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Activation of mammalian carcinogens to bacterial mutagens by microsomal enzymes from a pelecypod mollusk, Mercenaria mercenaria
116 (1983) 247-256 Elsevier BiomedicalPress Mutation Research,
247
Activation of mammalian carcinogens to bacterial mutagens by microsomal enzymes from a pelecypod mollusk, Mercenaria mercenaria Robert S. Anderson and Jessica E. D66s Sloan - Kettering Institute for Cancer Research, Donald S. Walker Laboratory, 145 Boston Post Road, Rye, N Y 10580 (U.S.A.)
(Received25 March 1982) (Revisionreceived6 July 1982) (Accepted6 August1982)
Summary Several kinds of neoplastic diseases have been described in mollusks collected from the field. The etiology of these lesions is unknown; however, the involvement of chemical carcinogens has been suggested. Experimental models for chemically-induced neoplasia in bivalves have yet to be developed. We have obtained data which suggest that aromatic amines may be more appropriate candidates than polycyclic aromatic hydrocarbons for putative molluscan carcinogens. Digestive gland enzymes from a bivalve mollusk, M e r c e n a r i a m e r c e n a r i a , were found to be able to convert aromatic amines to frameshift mutagens as detected by the Ames Salmonella tester strains. Extensive mutagenesis was obtained with 2aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene and 4 - a m i n o - t r a n s - s t i l bene which are all mammalian procarcinogens. Mutagenic activation of benzo[a]pyrene and 3-methylcholanthrene was minimal. Low levels of aromatic amine-activating enzyme(s) were found in all tissues examined, but activity was greatest in the digestive gland. Enzymatic activity was heat-labile, NADPH-dependent, and apparently not inducible by polychlorinated biphenyls (Aroclor 1254).
It has been suggested that in pelecypod mollusks there is little or no evidence for an enzyme system analogous to the mixed-function oxygenase (MFO) system of higher animals (Vandermeulen et al., 1977; Payne, 1977; Vandermeulen and Penrose, 1978); however, by the use of isotopicaUy-labeled substrate, metabolism of benzo[a]pyrene (BaP) has been quantified in oysters C r a s s o s t r e a v i r g i n i c a (Anderson, 1978), and mussels (Stegeman, 1981). Reactions catalyzed by MFO are not only involved in detoxification of xenobiotics, such as environmental hydrocarbons, but 0165-1218/83/0000-0000/$03.00 © ElsevierBiomedicalPress
248 also in their activation to carcinogenic proximate forms. Since many marine bivalves are reported to be subject to neoplastic diseases (e.g., see reviews by Farley and Sparks, 1970; Mix, 1976; Rosenfield, 1976; Brown et al., 1979), the study of MFO in these species takes on added interest. Although in some cases a possible correlation has been made between polynuclear aromatic hydrocarbon (PAH) exposure or body burden and the incidence of cellular proliferative disorders in bivalves (Lowe and Moore, 1978; Brown et al., 1979; Mix et al., 1979), the etiology of these lesions is controversial. PAHs may also play less direct roles in the development of neoplastic disease in bivalves, such as activation (Couch and Courtney, 1977) of viruses (Oprandy et al., 1981) in hematotopoietic neoplasia in Mya arenaria. PAH-induced lysosomal membrane destabilization, which has been implicated in neoplastic transformation (Allison and Paton, 1965), has been reported in bivalves (Moore et al., 1978). The possible relationship between xenobiotic-mediated impaired immunological surveillance mechanisms in Mercenaria (Fries and Tripp, 1980; Anderson et al., 1981) and expression of neoplastic disease has yet to be defined. To date, the majority of reports on molluscan neoplastic disease has been based on samples taken from the field. Few experimental studies of chemical carcinogenesis have been made with these species. Krieg (1972) induced epithelial tumors with 20-methylcholanthrene (MC) in a gastropod mollusk. Oysters chronically exposed to MC developed lesions in the mantle, characterized by cellular infiltration, resembling the sarcoma-like lesions previously seen in field-collected specimens (Couch et al., 1979). N-Nitroso compounds were reported to induce tumors in bivalves (Khudoley and Syrenko, 1978); nitrosamines had also been suggested as the causative agents of neoplasms seen in Macoma balthica (Christensen et al., 1974). A commonly used method to measure metabolic activation of carcinogens quantifies the production of metabolites that are mutagenic for various Salmonella typhimurium tester strains (Ames et al., 1975). In this study, we have used this technique to study the activation of BaP and some carcinogenic aromatic amines by enzyme preparations from the digestive gland of Mercenaria mercenaria. Metabolic activation of aromatic amines has not previously been studied using bivalve tissues.
Materials and methods
Hard clams (Mercenaria mercenaria) were obtained from a local commercial supplier and were held at - 12°C in recirculated artificial sea water (Instant Ocean). The clams were opened on ice and the digestive glands carefully removed from the visceral mass. The glands were minced and washed with 0.05 N Tris buffer (pH 7.2) containing 0.25 M sucrose. Some animals were injected with 6 mg Aroclor 1254 in 0.1 ml olive oil ( - 240 m g / k g body weight), 96-h prior to sacrifice. Injections or hemolymph withdrawal was made via the blood sinus in the anterior adductor muscle. 9000 g homogenate supernatants ($9) were prepared from digestive gland homogenates (20% in 0.25 M sucrose in 0.05 N Tris buffer, pH 7.2). In the studies reported here fresh $9, maintained on ice, was used, although enzymatic activity could be retained after longer term storage at - 8 0 ° C .
