCA to form DNA-reactive products has been more extensively evaluated. MOCA was one of the compounds included as part of the first International Study for the Evaluation of Potential Carcinogens (de Serres and Ashby, 1981) in which a variety of assays was utilized. MOCA has induced revertants in Salmonella strains TA98 and TA100 in a number of studies (Table 2). Mutagenicity was clearly dependent upon the presence of an $9 metabolizing system. The acetylated derivatives, mono- and diacetyl-MOCA were also mutagenic, although less so than the parent compound (Cocker et al., 1986). MOCA was mutagenic to B. subtilis (Kada, 1981) whereas both positive and negative responses were
observed with E. coil (Matsushimi et al., 1981; Rosenkranz et al., 1981; Ichinotsubo et al., 1981; Gatehouse, 1981). MOCA also was mutagenic to a mammalian cell, rat fibroblasts (Kugler-Steigmeier et al., 1989) (Table 3). The induction of D N A repair by MOCA has been investigated in HeLa cells and hepatocytes isolated from several species (Table 3). D N A repair was observed in rat, mouse, hamster and rabbit hepatocytes (McQueen et al., 1981, 1983; Williams et al., 1982; Moil et al., 1988). Differences in the response of hepatocytes from these species was observed: rat = hamster > mouse > rabbit. Species differences in the formation of DNA-damaging metabolites was also seen with bacterial mutation and fluctuation tests (Cocker et al., 1985). Only $9 preparations from uninduced mouse liver or Aroclor-induced rat liver had suffi-
TABLE2 SALMONELLA/MICROSOME
MUTAGENICITYOF
4,4'-METHYLENE-BIS-2-CHLOROANILINE
Strain a ( T A ) 98 + + +
Reference 100
+ +
1535
+ -
1537
1538
_
+ +
+
-
+ +
+ +
+ +
+ +
+ +
+
+
-
+
+
+
N a g a o and Takahashi, 1981 M a c D o n a l d , 1981 Ichinotsubo et al., 1981 Garner et al., 1981 Martire et al., 1981 -
_ _
Brooks and D e a n 1981 B a k e r a n d B o n i n , 1981 Richold and Jones, 1981
Venitt and Crofton-Sleigh, 1981 Hubbard et al., 1981 d -
_
+b + +
McCarm et al., 1975 R a o et al., 1 9 8 2 Trueman, 1981 Simon and Shepard, 1981 R o w l a n d and Severn, 1981
+ + +
a With rat-liver $9. b With mouse-liver $9. ¢ Negative in T A 9 2 .
d Fluctuation test. e A c M O C A and D i A c M O C A were also positive, but less mutagenic than M O C A .
Gatehouse, 1981 a Haworth et al., 1983 Cocker et al., 1985, 1 9 8 6 e Hesbert et al., 1 9 8 5 d Messerly et al., 1 9 8 7 Kugler-Steigmeier et al., 1 9 8 9
137 TABLE 3 GENOTOXICITY OF 4,4'-METHYLENE-BIS-2-CHLOROANILINE Test
Result a
Reference
Mutagenicity in S. typhimurium Mutagenicity in E. coli
+ +
Mutagenicity in B. subtilis
+
See Table 1 Matsushima et al., 1981; Rosenkranz et al., 1981; Inchinotsubo et al., 1981 Gatehouse, 1981 Kada, 1981
+ +
Sharp and Perry, 1981 Perry and Sharp, 1981
+
McQueen et al., 1981, 1983; Williams et al., 1982 Mori et al., 1988 Martin and McDermid, 1981 Kugler-Steigmeier et al., 1989 Styles, 1981; Daniel and Dehel, 1981 Perry and Thompson, 1981
In vitro Bacteria
Yeast
Mitotic gene conversion Mitotic aneuploidy Mammalian cells
Hepatocyte primary culture/DNA repair test (rat, mouse, hamster and rabbit) Unscheduled D N A synthesis in HeLa cells Mutagenicity in rat fibroblasts Cell transformation in BHK-12 cells Sister chromatid exchange in CHO cells
+ w +
In vivo Drosophila
Wing spot mutagenicity Sex-linked recessive lethal
w w
Kugler-Steigmeier et al., 1989 Vogel et al., 1981; Donner et al., 1983
+
Salamone et al., 1981 Tsuchimoto and Matter 1981
Mouse
Bone-marrow micronucleus
+ , positive; w, weakly positive; - , negative.
