Mutation Research, 262 0991) 189-193
189
© 1991Elsevier SciencePublishers B.V. (BiomedicalDivision)0165-7992/91/$03.50 ADONIS 016579929100020A MUTLET 0468
Inhibition by diacylmethane derivatives of mutagenicity in Salmonella typhimurium and tRNA-binding of chemical carcinogens* Ching Y. Wang, Mei-Sie Lee and Kim Zukowski Department of Chemical Carcinogenesis, Michigan CancerFoundation, 110 E. WarrenAvenue, Detroit, MI 48201 (U.S.A.)
(Received13 September1990) (Accepted29 October 1990)
Keywords: Diacylmethanes;Inhibition of mutation; Nucleicacid binding, inhibition
Summary Effects of diacylmethanes on the mutagenicity of 2-naphthohydroxamic acid, methylnitrosourea, benzo[a]pyrene and aflatoxin B, in S. t y p h i m u r i u m and the tRNA binding by benzo[a]pyrene and aflatoxin B, were investigated. Acetylacetone, benzoylacetone and dibenzoylmethane inhibited the mutagenicity of 2-naphthohydroxamic acid, and dibcnzoylmethane and 1,3-indandione inhibited that of methylnitrosourea, benzo[a]pyrene and aflatoxin B1. The binding to tRNA of benzo[a]pyrene and aflatoxin B, was inhibited by benzoylacetone and dibenzoylmethane, and dibenzoylmethane, 1,3-indandione and 1,1,1-trifluoroacetylacetone, respectively. The inhibition of methylnitrosourea mutagenicity was observed when the bacteria were exposed concomitantly to the inhibitors and the mutagen, but not when they were exposed to the inhibitors 1 h after exposure to the mutagen. These results demonstrate that active methylene compounds can inhibit mutagenicity and nucleic acid-binding of chemical carcinogens presumably by trapping carcinogenic electrophiles, and they are potential anti-carcinogenic agents during the initiation stage.
Most chemical carcinogens are electrophilic per se or they require metabolic activation to form electrophilic agents. Nucleophilic sites of DNA, particularly those present in guanine, are common
* Presented in part at the American Physiological Society/ American Society for Pharmacology and Therapeutics Joint Meeting, Montreal, Canada, 9-13 October 1988. Correspondence: Dr. ChingY. Wang, Departmentof Chemical Carcinogenesis, Michigan Cancer Foundation, 110 E. Warren Avenue, Detroit, MI 48201 (U.S.A.).
targets of electrophilic attack by chemical carcinogens (Miller and Miller, 1981). These nucleophilic sites may be protected by exogenous nucleophiles. Thus nucleophilic agents, such as sulfur containing compounds (van den Goorbergh et ai., 1987) and polyhydric phenols (Wood et al., 1982; Chang et al., 1985), have been shown to inhibit mutagenesis and carcinogenesis. Diacylmethanes possess an active methylene function. In neutral solutions, they form carbanions or enolic ions, as shown in the following equation, where R can be either alkyl or aryl groups:
190 R(CO-) = CHCOR ~- RCOCH2COR~ RCO(HC-)COR These ions are known to react with electrophilic agents (House, 1965). We have previously reported that several diacylmethanes inhibit the mutagenicity of 2-nitrofluorene and an enzyme-catalyzed binding of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to tRNA. At 37°C, pH 7.4, 1,1,1-trifluoroacetylacetone (TFAA) traps the acetamidonitrenium ion produced by N-acetoxy-2acetylaminofluorene (Wang et al., 1989). We now report the inhibitory activity of some diacylmethanes of the mutagenicities and nucleic acidbindings of other mutagens and carcinogens. Materials and methods
Chemicals. Aflatoxin B1 (AFB) was purchased from Aldrich Chem. Co., Milwaukee, WI, benzo[a]pyrene (B[a]P) from Eastman Kodak Co., Rochester, NY, methylnitrosourea (MNU) from Pfaultz and Bauer, Flushing, NY, NADPH from Sigma Chem. Co., St. Louis, MO, and Aroclor 1254 from Analabs, North Haven, CT. [Ring3H]AFB, 15 Ci/mmole, was purchased from Moravek Biochemicals, Inc., Brea, CA, and [ring-3HlB[a]P, 56 Ci/mmole, was from New England Nuclear Co., Boston, MA. They were diluted with unlabeled compounds to the desired specific activity. 2-Naphthohydroxamic acid (2-NHA) were synthesized as described (Wang, 1977). The other chemicals were purchased from Aldrich Chem. Co., Milwaukee, WI. 1,3-Indandione (IDD) and benzoylacetone (BA) were purified by recrystalization from acetone-ether-hexane and ether, respectively. Acetylacetone (AA), TFAA and dibenzoylmethane (DBM) were used without further purification. Preparation of microsomes. Male CD rats (Charles River Breeding Lab., Wilmington, MA), approximately 200 g, were injected i.p. with 500 mg/kg Aroclor 1254 5 days before sacrifice. The liver was removed, rinsed, and homogenized in 3 volumes of 0.15 M KCI solution using a Teflonglass homogenizer. The homogenate was centrifug-
ed at 9000 g for 10 min and the resultant supernatant fraction was further centrifuged at 105 000 g for 1 h to separate microsomes from cytosol. The microsomal pellets were suspended in the KCI solution. The microsomal fraction was sterile filtered through:0.45 #1 millipore filters. All the preparation procedures were performed at 4°C. The preparations were immediately used for the experiments described below.