249 The methods for mutagenicity testing were the same as those originally described by Ames et al. (1975). The Salmonella strain used (TA98)was periodically checked for histidine requirement, deep rough permeability character, and for the presence of the ampicillin-resistant R factor. Mercenaria $9 mix was prepared using the usual salts and cofactors, and usually contained 0.4 ml S9/ml. We also used MutazymeTM (Meloy Laboratories, Inc.), a lyophilized preparation of Aroclor 1254-induced rat-liver $9 mix, to enable comparison of the mutagenicity of the various substrates after metabolism by molluscan and mammalian enzymes. Clams were normally not pretreated with Aroclor because attemts to induce Mercenaria MFO were unsuccessful. The bacteria, substrate and $9 mix were combined in top agar and poured on minimal glucose agar plates, as described by Ames et al. (1975). The plates were protected from light until read to avoid photochemical reactions. The plates were incubated at 37°C for 48 h before counting his + revertants with an automated colony counter (BioTran II, New Brunswick Scientific Co., Inc.). All variations in this protocol are described as the data are presented. 2-Aminoanthracene (2AA), 4-amino-trans-stilbene (4ATS), N-acetylbenzidine (ABZ), and N, N'-diacetylbenzidine (DABZ), were obtained from ICN Pharmaceuticals, Inc. Benzidine (BZ), 2-acetylaminofluorene (2AAF), 4-aminobiphenyl (4ABP), and 3-methylcholanthrene (MC), were obtained from Sigma Chemical Co. Benzo[a]pyrene (BaP) and 2-aminofluorene (2AF) were supplied by the Aldrich Chemical Co. Aroclor 1254 was a gift of the Monsanto Co. The Salmonella tester strian was generously provided by Dr. Bruce N. Ames, University of California, Berkeley.
Results
Microsomal enzyme preparations from the digestive glands of Mercenaria were capable of producing mutagenic metabolites from several aromatic amines that are carcinogenic for vertebrates (Table 1). The data presented are from untreated clams because attempts to augment activity by standard inducers, such as Aroclor 1254, were ineffective. Mercenaria preparations were less active per mg protein than $9 derived from rat hepatic homogenates. The substrate concentrations reported in Table 1 gave responses on the linear portion of the dose-response curve for the particular enzyme preparation being tested. 5 bacterial tester strains were tested to determine the most sensitive for use in our assays. Both TA1538 and TA98 (TA1538 with R factor plasmids) were equally sensitive bacterial detector strains for aromatic amine activation, whereas TA100 was moderately sensitive, and TA 1535 and TA 1537 were not affected in the assay. Using TA98, marked activation of 2AA, 2AF, 2AAF and 4ATS by Mercenaria enzymes was seen; 4ABP, ABZ, and DABZ were slightly activated, and BZ was inactive. 2 carcinogenic PAH, BaP and MC~ were very slightly mutagenic for TA98 and TA100 in the presence of clam $9, but were strongly mutagenic in combination with rat $9 mix. . . . Several properties of the Mercenaria aromatic amine.activating enzyme system are presented in Fig. 1. Neither clam $9 in the absence of substrate, nor 2AA in the
250 TABLE 1 MUTAGENICITY OF C A R C I N O G E N I C AROMATIC AMINES AND PAH AFTER ACTIVATION BY MERCENARIA DIGESTIVE GLAND ENZYMES OR RAT $9 MIX Compound a
ktg/plate
$9 source b
His ÷
revertants/ plate c
Aromatic amines
2-Aminoanthracene (PC)
10 5
M R
626_+238 (8) 1 205 + 326 (4)
2-Aminofluorene (PC)
10 10
M R
423 ± 146 (8) 764_+ 202 (4)
100 5
M R
556_+201 (6) 343_+ 107 (4)
50 50
M R
21+ 10 (6) 330+ 94 (4)
4-Amino-trans-stilbene (PC)
100 50
M R
158_+ 52 (6) 396± 73 (4)
Benzidine (PC)
200 5O0 500
M M R
0 (6) 0 (4) 78_+ 20 (4)
N-Acetylbenzidine (NT)
50 200 10
M M R
12± 4 (6) 87+ 37 (4) 359_+ 85 (4)
N, N'-Diacetylbenzidine (NT)
50 200 50
M M R
12 + 7 (6) 73_+ 29 (4) 512_+223 (4)
Benzo[ a ]pyrene (PC)
10 5
M R
15± 7 (8) 382± 116 (4)
3-Methylcholanthrene (PC)
10 10
M R
11 ± 3 (8) 103± 32 (4)
2-Acetylaminofluorene (PC) 4-Aminobiphenyl (PC)
Polynuclear aromatic hydrocarbons
a PC, procarcinogen; NT, not adequately tested for carcinogen±city. b $9 source: M, Mercenaria mercenaria digestive gland, - 2 mg protein/plate; R, rat liver (Mutazyme, T M Meloy Laboratories), - 3 mg protein/plate. c TA98 His + revertants corrected for background, including direct toxic or mutagenic effects and spontaneous revertants (SR < 25). Mean + SD (N, number of animals tested; each experimental value used to calculate the mean was the average of duplicate plates).
a b s e n c e o f $9, p r o d u c e d s i g n i f i c a n t m u t a g e n e s i s . H o w e v e r , w h e n s u b s t r a t e a n d enzyme were combined, under adequate conditions, marked TA98 back-mutation w a s o b s e r v e d . S a t u r a t i o n o f t h e r e a c t i o n m i x t u r e w i t h C O h a d little e f f e c t o n mutagenesis; however, similar treatment of Mutazyme TM preparations virtually eliminated the response. Both enzyme preparations were inactive after heat treat-
251
800
-
co O~ 600
v (/1
400 O~ :>
200
+ m
m
m
SR
2AA
S-9
(no
S-9]
~J
2AA (no -I2AA) S-9
2AA S-9 +
co
m
2~1~ 2AA S-9 S-9 56% 10&,
50'
30'
~ S-9NADPH
4-
2AA
Fig. 1. Mutagenicity (total revertants/plate) of a representative aromatic amine, 2-aminoanthracene (2AA), mediated by Mercenaria digestive gland enzymes ($9). Substrate concentrations was 10 # g 2 A A / p l a t e ; enzyme concentration was 500 Pcl $9 ( - 2 m g protein)/plate. Other abbreviations used are SR, spontaneous revertants; CO, carbon monoxide; $9 minus N A D P H , no NADPH-generating system added.