cient activity to produce mutants in the plate-incorporation Ames test. However, when a fluctuation test was utilized a positive response was also observed with dog and human-liver $9. A limited number of in vivo genotoxicity studies on MOCA has been reported (Table 3). Both positive and negative results were observed in the mouse bone-marrow micronucleus test (Salamone et al., 1981; Kugler-Steigmeier et al., 1989). Binding of MOCA to D N A was demonstrated both in vivo and in vitro. Radiolabel was detected in liver D N A and hemoglobin of rats exposed to 14C MOCA (Cheever et al., 1988; Silk et al., 1989). In explant cultures of dog or human urinary bladder, incubation with 3H-MOCA resulted in concentration-dependent increases in 3H bound to D N A (Shivapurkur et al., 1987). Further characterization revealed 3 adducts in D N A isolated
from rat liver following the i.p. injection of 3HMOCA or incubation of this compound with liver slices (Silk et al., 1989). Two adducts were found when acetyl-MOCA, tritiated on the acetyl group, was used and three when ring-labelled compound was used, indicating that one adduct was deacetylated. The deacetylated adduct was the major one formed following exposure to MOCA or ringlabelled acetyl-MOCA and was characterized as N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, a monocyclic adduct. The reaction of synthetic N - O H - M O C A with D N A resulted in formation of this adduct as well as one of the other adducts formed from MOCA. The major adduct was also observed following incubation of D N A with N-hydroxy-4-arnino-3-chlorobenzyl alcohol the product resulting from cleavage of NO H - M O C A at the methylene bridge. The mecha-
138 nism for formation of this single ring adduct appears to involve N-hydroxylation; however, it is unclear whether ring cleavage occurs before or after this step. Thus, MDA and MOCA are clearly genotoxic. Both compounds, like other aromatic amines, require biotransformation to form reactive intermediates. However, it would appear there are significant differences between MOCA and aromatic amines such as benzidine. MOCA, unlike benzidine, is not activated by acetylation to form a reactive product, as shown by the fact that the major adduct was not acetylated (Silk et al., 1989). This adduct contains a single ring derived from MOCA. The methylene bridge of MOCA is cleaved during formation of this adduct by an unknown mechanism (Silk et al., 1989). Acetylated products of MOCA are less mutagenic in the Ames test (Hesbert et al., 1985; Cocker et al., 1986) and no differences were observed in the induction of DNA repair in heptocytes from either rapid or slow acetylator rabbits (McQueen et al., 1983). In contrast, the major benzidine adduct has been characterized as N-(deoxyguanosin-8-yl)-N'-acetylbenzidine (Martin et al., 1982). The two rings of benzidine are intact and the proposed pathway for benzidine activation involves formation of the monoacetyl derivative followed by N-hydroxylation (Martin et al., 1982; Kennelly et al., 1984). Cardnogenicity MDA has been evaluated by working groups of the International Agency for Research on Cancer (IARC) and judged to be carcinogenic to experimental animals inducing tumors primarily in liver and thyroid (IARC, 1974a, 1986). In mice and rats of both sexes exposed to up to 300 ppm MDA in the drinking water, thyroid and liver neoplasms were induced (NTP, 1983; Weisburger et al., 1984). MDA has been shown to have an enhancing effect on thyroid tumors in rats first exposed to Nbis(2-hydroxypropyl)nitrosamine (Hiasa et al., 1984). In contrast, the development of neoplasms in liver, kidney and bladder was inhibited by simultaneous or subsequent exposure to MDA (Fukushima et al., 1981; Ito et al., 1984; Masui et al., 1986; Tsuda et al., 1987).
Data concerning the human carcinogenicity of M D A were not available (IARC, 1986). MDA is toxic to liver and the human hepatotoxicity of M D A was first demonstrated when flour was contaminated with MDA (Koppelman et al., 1966). Toxic hepatitis has also resulted from occupational exposure (McGill and Moto, 1974; Williams et al., 1974). MOCA has been evaluated by a working group of the IARC and judged to be carcinogenic in rats and mice (IARC, 1974b). Oral administration of up to 2000 ppm MOCA in the diet induced vascular and liver neoplasms in mice (Russfield et al., 1975). In rats, MOCA has been administered with protein-deficient diets as well as diets of normal protein content. Lung, liver and mammary tumors were reported (Stula et al., 1975; Russfield et al., 1975; Kommineni et al., 1978). Dogs exposed to MOCA for up to 9 years developed urinary bladder tumors (Stula et al., 1977). Only limited information is available concerning the carcinogenicity of MOCA in humans and the lack of adequate epidemiologic studies prevented the evaluation of human carcinogenicity of this compound by IARC (1974b). No evidence of bladder cancer was observed in a study of 31 workers exposed to MOCA for periods of 6 months to 16 years (Linch et al., 1971); however, the usefulness of this study was limited by the small sample size and the fact that only 2 workers had exposures of 16 years. Recently, cystoscopic examination of 203 workers exposed to MOCA led to the discovery of 2 individuals with grade 1-2 papillary neoplasms (Ward et al., 1988). Both workers were nonsmoking males under 30 years of age. Conclusions