Mutation assays. The protocol of Maron and Ames (1983) was used with slight modifications. The experiments were performed using either Salmonella typhimurium TA98 or TAI00. 0.5 ml of mixtures contained 0.1 ml overnight culture of the Salmonella strains, the test mutagens and antimutagens in 20 #1 of DMSO, and 25 #moles Na phosphate, pH 7.4. When microsomes were needed, approximately 0.3 mg microsomal protein, 2 #moles NADPH and 4 #moles MgC12 were added. The mixtures were kept at room temperature for 30 min and then plated. The plates were incubated 48 h at 37°C in the dark, and revertant colonies were enumerated with an Artek Systems model 880 colony counter (Artek Systems Corp., Farmingdale, NY). All experiments were performed in triplicate. RNA-binding assays. Yeast tRNA was used as a trapping agent for the binding assays. A 0.5-ml incubation mixture containing 0.2 #moles NADI~H, 25 mmoles Na phosphate, pH 7.4, 15 A26o units yeast tRNA, approximately 0.3 mg microsomal protein, l0 nmoles [ring-3H]B[a]P (536 mCi/ mmole) or [ring-3H]AFB (267 mCi/mmole), and 20 #l DMSO with or without test inhibitors was incubated at 37°C for l0 min. The binding of the labeled compound to tRNA was determined as described (King, 1974). Results
The diacylmethanes were tested at concentrations that did not produce observable toxicity. Because of its insolubility in aqueous solutions, the concentration of DBM used was lower than those of the other compounds. The diacylmethanes were
191 1200
A
S~
800 uJ I-
189
© 1991Elsevier SciencePublishers B.V. (BiomedicalDivision)0165-7992/91/$03.50 ADONIS 016579929100020A MUTLET 0468
Inhibition by diacylmethane derivatives of mutagenicity in Salmonella typhimurium and tRNA-binding of chemical carcinogens* Ching Y. Wang, Mei-Sie Lee and Kim Zukowski Department of Chemical Carcinogenesis, Michigan CancerFoundation, 110 E. WarrenAvenue, Detroit, MI 48201 (U.S.A.)
(Received13 September1990) (Accepted29 October 1990)
Keywords: Diacylmethanes;Inhibition of mutation; Nucleicacid binding, inhibition
Summary Effects of diacylmethanes on the mutagenicity of 2-naphthohydroxamic acid, methylnitrosourea, benzo[a]pyrene and aflatoxin B, in S. t y p h i m u r i u m and the tRNA binding by benzo[a]pyrene and aflatoxin B, were investigated. Acetylacetone, benzoylacetone and dibenzoylmethane inhibited the mutagenicity of 2-naphthohydroxamic acid, and dibcnzoylmethane and 1,3-indandione inhibited that of methylnitrosourea, benzo[a]pyrene and aflatoxin B1. The binding to tRNA of benzo[a]pyrene and aflatoxin B, was inhibited by benzoylacetone and dibenzoylmethane, and dibenzoylmethane, 1,3-indandione and 1,1,1-trifluoroacetylacetone, respectively. The inhibition of methylnitrosourea mutagenicity was observed when the bacteria were exposed concomitantly to the inhibitors and the mutagen, but not when they were exposed to the inhibitors 1 h after exposure to the mutagen. These results demonstrate that active methylene compounds can inhibit mutagenicity and nucleic acid-binding of chemical carcinogens presumably by trapping carcinogenic electrophiles, and they are potential anti-carcinogenic agents during the initiation stage.