m e n t ( > 56 °, 30 re_in), or if the standard NADPH-generating system was not added to the reaction mixture. The rate of mutagenesis was approximately linear at given enzyme concentrations for 2AA concentrations up to 10 # g / p l a e (Fig. 2). The response reached a plateau or declined at higher substrate concentrations. This dose--response relationship could be seen for all the aromatic amines tested. Similarly, the rate of reaction was
] ,000
(D
aoo 600
9
g ~)
400
0 ~
, 5
,
IO
I
25 P-9 2- aminoonthracene
I
50
Fig. 2. Effect of substrate concentration (peg 2AA/plate) on revertamts/p]ate using 200 pc] ( - 0.8 mg protein) and 500 FI ( - 2 mg protein) S9/p|ate. Data are corrected for spontaneous revertants and other background. Digestive glands were pooled from 3 Mercenada to prepare $9; each point is the average of tripficate determinations.
252
800 o~
2-A 600-
c-
/
/
2-AF
400 -
(D >
zoo +~1
~
-,-,
0
2(~0
n a
,
500
700
/zl S-9
BoP 1000
mix
Fig. 3. Effect of enzyme concentration on TA98 reversion using 2-aminoanthracene (2AA, 10 btg/plate), 2-aminofluorene (2AF, 10 ~g/plate), 2-acetylaminofluorene (2AAF, 100/xg/plate) and benzo[a]pyrene (BaP, 10/xg/plate). The $9 used was from a c o m m o n pool. In the case of the aromatic amines, - 2 mg p r o t e i n / m l $9 mix was added, twice as much $9 was used in the BaP assay. Data are corrected for spontaneous revertants and other background.
dependent on enzyme concentration for both aromatic amines and BaP (Fig. 3). The tissue distribution of the enzyme is given using the substrate 2AF (Fig. 4). Clearly, the digestive glands provided the most active preparations for the activation of aromatic amines. However, some activity was consistently recorded in all tissues examined, including mantle, gill, intestine, remaining visceral mass and the hemocytes. Essentially the same results were obtained using 2AA, except that slightly 800
-
G0 03
600 v
t-
400
>
200 + iF#3 Dig. Mantle Gland
Gills
Gut
• 7 •
m
Visceral HemoMoss cytes
Fig. 4. Tissue distribution of 2-aminofluorene-activating enzyme in Mercenaria, $9 from each tissue prepared from the corresponding 20% homogenate. Hemocytes were obtained from 10 ml hemolymph, washed, and homogenized in 2 ml buffer; 500 #1 of this hemocyte preparation was added per plate. $9 mixes contained 0.4 ml S 9 / m l and 500 VI $9 mix was added per plate. The actual a m o u n t of protein added per plate of each tissue was: digestive gland, 2.1 mg; mantle, 1.2 mg; gills, 1.1 mg; intestine, 1.0 mg; visceral amss (excluding digestive gland, intestine, mantle), 1.6 mg; and hemocytes, 1.1 mg.
253 more activity was seen in t h e gill, ( - 9 0 revertants/plate) and intestine ( - 7 0 revertants/plate) in relation to the digestive gland ( - 8 0 0 revertants/plate), than was the case for 2AF.
Discussion Bacterial mutation assays are often used to screen for potential carcinogens. The most widely used of these assays is that of Ames et al. (1973) in which rodent-liver homogenates are used for metabolic activation of carcinogens, resulting in the reversion of specially constructed mutant strains of Salmonella typhimurium from a histidine requirement to prototrophy. The oncogenetic potential of the major classes of vertebrate carcinogens for bivalve mollusks has received little experimental study. Therefore, we modified the Ames assay to determine the ability of the hepatic equivalent in a clam (Mercernaria mercenaria) to produce mutagenic metabolites from some well-known mammalian procarcinogens. The chemicals tested included the environmental carcinogen BaP and several aromatic amines, a class of carcinogens not previously tested for activity using a molluscan system. Mercenaria $9 readily converted certain aromatic amines, such as 2AA, 2AF, 2AAF and 4ATS, into frameshift mutagens that were detected using tester strains TA1538 and TA98. Strain TA100 was also reverted by these agents, since it responds to both frameshift and base-pair substitution mutagens (McMahan et al., 1979). Weak mutagenssis was observed with 4ABP, ABZ and DABZ. Benzidine was apparently not activated by Mercenaria $9. All of the above aromatic amines, with the exception of benzidine, were strongly mutagenic in the presence of rat $9. However, some mutagenesis was observed after incubation of benzidine with rat $9. Benzidine activation probably involves a 2-step acetylation (BZ ~ ABZ ~ DABZ), DABZ is N-hydroxylated by liver microsomes to give 3-hydroxy-N,N'-diacetylbenzidine; 3 0 H - D A B Z can bind to DNA and is mutagenic (Morton et al., 1979). The acetylated benzidine metabolites were much more active than the parent compound in Salmonella mutagenesis assays using Mutazyme TM. Similarly, monoand diacetylbenzidine showed evidence of activation by Mercenaria $9, indicating the ability of clam enzymes to N-hydroxylate benzidine metabolites, as well as the other aromatic amines tested. Clearly, the observed activation of aromatic amines was mediated by the Mercenaria $9, and was not an experimental artifact. It was shown for each concentration tested that the substrate alone produced no significant mutagenesis. Fresh clam $9 alone was not mutagenic and was completely inactivated if heated to 56°C (or above) prior to the addition of substrate. In higher animals and insects, M F O activity is NADPH-dependent and is markedly inhibited by carbon monoxide. Mercenaria aromatic amine activation is also much reduced in the absence of N A D P H ; however, carbon monoxide treatment failed to affect mutagenesis. We showed that Salmonella mutagenicity was directly proportional to substrate concentration over a given range of values; the magnitude of response at any given substrate concentration was dependent on enzyme concentration.