Many polycyclic aromatic amines have been shown to be carcinogens in animals and two of these, benzidine and 4-aminobiphenyl, have been found to be human carcinogens. MDA and MOCA are polycyclic aromatic amines. Both are genotoxic and a MOCA-derived DNA adduct has been characterized. Both compounds are also carcinogenic in several animal species. While epidemiologic proof of the human carcinogenicity of MDA and MOCA is lacking, human tissue can metabo-
139 lize M O C A to p r o d u c t s t h a t b i n d to D N A a n d a r e m u t a g e n i c . A d d i t i o n a l l y , t w o y o u n g w o r k e r s exp o s e d to M O C A w e r e d i a g n o s e d w i t h u r i n a r y bladder papillary neoplasms. These data corres p o n d to t h e p r o f i l e o f a c h e m i c a l r e p r e s e n t i n g a h u m a n c a n c e r h a z a r d ( W e i s b u r g e r , 1985; W i l l i a m s , 1987) a n d a c c o r d i n g l y , M D A a n d M O C A s h o u l d b e c o n s i d e r e d as p u t a t i v e h u m a n c a r c i n o gens. References
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coded chemicals, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 285-297. Manis, M.O., and W.E. Braselton (1984) Structure elucidation and in vitro reactivity of the major metabolite of 4,4'-methylenebis-(2-chloroaniline) (MBOCA) in canine urine, Fund. Appl. Toxicol., 4, 1000-1008. Manis, M.O., and W.E. Braselton (1986) Metabolism of 4,4'methylene-bis-(2-chloroaniline) by canine liver and kidney slices, Drug Metab. Dispo., 14, 166-174. Martin, C.N., and A.C. McDermid (1981) Testing of 42 coded compounds for their ability to induce unscheduled DNA repair synthesis in HeLa cells, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 533-537. Martin, C.N., F.A. Beland, R.W. Roth and F.F. Kadlubar (1982) Covalent binding of benzidine and N-acetylbenzidine to DNA at the C-8 atom of deoxyguanosine in vivo and in vitro, Cancer Res., 42, 2678-2696. Masui, T., H. Tsuda, K. Inoue, T. Ogiso and N. Ito (1986) Inhibitory effects of ethoxyquin, 4,4'-diaminodiphenylmethane and acetaminophen on rat hepatocarcinogenesis, Jpn. J. Cancer Res., 77, 231-237. Matire, G., G. Vricella, A.M. Perfumo and F. de Lorenzo (1981) Evaluation of the mutagenic activity of coded compounds in the Salmonella test, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 221-279. Matsushima, T., Y. Takamoto, A. Shirai, M. Sawamura and T. Sugimura (1981) Reverse mutation test on 42 coded compounds with the E. coli WP2 system, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 387-395. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. McCarthy, D.J., R.F. Struck, T. Shih, W.J. Suling, D.L. Hill and S.E. Enke (1982) Disposition and metabolism of the carcinogen reduced Michler's ketone in rats, Cancer Res., 42, 3475-3479. McGill, D.B., and J.D. Motto (1974) An industrial outbreak of toxic hepatitis due to methylenedianiline, N. Engl. J. Med., 291,278-282. McQueen, C.A., C.J. Maslansky, S.B. Crescensi and G.M. Williams (1981) The genotoxicity of 4,4'-methylenebis-2chloroaniline in rat, mouse and hamster liepatocytes, Toxicol. Appl. Pharmacol., 58, 231-235. McQueen, C.A., C.J. Maslansky and G.M. Williams (1983) Role of the acetylation polymorphism in determining susceptibility of cultured rabbit hepatocytes to DNA damage by aromatic amines, Cancer Res., 43, 3120-3123. Messerly, E.A., J.E. Fekete, D.R. Wade and J.E. Sinsheimer (1987) Structure mutagenicity relationships of benzidine analogues, Environ. Mol. Mutagen., 10, 263-274. Mori, H., N. Yoshimi, S. Sugie, H. Iwata, K. Kawai, N. Mashizu and H. Shimizu (1988) Genotoxicity of epoxy resin hardeners in the hepatocyte primary culture/DNA repair test, Mutation Res., 204, 683-688. Morton, K.C., M.S. Lee, P. Siedlik and R. Chapman (1988)
141 Metabolism of 4,4'-methylene-bis-2-chloroaniline (MOCA) by rats in vivo and the formation of N-hydroxy MOCA by rat and human liver microsomes, Carcinogenesis, 9, 731739. Nagao, M., and Y. Takahashi (1981) Mutagenic activity of 42 coded compounds in the Salmonella/microsome assay, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 302-313. National Toxicology Program (1983) Technical report 248 on the carcinogenesis bioassay of 4,4'-methylenedianiline hydrochloride in F344/N rats and B6C3F1/N mice (NIH No. 83-2504). Parodi, S., M. Taningher, P. Russo, M. Pala, M. Tamaro and C. Monti-Bragadin (1981) DNA damaging activity in vivo and bacterial mutagenicity of sixteen aromatic amines and azoderivatives, as related quantitatively to their carcinogenicity, Carcinogenesis, 2, 1317-1326. Parodi, S., A. Zunino, L. Ottaggio, M. DeFerrari and L. Santi (1983) Lack of correlation between the capability of inducing sister chromatid exchanges in vivo and carcinogenic potency for 16 aromatic amine and azo derivatives, Mutation Res., 108, 