Most chemical carcinogens are electrophilic per se or they require metabolic activation to form electrophilic agents. Nucleophilic sites of DNA, particularly those present in guanine, are common
* Presented in part at the American Physiological Society/ American Society for Pharmacology and Therapeutics Joint Meeting, Montreal, Canada, 9-13 October 1988. Correspondence: Dr. ChingY. Wang, Departmentof Chemical Carcinogenesis, Michigan Cancer Foundation, 110 E. Warren Avenue, Detroit, MI 48201 (U.S.A.).
targets of electrophilic attack by chemical carcinogens (Miller and Miller, 1981). These nucleophilic sites may be protected by exogenous nucleophiles. Thus nucleophilic agents, such as sulfur containing compounds (van den Goorbergh et ai., 1987) and polyhydric phenols (Wood et al., 1982; Chang et al., 1985), have been shown to inhibit mutagenesis and carcinogenesis. Diacylmethanes possess an active methylene function. In neutral solutions, they form carbanions or enolic ions, as shown in the following equation, where R can be either alkyl or aryl groups:
190 R(CO-) = CHCOR ~- RCOCH2COR~ RCO(HC-)COR These ions are known to react with electrophilic agents (House, 1965). We have previously reported that several diacylmethanes inhibit the mutagenicity of 2-nitrofluorene and an enzyme-catalyzed binding of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to tRNA. At 37°C, pH 7.4, 1,1,1-trifluoroacetylacetone (TFAA) traps the acetamidonitrenium ion produced by N-acetoxy-2acetylaminofluorene (Wang et al., 1989). We now report the inhibitory activity of some diacylmethanes of the mutagenicities and nucleic acidbindings of other mutagens and carcinogens. Materials and methods
Chemicals. Aflatoxin B1 (AFB) was purchased from Aldrich Chem. Co., Milwaukee, WI, benzo[a]pyrene (B[a]P) from Eastman Kodak Co., Rochester, NY, methylnitrosourea (MNU) from Pfaultz and Bauer, Flushing, NY, NADPH from Sigma Chem. Co., St. Louis, MO, and Aroclor 1254 from Analabs, North Haven, CT. [Ring3H]AFB, 15 Ci/mmole, was purchased from Moravek Biochemicals, Inc., Brea, CA, and [ring-3HlB[a]P, 56 Ci/mmole, was from New England Nuclear Co., Boston, MA. They were diluted with unlabeled compounds to the desired specific activity. 2-Naphthohydroxamic acid (2-NHA) were synthesized as described (Wang, 1977). The other chemicals were purchased from Aldrich Chem. Co., Milwaukee, WI. 1,3-Indandione (IDD) and benzoylacetone (BA) were purified by recrystalization from acetone-ether-hexane and ether, respectively. Acetylacetone (AA), TFAA and dibenzoylmethane (DBM) were used without further purification. Preparation of microsomes. Male CD rats (Charles River Breeding Lab., Wilmington, MA), approximately 200 g, were injected i.p. with 500 mg/kg Aroclor 1254 5 days before sacrifice. The liver was removed, rinsed, and homogenized in 3 volumes of 0.15 M KCI solution using a Teflonglass homogenizer. The homogenate was centrifug-
ed at 9000 g for 10 min and the resultant supernatant fraction was further centrifuged at 105 000 g for 1 h to separate microsomes from cytosol. The microsomal pellets were suspended in the KCI solution. The microsomal fraction was sterile filtered through:0.45 #1 millipore filters. All the preparation procedures were performed at 4°C. The preparations were immediately used for the experiments described below.
Mutation assays. The protocol of Maron and Ames (1983) was used with slight modifications. The experiments were performed using either Salmonella typhimurium TA98 or TAI00. 0.5 ml of mixtures contained 0.1 ml overnight culture of the Salmonella strains, the test mutagens and antimutagens in 20 #1 of DMSO, and 25 #moles Na phosphate, pH 7.4. When microsomes were needed, approximately 0.3 mg microsomal protein, 2 #moles NADPH and 4 #moles MgC12 were added. The mixtures were kept at room temperature for 30 min and then plated. The plates were incubated 48 h at 37°C in the dark, and revertant colonies were enumerated with an Artek Systems model 880 colony counter (Artek Systems Corp., Farmingdale, NY). All experiments were performed in triplicate. RNA-binding assays. Yeast tRNA was used as a trapping agent for the binding assays. A 0.5-ml incubation mixture containing 0.2 #moles NADI~H, 25 mmoles Na phosphate, pH 7.4, 15 A26o units yeast tRNA, approximately 0.3 mg microsomal protein, l0 nmoles [ring-3H]B[a]P (536 mCi/ mmole) or [ring-3H]AFB (267 mCi/mmole), and 20 #l DMSO with or without test inhibitors was incubated at 37°C for l0 min. The binding of the labeled compound to tRNA was determined as described (King, 1974). Results
The diacylmethanes were tested at concentrations that did not produce observable toxicity. Because of its insolubility in aqueous solutions, the concentration of DBM used was lower than those of the other compounds. The diacylmethanes were
191 1200
A
S~
800 uJ I-