254 Aromatic amine-activating enzyme was most concentrated, on the basis of activity/mg protein, in the digestive gland of Mercenaria. This tissue was darker in color than the surrounding visceral mass and was routinely excised and used as an enzyme source for these assays. Minimal activity was also detected in the other tissues, including a very low amount in the hemocytes. Moore (1979) reported the presence of the MFO-linked microsomal enzyme N A D P H neotetrazolium reductase in the blood cells, intestinal epithelial cells, and developing oocytes of another bivalve, Mytilus edulis. This histochemical staining reaction was most intense in the hemocytes, suggesting elevated M F O activity. This did not appear to be the case for Mercenaria, since hemocytes were a relatively poor source of aromatic amine-activating enzymes, compared to the digestive gland. Carcinogenic PAH are also frameshift mutagens after activation and can be detected readily with TA98. Typically, rat $9 converted both BaP and MC to mutagenic metabolites, giving strong responses in the Salmonella system. However, clam enzymes produced a very weak, but consistent, response to these carcinogens. Perhaps this reflected the relatively low rate of PAH metabolism reported for bivalves (Anderson, 1978; Stegeman, 1981). It is interesting that similar patterns of carcinogen mutagenesis to that reported here for Mercenaria (substantial aromatic amine activation, but little or no PAH activation) have also been observed in other lower animals, such as insects (Baars et al., 1980) and a marine ciliate (Lindmark, 1981). The data suggest that aromatic amines may prove more useful than PAH in developing experimental bivalve models of chemical carcinogenesis. However, one must bear in mind the results of McGregor (1975), who found that hepatic samples from all of the mammalian species studied showed activation of 2AAF in the Ames test, irrespective of their susceptibility to 2AAF carcinogenesis.
Acknowledgments This work was supported by Grants OCE-7723443 and OCE-8016186 from the National Science Foundation, Grant CA-08748 from the National Cancer Institute, and a Grant from the Whitehall Foundation.
References Allison, A.C., and G.R. Paton (1965) Chromosomedamage in human diploid cells followingactivation of lysosomal enzymes, Nature (London), 207, 1170-1173. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973) Carcinogens are mutagens: a simple test system combiningliver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70, 2281-2285. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsomemutagenicity test, Mutation Res., 31,346-364. Anderson, R.S. (1978) Benzo[a]pyrenemetabolism in the American oyster, Crassostrea virginica, EPA Ecol. Res. Ser. Monogr. (EPA-600/3-78-009),pp. 1-18.
255 Anderson, R.S., C.S. Giam, L.E. Ray and M.R. "Fripp (1981) Effects of environmental pollutants on immunological competency of the clam Mercenaria mercenaria: impaired bacterial clearance, Aquatic Toxicol., !, 187-195. Baars, A.J., W.G.H. Blijleven, G.R. Mohn, A.T. Natarajan and D.D. Breimer (1980) Preliminary studies on the ability of Drosophila microsomal preparations to activate mutagens and carcinogens, Mutation Res., 72, 257-264. Brown, R.S., R.E. Wolke, C.W. Brown and S.B. Saila (1979) Hydrocarbon pollution and the prevalence of neoplasia in New England soft-shell clams (Mya arenaria), in: Animals as Monitors of Environmental Pollutants, National Academy of Sciences, Washington, pp. 41-50. Christensen, D.J., C.A. Farley and F.G. Kern (1974) Epizootic neoplasms in the clam Macoma balthica (L.) from Chesapeake Bay, J. Natl. Cancer Inst., 52, 1739-1749. Couch, J.A., and L. Courtney (1977) Interaction of chemical pollutants and virus in a crustacean: a novel bioassay system, Ann. N.Y. Acad. Sci., 298, 497-504. Couch, J.A., L.A. Courtney, J.T. Winstead and S.S. Foss (1979) The American oyster (Crassostrea virginica) as an indicator of carcinogens in the aquatic environment, in: Animals as Monitors of Environmental Pollutant, National Academy of Sciences, Washington, pp. 65-82. Farley, C.A.. and A.K. Sparks (1970) Proliferative disorders of hemocytes, endothelial cells and connective tissue cells in molluscs, Bibl. Haematol. (Basel), 36, 610-617. Fries, C.R., and M.R. Tripp (1980) Depression of phagocytosis in Mercenaria following chemical stress, Develop. Comp. Immunol., 4, 233-244. Khudoley, V.V., and O.A. Syrenko (1978) Tumor induction by N-nitroso compounds in bivalve mollusks Unio pictorum, Cancer Lett., 4, 349-354. Krieg, K. (1972) Ampullarius