225-238. Parris, G.E., G.W. Diachenko, R.C. Entz, J.A. Poppiti, P. Lombardo, T.R. Rohrer and J.L. Hesse (1980) Waterborne methylene bis(2-chloroaniline) and 2-chloroaniline contamination around Adrian, Mich. Bull. Environ. Contam. Toxicol., 24, 497-503. Perry, J.M., and D. Sharp (1981) Induction of mitotic aneuploidy in the yeast strain D6 by 42 coded compounds, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 468-480. Rao, T.K., G.F. Dorsay, B.E. Allen and J.L. Epler (1982) Mutagenicity of 4,4'-methylenedianiline derivatives in the Salmonella histidine reversion assay, Arch. Toxicol., 49, 185-190. Richold, M., and E. Jones (1981) Mutagenic activity of 42 coded compounds in the Salmonella/microsome assay, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 314-322. Rosenkranz, H.S., J. Hyman and Z. Leifer (1981) DNA polymerase deficient assay, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 210-218. Rowland, I., and B. Severn (1981) Mutagenicity of carcinogens and noncarcinogens in the Salmonella/microsome test, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 314-322. Russfield, A., F. Homburger, E. Boger, C.G. Van Dongen, E.K. Weisburger and J.H. Weisburger (1975) The carcinogenic effect of 4,4'-methylene-bis-(2-chloroaniline) in mice and rats, Toxicol. Appl. Pharmacol., 31, 47-54. Salamone, M.F., J.A. Heddle and M. Katz (1981) Mutagenic activity of 41 compounds in the in vivo micronucleus test, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of ShortTerm Tests for Carcinogens, Elsevier, New York, pp. 686697. Sharp, D.C. and J.M. Perry (1981) Induction of mitotic gene conversion by 41 coded compounds using the yeast culture
JD1, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 491-501. Shintani, H., and A. Nakamura (1989) Analysis of a carcinogen, 4,4'-methylenedianiline, from thermosetting polyurethane during sterilization, J. Anal. Toxicol., 13, 354-357. Shivapurkar, N., T.A. Lehman, H.A.J. Schut and G.D. Stoner (1987) DNA binding of 4,4'-methylene-bis(2-chloroaniline) MOCA in explant cultures of human and dog bladder, Cancer Lett., 38, 41-48. Silk, N.A., J.O. Lay and C.N. Martin (1989) Covalent binding of 4,4'-methylene-bis-(2-chloroaniline) to rat liver DNA in vivo and of its N-hydroxylated derivative to DNA in vitro, Biochem. Pharmacol., 38, 279-287. Simmon, V.F., and G.F. Shepherd (1981) Mutagenic activity of 42 coded compounds in the Salmonella/microsome assay, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of ShortTerm Tests for Carcinogens, Elsevier, New York, pp. 333342. Stula, E.F., H. Sherman, J.A. Zapp and J.W. Clayton (1975) Experimental neoplasia in rats from oral administration of 3,3 '-dichlorobenzidine, 4,4'-methylene-bis-(2-chloroaniline and 4,4'-methylene-bis-(2-methylaniline), Toxicol. Appl. Pharmacol., 31, 159-176. Stula, E.F., J.R. Barnes, H. Sherman, C.F. Reinhardt and J.A. Zapp (1977) Urinary bladder tumors in dogs from 4,4'methylene-his-(2-chloroaniline) (MOCA), J. Environ. Pathol. Toxicol., 1, 31-50. Styles, J.A. (1981) Activity of 42 coded compounds in the BHK-21 cell transformation, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 638-646. Swenberg, J.A. (1981) Utilization of the alkaline elution assay as a short-term test for chemical carcinogens, in: H.F. Stich and R.H.C. San (Eds.), Short-Term Tests for Chemical Carcinogens, Springer, New York, pp. 48-58. Tanaka, K., T. Ino, T. Sawahata, S. Marui, H. Igaki and H. Yashima (1985) Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy resin hardeners, Mutation Res., 143, 11-15. Trneman, R.W. (1981) Activity of 42 coded compounds in the Salmonella reverse mutation test, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 343-350. Tsuchimoto, T., and B.E. Matter (1981) Activity of coded compounds in the micronucleus test, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 705-711. Tsuda, H., T. Ogiso, R. Hasegawa, K. Imaida, T. Masui and N. Ito (1987) Inhibition of neoplastic development in rat liver, kidney, oesophagus and forestomach by 4,4'-diaminodiphenylmethane administration, Carcinogenesis, 8, 719722. Venitt, S., and C. Crofton-Sleigh (1981) Mutagenicity of 42 coded compounds in the bacterial assay using Escherichia coli and Salmonella typhimurium, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, New York, pp. 351-360.
142 Vogel, E., W.G.H. Blijleven, M.J.H. Kortselius and J.A. Zijlstra (1981) Mutagenic activity of 17 coded compounds in the sex-linked recessive lethal test in Drosophila rnelanogaster, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of ShortTerm Tests for Carcinogens, Elsevier, New York, pp. 660665. Ward, E., A.B. Smith and W. Halperin (1987) 4,4'-Methylene bis-(2-chloroaniline): an unregulated carcinogen, Am. J. Indust. Med., 12, 537-549. Ward, E., W. Halperin, M. Thun, H.B. Grossman, B. Fink, L. Koss, A.M. Osorio and P. Schultz (1988) Bladder tumors in two young males occupationally exposed to MBOCA, Am. J. Ind. Med., 14, 267-272. Weber, W.W., and D.W. Hein (1985) N-Acetylation pharmacogenetics, Pharmacol. Rev., 737, 25-79. Weisburger, J.H. (1985) Definition of a carcinogen as a potential human carcinogenic risk, Jpn. J. Cancer Res., 76, 1244-1246.