australis d'Orbigny (Mollusca, Gastropoda) as experimental animal in oncological research, A contribution to the study of cancerogenesis in invertebrates, Neoplasma, 19, 41-49. Lindmark, D.G. (1981) Activation of polynuclear aromatic hydrocarbons to mutagens by the marine ciliate Parauronema acutum, Appl. Environ. Microbiol., 41, 1238-1242. Lowe, D.M., and M.N. Moore (1978) Cytology and quantitative cytochemistry of a proliferative atypical hemocytic condition in Mytilus edulis (Bivalvia, Mollusca), J. Natl. Cancer Inst., 60, 1455-1459. McGregor, D. (1975) The relationship of 2-acetamidofluorene mutagenicity in plate tests with its in vivo liver cell component distribution and its carcinogenic potential, Mutation Res., 30, 305-316. McMahan, R.E., J.C. Cline and C.Z. Thompson (1979) Assay of 885 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens, Cancer Res., 39, 682-693. Mix, M.C. (1976) A review of the cellular proliferative disorders of oysters (Ostrea lurida) from Yaquina Bay, Oregon, Prog. Exp. Tumor Res., 20, 275-282. Mix, M.C., S.R. Trenholm and K.I. King (1979) Benzo[a]pyrene body burdens and the prevalence of proliferative disorders in mussels (Mytilus edulis) in Oregon, in: Animals as Monitors of Environmental Pollutants, National Academy of Sciences, Washington, pp. 52-62. Moore, M.n. (1979) Cellular responses to polycyclic aromatic hydrocarbons and phenobarbital in Mytilus edulis, Mar. Environ. Res., 2, 255-264. Moore, M.N., D.M. Lowe and P.E.M. Fieth (1978) Lysosomal responses to experimentally injected anthracene in digestive cells of mytilus edulis, Mar. Biol., 48, 297-302. Morton, K.C., C.M. King and K.P. Baetcke (1979) Metabolism of benzidine to N-hydroxy-N,N'-diacetylbenzidine and subsequent nucleic acid binding and mutagenicity, Cancer Res., 39, 3107-3113. Oprandy, J.J., P.W. Chang, A.D. Pronovost, K.R. Cooper, R.S. Brown and V.J. Yates (1981) Isolation of a viral agent causing hematopoietic neoplasia in the soft-shell clam, Mya arenaria, J. Invert. Pathol., 38, 45-51. Payne, J.F. (1977) Mixed function oxidases in marine organisms in relation to petroleum hydrocarbon metabolism and detection, Marine Pollution Bull., 8, 112-116. Rosenfield, A. (1976) Recent environmental studies of neoplasms in marine shellfish, Prog. Exp. Tumor Res., 20, 263-274. Stegeman, J.J. (1981) Polynuclear aromatic hydrocarbons and their metabolism in the marine environment, in: H.V. Gelboin and P.O.P. Ts'o (Eds.), Polycyclic Hydrocarbons and Cancer, Vol. 3, Academic Press, New York, pp. 1-60.
256 Vandermeulen, J.H., and W.R. Penrose (1978) Absence of aryl hydrocarbon hydroxylase (AHH) in three marine bivalves, J. Fish. Res. Board Can., 35,643-647. Vandermeulen, J.H., P.D. Keizer and W.R. Penrose (1977) Persistence of non-alkane components of bunker C oil in beach sediments of Chedabucto Bay, and lack of their metabolism by molluscs, in: Proceedings, 1977 Oil Spill Conference, American Petroleum Institute, Washington, pp. 469-473.
247
Activation of mammalian carcinogens to bacterial mutagens by microsomal enzymes from a pelecypod mollusk, Mercenaria mercenaria Robert S. Anderson and Jessica E. D66s Sloan - Kettering Institute for Cancer Research, Donald S. Walker Laboratory, 145 Boston Post Road, Rye, N Y 10580 (U.S.A.)
(Received25 March 1982) (Revisionreceived6 July 1982) (Accepted6 August1982)
Summary Several kinds of neoplastic diseases have been described in mollusks collected from the field. The etiology of these lesions is unknown; however, the involvement of chemical carcinogens has been suggested. Experimental models for chemically-induced neoplasia in bivalves have yet to be developed. We have obtained data which suggest that aromatic amines may be more appropriate candidates than polycyclic aromatic hydrocarbons for putative molluscan carcinogens. Digestive gland enzymes from a bivalve mollusk, M e r c e n a r i a m e r c e n a r i a , were found to be able to convert aromatic amines to frameshift mutagens as detected by the Ames Salmonella tester strains. Extensive mutagenesis was obtained with 2aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene and 4 - a m i n o - t r a n s - s t i l bene which are all mammalian procarcinogens. Mutagenic activation of benzo[a]pyrene and 3-methylcholanthrene was minimal. Low levels of aromatic amine-activating enzyme(s) were found in all tissues examined, but activity was greatest in the digestive gland. Enzymatic activity was heat-labile, NADPH-dependent, and apparently not inducible by polychlorinated biphenyls (Aroclor 1254).