Weisburger, E.K., A.S.K. Murthy, H.S. Lilja and J.C. Lamb (1984) Neoplastic response of F344 rats and B6C3F1 mice to the polymer and dyestuff intermediates 4,4'-methylene bis(N,N-dimethyl)-benzamine, 4,4'-oxydianiline and 4,4'methylenedianiline, J. Natl. Cancer Inst., 72, 1457-1463. Williams, G.M. (1987) Definition of a Human Cancer Hazard, in: Banbury Report 25: Nongenotoxic Mechanisms in Carcinogenesis, Cold Spring Harbor Laboratory, pp. 367380. Williams, G.M., M.F. Laspia and V.C. Dunkel (1982) Reliability of the hepatocte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens, Mutation Res., 97, 359-370. Williams, S.V., J.A. Bryan, J.R. Burk and F.S. Wolf (1974) Toxic hepatitis due to methylenedianiline, N. Engl. J. Med., 291, 1256.
133
Elsevier MUTREV 07283
Review of the genotoxicity and carcinogenicity of 4,4'-methylene-dianiline and 4,4'-methylene-bis-2-chloroaniline Charlene A. McQueen and Gary M. Williams American Health Foundation, One Dana Road, Valhalla, N Y 10595 (U.S.A.)
(Received4 December 1989) (Accepted 5 February 1990)
Keywords: 4,4'-Methylene-dianiline;4,4'-Methylene-bis-2-chloroaniline;Polycyclicaromatic amines
Summary 4,4'-Methylene-dianiline (MDA) and 4,4'-methylene-bis-2-chloroaniline (MOCA) are polycyclic aromatic amines that are currently used in industry. Both compounds have been found to be bacterial mutagens and to be positive in a number of assays for genotoxicity. In animal studies, MDA has induced thyroid and liver neoplasms while exposure to MOCA resulted in a variety of tumors including those of the liver, mammary gland and bladder. Epidemiologic proof of human carcinogenicity of both compounds is lacking; however, there is evidence that MOCA can be metabolized to mutagenic products by human tissue. In this paper, the major findings concerning the biotransformation, genotoxicity and carcinogenicity of MDA and MOCA are reviewed.
4,4'-Methylene-dianiline (MDA) and 4,4'methylene-bis-2-chloroaniline (MOCA) are structurally related polycyclic aromatic amines in which two aniline rings are linked by a methylene bridge (Fig. 1). MDA has been commercially produced for more than 50 years. It is in wide use mainly in the manufacture of polyurethane foams and resins and in the production of polyurethane for medical devices. MDA has been identified as the N-demethylation product of Michler's ketone, which is 4,4'-methylene-bis-(N, N'-dimethyl)aniline (McCarthy et al., 1982). The main source of exposure to MDA is occupational although it has been suggested that exposure could occur in patients
Correspondence: Dr. Charlene A. McQueen, American Health Foundation, One Dana Road, Valhalla, NY 10595 (U.S.A.).
due to the formation of MDA during sterilization of medical devices (Shintani and Nakamura, 1989). MOCA has been commercially available for more than 30 years, but is no longer produced in the United States. It is used as a curing agent for
4,4'-methylene dianiline (MDA)
4,4'-methylene bis-2-chloroaniline (MOCA) Fig. 1. Chemical structures.
0165-1110/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
134
Biotransformation
isocyanate-containing polymers such as polyurethane resins and in a number of other industrial processes. Exact usage of MOCA is difficult to determine because of the fact that many different curing agents are used by polyurethane manufacturers, but it has been estimated that up to 33000 workers are potentially exposed (Ward et al., 1987). Environmental contamination was reported in a Michigan town where MOCA was manufactured (Parris et al., 1980). Both compounds have structural similarities to benzidine and 4-aminobiphenyl, two compounds identified as human carcinogens (International Agency for Research on Cancer, 1971); hence the potential human effects of MDA and MOCA are of particular interest. The major findings from literature reports of the biotransformation, genotoxicity and carcinogenicity of MDA and MOCA are reviewed in this paper.
The biotransformation of polycyclic aromatic amines has been well documented (see review, Garner et al., 1984). In general, aromatic amines can undergo enzymatic ring and N-hydroxylation followed by the formation of sulfate and glucuronide conjugates. The extra-cyclic amino group of the parent aromatic amine can be acetylated and the acetylated product can be deacetylated. N-Hydroxylation is considered to be an activation process leading to the formation of reactive compounds. Liver is the major site of biotransformation although metabolism can also occur in tissues such as mammary gland and urinary bladder that are targets for development of aromatic amine induced tumors. Little has been specifically reported on the biotransformation of MDA. Clearly, acetylation of the free amino group occurs since N-acetyl-MDA
CI
.CI
H H H3COC-N-~CH--~N-COCH3 DiacetylMOCA(V)
?. 4,4'-Diamino-3,3'-dichlofobenzhydrol (VII Cl
.el
°' 5-Hydroxy-MOCA(111)
T
_@
Cl CI H2N--~CH2 --~H--cOCH3 AcetylMOCA(IV)
MOCA(I)
H2N-~CHz~HOH N-Hydrow-M0CA(11) Fig. 2. Some metabolitesof 4,4'-methylene-bis-2-chloroaniline.
135
and N, N'-diacetyl-MDA were identified as urinary excretion products in rats exposed to MDA (Tanaka et al., 1985) and N-acetyl-MDA has been detected following occupational exposure to MDA (Cocker et al., 1986b). Other products have not been reported. The biotransformation pathways for MOCA have been elucidated through in vivo and in vitro studies in a number of species. In rats exposed to MOCA, less than 1% of the parent compound was recovered unchanged in urine (Farmer et al., 1981; Groth et al., 1984; Morton et al., 1988). Both sulfate and glucuronide conjugates were present. 9 metabolites were separated by HPLC but not subsequently identified (Farmer et al., 1981). Biotransformation products of MOCA have been identified using rat-liver microsomes (Fig. 2). N-Hydroxy-MOCA (II), N-nitroso-MOCA, 5-hydroxy-MOCA (III) and oxidation products of the methylene carbon were identified (Morton et al., 1988; Chen et al., 1989). In rat, N-hydroxylation of MOCA was catalyzed by the phenobarbital inducible forms of cytochrome P450, P-450ps_ B and P-450pB.D (Butler et al., 1989). C-Hydroxylation of MOCA was catalyzed by 10 cytochromes P-450 (Butler et al., 1989). The major urinary metabolite in dogs given M O C A was 5-hydroxy-3,3'-dichloro-4,4'-diaminodiphenylmethane-5-sulfate, the sulfate conjugate of 5-hydroxy-MOCA (III) (Manis and Braselton, 1984). Incubation of MOCA with dogliver slices yielded the sulfate as well as a glucose conjugate and O- and N-glucuronides (Manis and Braselton, 1986). Recently, N-hydroxy and nitroso metabolites of MOCA have been identified using dog-liver microsomes (Chen et al., 1989). Exposure of workers to MOCA has been monitored by its urinary excretion (Linch et al., 1971; Ducos et al., 1985). The detection of acetylated metabolites (IV, V) in urine has been reported although N-acetyl-MOCA (IV) appears to be a minor metabolite (Ducos et al., 1985; Cocker et al., 1988). Human-fiver microsomes catalyzed the formation of N-hydroxy-MOCA (II), 5-hydroxyMOCA (III) and 4,4'-diamino-3,3'-dichiorobenzhydrol (VI), which is produced by hydroxylation of the methylene carbon (Morton et al., 1988; Butler et al., 1989). Homogenates of human liver also have been shown to N-acetylate MOCA
(Glowinski et al., 1978). For this reaction, catalyzed by a polymorphic N-acetyltransferase, individuals are classified as being rapid or slow acetylators (see review, Weber and Hein, 1985). An association between acetylator phenotype and susceptibility to the carcinogenicity of benzidine and 2-naphthylamine has been described; among exposed workers, more cases of urinary bladder cancer were found in slow acetylators than in rapid acetylators (Cartwright et al., 1982). Genotoxicity The genotoxicity of MDA has been investigated both in vitro and in vivo. MDA was mutagenic to Salmonella typhimurium, strains TA98 and TA100, in the presence of an $9 activating system (Table 1). Two metabolites of MDA, Nacetyl-MDA and N,N'-diacetyl-MDA were not mutagenic in these strains (Tanaka et al., 1985; Cocker et al., 1986a). DNA-strand breaks were induced in V79 cells by MDA with exogenous activation (Swenburg, 1981) and in rat liver following in vivo exposure (Parodi et al., 1981). A small but significant increase in sister-chromatid exchanges in mouse bone marrow following exposure to MDA also has been reported (Parodi et al., 1983). DNA repair in rat hepatocytes was induced by MDA (Moil et al., 1988) and Michler's ketone, the N,N'-dimethyl derivative of MDA
TABLE 1 SALMONELLA/MICROSOME 4,4'-METHYLENE DIANILINE Strain a (TA) 98
100
+ +/-
+ +
+
+
+ +b
+
+
1537
+ +
OF
Reference 1535
+ -
MUTAGENICITY
+
a With rat-liver $9. b With mouse-liver $9.