It has been suggested that in pelecypod mollusks there is little or no evidence for an enzyme system analogous to the mixed-function oxygenase (MFO) system of higher animals (Vandermeulen et al., 1977; Payne, 1977; Vandermeulen and Penrose, 1978); however, by the use of isotopicaUy-labeled substrate, metabolism of benzo[a]pyrene (BaP) has been quantified in oysters C r a s s o s t r e a v i r g i n i c a (Anderson, 1978), and mussels (Stegeman, 1981). Reactions catalyzed by MFO are not only involved in detoxification of xenobiotics, such as environmental hydrocarbons, but 0165-1218/83/0000-0000/$03.00 © ElsevierBiomedicalPress
248 also in their activation to carcinogenic proximate forms. Since many marine bivalves are reported to be subject to neoplastic diseases (e.g., see reviews by Farley and Sparks, 1970; Mix, 1976; Rosenfield, 1976; Brown et al., 1979), the study of MFO in these species takes on added interest. Although in some cases a possible correlation has been made between polynuclear aromatic hydrocarbon (PAH) exposure or body burden and the incidence of cellular proliferative disorders in bivalves (Lowe and Moore, 1978; Brown et al., 1979; Mix et al., 1979), the etiology of these lesions is controversial. PAHs may also play less direct roles in the development of neoplastic disease in bivalves, such as activation (Couch and Courtney, 1977) of viruses (Oprandy et al., 1981) in hematotopoietic neoplasia in Mya arenaria. PAH-induced lysosomal membrane destabilization, which has been implicated in neoplastic transformation (Allison and Paton, 1965), has been reported in bivalves (Moore et al., 1978). The possible relationship between xenobiotic-mediated impaired immunological surveillance mechanisms in Mercenaria (Fries and Tripp, 1980; Anderson et al., 1981) and expression of neoplastic disease has yet to be defined. To date, the majority of reports on molluscan neoplastic disease has been based on samples taken from the field. Few experimental studies of chemical carcinogenesis have been made with these species. Krieg (1972) induced epithelial tumors with 20-methylcholanthrene (MC) in a gastropod mollusk. Oysters chronically exposed to MC developed lesions in the mantle, characterized by cellular infiltration, resembling the sarcoma-like lesions previously seen in field-collected specimens (Couch et al., 1979). N-Nitroso compounds were reported to induce tumors in bivalves (Khudoley and Syrenko, 1978); nitrosamines had also been suggested as the causative agents of neoplasms seen in Macoma balthica (Christensen et al., 1974). A commonly used method to measure metabolic activation of carcinogens quantifies the production of metabolites that are mutagenic for various Salmonella typhimurium tester strains (Ames et al., 1975). In this study, we have used this technique to study the activation of BaP and some carcinogenic aromatic amines by enzyme preparations from the digestive gland of Mercenaria mercenaria. Metabolic activation of aromatic amines has not previously been studied using bivalve tissues.
Materials and methods
Hard clams (Mercenaria mercenaria) were obtained from a local commercial supplier and were held at - 12°C in recirculated artificial sea water (Instant Ocean). The clams were opened on ice and the digestive glands carefully removed from the visceral mass. The glands were minced and washed with 0.05 N Tris buffer (pH 7.2) containing 0.25 M sucrose. Some animals were injected with 6 mg Aroclor 1254 in 0.1 ml olive oil ( - 240 m g / k g body weight), 96-h prior to sacrifice. Injections or hemolymph withdrawal was made via the blood sinus in the anterior adductor muscle. 9000 g homogenate supernatants ($9) were prepared from digestive gland homogenates (20% in 0.25 M sucrose in 0.05 N Tris buffer, pH 7.2). In the studies reported here fresh $9, maintained on ice, was used, although enzymatic activity could be retained after longer term storage at - 8 0 ° C .
249 The methods for mutagenicity testing were the same as those originally described by Ames et al. (1975). The Salmonella strain used (TA98)was periodically checked for histidine requirement, deep rough permeability character, and for the presence of the ampicillin-resistant R factor. Mercenaria $9 mix was prepared using the usual salts and cofactors, and usually contained 0.4 ml S9/ml. We also used MutazymeTM (Meloy Laboratories, Inc.), a lyophilized preparation of Aroclor 1254-induced rat-liver $9 mix, to enable comparison of the mutagenicity of the various substrates after metabolism by molluscan and mammalian enzymes. Clams were normally not pretreated with Aroclor because attemts to induce Mercenaria MFO were unsuccessful. The bacteria, substrate and $9 mix were combined in top agar and poured on minimal glucose agar plates, as described by Ames et al. (1975). The plates were protected from light until read to avoid photochemical reactions. The plates were incubated at 37°C for 48 h before counting his + revertants with an automated colony counter (BioTran II, New Brunswick Scientific Co., Inc.). All variations in this protocol are described as the data are presented. 2-Aminoanthracene (2AA), 4-amino-trans-stilbene (4ATS), N-acetylbenzidine (ABZ), and N, N'-diacetylbenzidine (DABZ), were obtained from ICN Pharmaceuticals, Inc. Benzidine (BZ), 2-acetylaminofluorene (2AAF), 4-aminobiphenyl (4ABP), and 3-methylcholanthrene (MC), were obtained from Sigma Chemical Co. Benzo[a]pyrene (BaP) and 2-aminofluorene (2AF) were supplied by the Aldrich Chemical Co. Aroclor 1254 was a gift of the Monsanto Co. The Salmonella tester strian was generously provided by Dr. Bruce N. Ames, University of California, Berkeley.