1538
+/-
Darby et al., 1978 LaVoie et al., 1979 Andersen et al., 1980 Parodi et al., 1981 Rao et al., 1982 McCarthy et al., 1982 Tanaka et al., 1985 Cocker et al., 1986a Messerly et al., 1987
136
(Williams et al., 1982) while the isocyanate of MDA, methylene diphenylisocyanate was mutagenic in TA98 and TA100 (Andersen et al., 1980). The potential of MOCA to form DNA-reactive products has been more extensively evaluated. MOCA was one of the compounds included as part of the first International Study for the Evaluation of Potential Carcinogens (de Serres and Ashby, 1981) in which a variety of assays was utilized. MOCA has induced revertants in Salmonella strains TA98 and TA100 in a number of studies (Table 2). Mutagenicity was clearly dependent upon the presence of an $9 metabolizing system. The acetylated derivatives, mono- and diacetyl-MOCA were also mutagenic, although less so than the parent compound (Cocker et al., 1986). MOCA was mutagenic to B. subtilis (Kada, 1981) whereas both positive and negative responses were
observed with E. coil (Matsushimi et al., 1981; Rosenkranz et al., 1981; Ichinotsubo et al., 1981; Gatehouse, 1981). MOCA also was mutagenic to a mammalian cell, rat fibroblasts (Kugler-Steigmeier et al., 1989) (Table 3). The induction of D N A repair by MOCA has been investigated in HeLa cells and hepatocytes isolated from several species (Table 3). D N A repair was observed in rat, mouse, hamster and rabbit hepatocytes (McQueen et al., 1981, 1983; Williams et al., 1982; Moil et al., 1988). Differences in the response of hepatocytes from these species was observed: rat = hamster > mouse > rabbit. Species differences in the formation of DNA-damaging metabolites was also seen with bacterial mutation and fluctuation tests (Cocker et al., 1985). Only $9 preparations from uninduced mouse liver or Aroclor-induced rat liver had suffi-
TABLE2 SALMONELLA/MICROSOME
MUTAGENICITYOF
4,4'-METHYLENE-BIS-2-CHLOROANILINE
Strain a ( T A ) 98 + + +
Reference 100
+ +
1535
+ -
1537
1538
_
+ +
+
-
+ +
+ +
+ +
+ +
+ +
+
+
-
+
+
+
N a g a o and Takahashi, 1981 M a c D o n a l d , 1981 Ichinotsubo et al., 1981 Garner et al., 1981 Martire et al., 1981 -
_ _
Brooks and D e a n 1981 B a k e r a n d B o n i n , 1981 Richold and Jones, 1981
Venitt and Crofton-Sleigh, 1981 Hubbard et al., 1981 d -
_
+b + +
McCarm et al., 1975 R a o et al., 1 9 8 2 Trueman, 1981 Simon and Shepard, 1981 R o w l a n d and Severn, 1981
+ + +
a With rat-liver $9. b With mouse-liver $9. ¢ Negative in T A 9 2 .
d Fluctuation test. e A c M O C A and D i A c M O C A were also positive, but less mutagenic than M O C A .
Gatehouse, 1981 a Haworth et al., 1983 Cocker et al., 1985, 1 9 8 6 e Hesbert et al., 1 9 8 5 d Messerly et al., 1 9 8 7 Kugler-Steigmeier et al., 1 9 8 9
137 TABLE 3 GENOTOXICITY OF 4,4'-METHYLENE-BIS-2-CHLOROANILINE Test
Result a
Reference
Mutagenicity in S. typhimurium Mutagenicity in E. coli
+ +
Mutagenicity in B. subtilis
+
See Table 1 Matsushima et al., 1981; Rosenkranz et al., 1981; Inchinotsubo et al., 1981 Gatehouse, 1981 Kada, 1981
+ +
Sharp and Perry, 1981 Perry and Sharp, 1981
+
McQueen et al., 1981, 1983; Williams et al., 1982 Mori et al., 1988 Martin and McDermid, 1981 Kugler-Steigmeier et al., 1989 Styles, 1981; Daniel and Dehel, 1981 Perry and Thompson, 1981
In vitro Bacteria
Yeast
Mitotic gene conversion Mitotic aneuploidy Mammalian cells
Hepatocyte primary culture/DNA repair test (rat, mouse, hamster and rabbit) Unscheduled D N A synthesis in HeLa cells Mutagenicity in rat fibroblasts Cell transformation in BHK-12 cells Sister chromatid exchange in CHO cells
+ w +
In vivo Drosophila
Wing spot mutagenicity Sex-linked recessive lethal
w w
Kugler-Steigmeier et al., 1989 Vogel et al., 1981; Donner et al., 1983
+
Salamone et al., 1981 Tsuchimoto and Matter 1981
Mouse
Bone-marrow micronucleus
+ , positive; w, weakly positive; - , negative.