Results
Microsomal enzyme preparations from the digestive glands of Mercenaria were capable of producing mutagenic metabolites from several aromatic amines that are carcinogenic for vertebrates (Table 1). The data presented are from untreated clams because attempts to augment activity by standard inducers, such as Aroclor 1254, were ineffective. Mercenaria preparations were less active per mg protein than $9 derived from rat hepatic homogenates. The substrate concentrations reported in Table 1 gave responses on the linear portion of the dose-response curve for the particular enzyme preparation being tested. 5 bacterial tester strains were tested to determine the most sensitive for use in our assays. Both TA1538 and TA98 (TA1538 with R factor plasmids) were equally sensitive bacterial detector strains for aromatic amine activation, whereas TA100 was moderately sensitive, and TA 1535 and TA 1537 were not affected in the assay. Using TA98, marked activation of 2AA, 2AF, 2AAF and 4ATS by Mercenaria enzymes was seen; 4ABP, ABZ, and DABZ were slightly activated, and BZ was inactive. 2 carcinogenic PAH, BaP and MC~ were very slightly mutagenic for TA98 and TA100 in the presence of clam $9, but were strongly mutagenic in combination with rat $9 mix. . . . Several properties of the Mercenaria aromatic amine.activating enzyme system are presented in Fig. 1. Neither clam $9 in the absence of substrate, nor 2AA in the
250 TABLE 1 MUTAGENICITY OF C A R C I N O G E N I C AROMATIC AMINES AND PAH AFTER ACTIVATION BY MERCENARIA DIGESTIVE GLAND ENZYMES OR RAT $9 MIX Compound a
ktg/plate
$9 source b
His ÷
revertants/ plate c
Aromatic amines
2-Aminoanthracene (PC)
10 5
M R
626_+238 (8) 1 205 + 326 (4)
2-Aminofluorene (PC)
10 10
M R
423 ± 146 (8) 764_+ 202 (4)
100 5
M R
556_+201 (6) 343_+ 107 (4)
50 50
M R
21+ 10 (6) 330+ 94 (4)
4-Amino-trans-stilbene (PC)
100 50
M R
158_+ 52 (6) 396± 73 (4)
Benzidine (PC)
200 5O0 500
M M R
0 (6) 0 (4) 78_+ 20 (4)
N-Acetylbenzidine (NT)
50 200 10
M M R
12± 4 (6) 87+ 37 (4) 359_+ 85 (4)
N, N'-Diacetylbenzidine (NT)
50 200 50
M M R
12 + 7 (6) 73_+ 29 (4) 512_+223 (4)
Benzo[ a ]pyrene (PC)
10 5
M R
15± 7 (8) 382± 116 (4)
3-Methylcholanthrene (PC)
10 10
M R
11 ± 3 (8) 103± 32 (4)
2-Acetylaminofluorene (PC) 4-Aminobiphenyl (PC)
Polynuclear aromatic hydrocarbons
a PC, procarcinogen; NT, not adequately tested for carcinogen±city. b $9 source: M, Mercenaria mercenaria digestive gland, - 2 mg protein/plate; R, rat liver (Mutazyme, T M Meloy Laboratories), - 3 mg protein/plate. c TA98 His + revertants corrected for background, including direct toxic or mutagenic effects and spontaneous revertants (SR < 25). Mean + SD (N, number of animals tested; each experimental value used to calculate the mean was the average of duplicate plates).
a b s e n c e o f $9, p r o d u c e d s i g n i f i c a n t m u t a g e n e s i s . H o w e v e r , w h e n s u b s t r a t e a n d enzyme were combined, under adequate conditions, marked TA98 back-mutation w a s o b s e r v e d . S a t u r a t i o n o f t h e r e a c t i o n m i x t u r e w i t h C O h a d little e f f e c t o n mutagenesis; however, similar treatment of Mutazyme TM preparations virtually eliminated the response. Both enzyme preparations were inactive after heat treat-
251
800
-
co O~ 600
v (/1
400 O~ :>
200
+ m
m
m
SR
2AA
S-9
(no
S-9]
~J
2AA (no -I2AA) S-9
2AA S-9 +
co
m
2~1~ 2AA S-9 S-9 56% 10&,
50'
30'
~ S-9NADPH
4-
2AA
Fig. 1. Mutagenicity (total revertants/plate) of a representative aromatic amine, 2-aminoanthracene (2AA), mediated by Mercenaria digestive gland enzymes ($9). Substrate concentrations was 10 # g 2 A A / p l a t e ; enzyme concentration was 500 Pcl $9 ( - 2 m g protein)/plate. Other abbreviations used are SR, spontaneous revertants; CO, carbon monoxide; $9 minus N A D P H , no NADPH-generating system added.
m e n t ( > 56 °, 30 re_in), or if the standard NADPH-generating system was not added to the reaction mixture. The rate of mutagenesis was approximately linear at given enzyme concentrations for 2AA concentrations up to 10 # g / p l a e (Fig. 2). The response reached a plateau or declined at higher substrate concentrations. This dose--response relationship could be seen for all the aromatic amines tested. Similarly, the rate of reaction was
] ,000
(D
aoo 600
9
g ~)
400
0 ~
, 5
,
IO
I
25 P-9 2- aminoonthracene
I
50
Fig. 2. Effect of substrate concentration (peg 2AA/plate) on revertamts/p]ate using 200 pc] ( - 0.8 mg protein) and 500 FI ( - 2 mg protein) S9/p|ate. Data are corrected for spontaneous revertants and other background. Digestive glands were pooled from 3 Mercenada to prepare $9; each point is the average of tripficate determinations.
252
800 o~
2-A 600-
c-
/
/
2-AF
400 -
(D >
zoo +~1
~
-,-,
0
2(~0
n a
,
500
700
/zl S-9
BoP 1000
mix
Fig. 3. Effect of enzyme concentration on TA98 reversion using 2-aminoanthracene (2AA, 10 btg/plate), 2-aminofluorene (2AF, 10 ~g/plate), 2-acetylaminofluorene (2AAF, 100/xg/plate) and benzo[a]pyrene (BaP, 10/xg/plate). The $9 used was from a c o m m o n pool. In the case of the aromatic amines, - 2 mg p r o t e i n / m l $9 mix was added, twice as much $9 was used in the BaP assay. Data are corrected for spontaneous revertants and other background.