cient activity to produce mutants in the plate-incorporation Ames test. However, when a fluctuation test was utilized a positive response was also observed with dog and human-liver $9. A limited number of in vivo genotoxicity studies on MOCA has been reported (Table 3). Both positive and negative results were observed in the mouse bone-marrow micronucleus test (Salamone et al., 1981; Kugler-Steigmeier et al., 1989). Binding of MOCA to D N A was demonstrated both in vivo and in vitro. Radiolabel was detected in liver D N A and hemoglobin of rats exposed to 14C MOCA (Cheever et al., 1988; Silk et al., 1989). In explant cultures of dog or human urinary bladder, incubation with 3H-MOCA resulted in concentration-dependent increases in 3H bound to D N A (Shivapurkur et al., 1987). Further characterization revealed 3 adducts in D N A isolated
from rat liver following the i.p. injection of 3HMOCA or incubation of this compound with liver slices (Silk et al., 1989). Two adducts were found when acetyl-MOCA, tritiated on the acetyl group, was used and three when ring-labelled compound was used, indicating that one adduct was deacetylated. The deacetylated adduct was the major one formed following exposure to MOCA or ringlabelled acetyl-MOCA and was characterized as N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, a monocyclic adduct. The reaction of synthetic N - O H - M O C A with D N A resulted in formation of this adduct as well as one of the other adducts formed from MOCA. The major adduct was also observed following incubation of D N A with N-hydroxy-4-arnino-3-chlorobenzyl alcohol the product resulting from cleavage of NO H - M O C A at the methylene bridge. The mecha-
138 nism for formation of this single ring adduct appears to involve N-hydroxylation; however, it is unclear whether ring cleavage occurs before or after this step. Thus, MDA and MOCA are clearly genotoxic. Both compounds, like other aromatic amines, require biotransformation to form reactive intermediates. However, it would appear there are significant differences between MOCA and aromatic amines such as benzidine. MOCA, unlike benzidine, is not activated by acetylation to form a reactive product, as shown by the fact that the major adduct was not acetylated (Silk et al., 1989). This adduct contains a single ring derived from MOCA. The methylene bridge of MOCA is cleaved during formation of this adduct by an unknown mechanism (Silk et al., 1989). Acetylated products of MOCA are less mutagenic in the Ames test (Hesbert et al., 1985; Cocker et al., 1986) and no differences were observed in the induction of DNA repair in heptocytes from either rapid or slow acetylator rabbits (McQueen et al., 1983). In contrast, the major benzidine adduct has been characterized as N-(deoxyguanosin-8-yl)-N'-acetylbenzidine (Martin et al., 1982). The two rings of benzidine are intact and the proposed pathway for benzidine activation involves formation of the monoacetyl derivative followed by N-hydroxylation (Martin et al., 1982; Kennelly et al., 1984). Cardnogenicity MDA has been evaluated by working groups of the International Agency for Research on Cancer (IARC) and judged to be carcinogenic to experimental animals inducing tumors primarily in liver and thyroid (IARC, 1974a, 1986). In mice and rats of both sexes exposed to up to 300 ppm MDA in the drinking water, thyroid and liver neoplasms were induced (NTP, 1983; Weisburger et al., 1984). MDA has been shown to have an enhancing effect on thyroid tumors in rats first exposed to Nbis(2-hydroxypropyl)nitrosamine (Hiasa et al., 1984). In contrast, the development of neoplasms in liver, kidney and bladder was inhibited by simultaneous or subsequent exposure to MDA (Fukushima et al., 1981; Ito et al., 1984; Masui et al., 1986; Tsuda et al., 1987).
Data concerning the human carcinogenicity of M D A were not available (IARC, 1986). MDA is toxic to liver and the human hepatotoxicity of M D A was first demonstrated when flour was contaminated with MDA (Koppelman et al., 1966). Toxic hepatitis has also resulted from occupational exposure (McGill and Moto, 1974; Williams et al., 1974). MOCA has been evaluated by a working group of the IARC and judged to be carcinogenic in rats and mice (IARC, 1974b). Oral administration of up to 2000 ppm MOCA in the diet induced vascular and liver neoplasms in mice (Russfield et al., 1975). In rats, MOCA has been administered with protein-deficient diets as well as diets of normal protein content. Lung, liver and mammary tumors were reported (Stula et al., 1975; Russfield et al., 1975; Kommineni et al., 1978). Dogs exposed to MOCA for up to 9 years developed urinary bladder tumors (Stula et al., 1977). Only limited information is available concerning the carcinogenicity of MOCA in humans and the lack of adequate epidemiologic studies prevented the evaluation of human carcinogenicity of this compound by IARC (1974b). No evidence of bladder cancer was observed in a study of 31 workers exposed to MOCA for periods of 6 months to 16 years (Linch et al., 1971); however, the usefulness of this study was limited by the small sample size and the fact that only 2 workers had exposures of 16 years. Recently, cystoscopic examination of 203 workers exposed to MOCA led to the discovery of 2 individuals with grade 1-2 papillary neoplasms (Ward et al., 1988). Both workers were nonsmoking males under 30 years of age. Conclusions
Many polycyclic aromatic amines have been shown to be carcinogens in animals and two of these, benzidine and 4-aminobiphenyl, have been found to be human carcinogens. MDA and MOCA are polycyclic aromatic amines. Both are genotoxic and a MOCA-derived DNA adduct has been characterized. Both compounds are also carcinogenic in several animal species. While epidemiologic proof of the human carcinogenicity of MDA and MOCA is lacking, human tissue can metabo-
139 lize M O C A to p r o d u c t s t h a t b i n d to D N A a n d a r e m u t a g e n i c . A d d i t i o n a l l y , t w o y o u n g w o r k e r s exp o s e d to M O C A w e r e d i a g n o s e d w i t h u r i n a r y bladder papillary neoplasms. These data corres p o n d to t h e p r o f i l e o f a c h e m i c a l r e p r e s e n t i n g a h u m a n c a n c e r h a z a r d ( W e i s b u r g e r , 1985; W i l l i a m s , 1987) a n d a c c o r d i n g l y , M D A a n d M O C A s h o u l d b e c o n s i d e r e d as p u t a t i v e h u m a n c a r c i n o gens. References
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