dependent on enzyme concentration for both aromatic amines and BaP (Fig. 3). The tissue distribution of the enzyme is given using the substrate 2AF (Fig. 4). Clearly, the digestive glands provided the most active preparations for the activation of aromatic amines. However, some activity was consistently recorded in all tissues examined, including mantle, gill, intestine, remaining visceral mass and the hemocytes. Essentially the same results were obtained using 2AA, except that slightly 800
-
G0 03
600 v
t-
400
>
200 + iF#3 Dig. Mantle Gland
Gills
Gut
• 7 •
m
Visceral HemoMoss cytes
Fig. 4. Tissue distribution of 2-aminofluorene-activating enzyme in Mercenaria, $9 from each tissue prepared from the corresponding 20% homogenate. Hemocytes were obtained from 10 ml hemolymph, washed, and homogenized in 2 ml buffer; 500 #1 of this hemocyte preparation was added per plate. $9 mixes contained 0.4 ml S 9 / m l and 500 VI $9 mix was added per plate. The actual a m o u n t of protein added per plate of each tissue was: digestive gland, 2.1 mg; mantle, 1.2 mg; gills, 1.1 mg; intestine, 1.0 mg; visceral amss (excluding digestive gland, intestine, mantle), 1.6 mg; and hemocytes, 1.1 mg.
253 more activity was seen in t h e gill, ( - 9 0 revertants/plate) and intestine ( - 7 0 revertants/plate) in relation to the digestive gland ( - 8 0 0 revertants/plate), than was the case for 2AF.
Discussion Bacterial mutation assays are often used to screen for potential carcinogens. The most widely used of these assays is that of Ames et al. (1973) in which rodent-liver homogenates are used for metabolic activation of carcinogens, resulting in the reversion of specially constructed mutant strains of Salmonella typhimurium from a histidine requirement to prototrophy. The oncogenetic potential of the major classes of vertebrate carcinogens for bivalve mollusks has received little experimental study. Therefore, we modified the Ames assay to determine the ability of the hepatic equivalent in a clam (Mercernaria mercenaria) to produce mutagenic metabolites from some well-known mammalian procarcinogens. The chemicals tested included the environmental carcinogen BaP and several aromatic amines, a class of carcinogens not previously tested for activity using a molluscan system. Mercenaria $9 readily converted certain aromatic amines, such as 2AA, 2AF, 2AAF and 4ATS, into frameshift mutagens that were detected using tester strains TA1538 and TA98. Strain TA100 was also reverted by these agents, since it responds to both frameshift and base-pair substitution mutagens (McMahan et al., 1979). Weak mutagenssis was observed with 4ABP, ABZ and DABZ. Benzidine was apparently not activated by Mercenaria $9. All of the above aromatic amines, with the exception of benzidine, were strongly mutagenic in the presence of rat $9. However, some mutagenesis was observed after incubation of benzidine with rat $9. Benzidine activation probably involves a 2-step acetylation (BZ ~ ABZ ~ DABZ), DABZ is N-hydroxylated by liver microsomes to give 3-hydroxy-N,N'-diacetylbenzidine; 3 0 H - D A B Z can bind to DNA and is mutagenic (Morton et al., 1979). The acetylated benzidine metabolites were much more active than the parent compound in Salmonella mutagenesis assays using Mutazyme TM. Similarly, monoand diacetylbenzidine showed evidence of activation by Mercenaria $9, indicating the ability of clam enzymes to N-hydroxylate benzidine metabolites, as well as the other aromatic amines tested. Clearly, the observed activation of aromatic amines was mediated by the Mercenaria $9, and was not an experimental artifact. It was shown for each concentration tested that the substrate alone produced no significant mutagenesis. Fresh clam $9 alone was not mutagenic and was completely inactivated if heated to 56°C (or above) prior to the addition of substrate. In higher animals and insects, M F O activity is NADPH-dependent and is markedly inhibited by carbon monoxide. Mercenaria aromatic amine activation is also much reduced in the absence of N A D P H ; however, carbon monoxide treatment failed to affect mutagenesis. We showed that Salmonella mutagenicity was directly proportional to substrate concentration over a given range of values; the magnitude of response at any given substrate concentration was dependent on enzyme concentration.
254 Aromatic amine-activating enzyme was most concentrated, on the basis of activity/mg protein, in the digestive gland of Mercenaria. This tissue was darker in color than the surrounding visceral mass and was routinely excised and used as an enzyme source for these assays. Minimal activity was also detected in the other tissues, including a very low amount in the hemocytes. Moore (1979) reported the presence of the MFO-linked microsomal enzyme N A D P H neotetrazolium reductase in the blood cells, intestinal epithelial cells, and developing oocytes of another bivalve, Mytilus edulis. This histochemical staining reaction was most intense in the hemocytes, suggesting elevated M F O activity. This did not appear to be the case for Mercenaria, since hemocytes were a relatively poor source of aromatic amine-activating enzymes, compared to the digestive gland. Carcinogenic PAH are also frameshift mutagens after activation and can be detected readily with TA98. Typically, rat $9 converted both BaP and MC to mutagenic metabolites, giving strong responses in the Salmonella system. However, clam enzymes produced a very weak, but consistent, response to these carcinogens. Perhaps this reflected the relatively low rate of PAH metabolism reported for bivalves (Anderson, 1978; Stegeman, 1981). It is interesting that similar patterns of carcinogen mutagenesis to that reported here for Mercenaria (substantial aromatic amine activation, but little or no PAH activation) have also been observed in other lower animals, such as insects (Baars et al., 1980) and a marine ciliate (Lindmark, 1981). The data suggest that aromatic amines may prove more useful than PAH in developing experimental bivalve models of chemical carcinogenesis. However, one must bear in mind the results of McGregor (1975), who found that hepatic samples from all of the mammalian species studied showed activation of 2AAF in the Ames test, irrespective of their susceptibility to 2AAF carcinogenesis.
Acknowledgments This work was supported by Grants OCE-7723443 and OCE-8016186 from the National Science Foundation, Grant CA-08748 from the National Cancer Institute, and a Grant from the Whitehall Foundation.
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