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Metabolic activation of 2- and 3-nitrodibenzopyranone isomers and related compounds by rat liver S9 and the effect of S9 on the mutational specificity of nitrodibenzopyranones
Mutation Research 388 Ž1997. 67–78
Metabolic activation of 2- and 3-nitrodibenzopyranone isomers and related compounds by rat liver S9 and the effect of S9 on the mutational specificity of nitrodibenzopyranones Tetsushi Watanabe a,) , Hideaki Kaji a , Mari Takashima a , Terue Kasai a , Joellen Lewtas b, Teruhisa Hirayama a a
b
Kyoto Pharmaceutical UniÕersity, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607, Japan National Health and EnÕironmental Effects Research Laboratory, U.S. EnÕironmental Protection Agency, Research, Triangle Park, NC 27711, USA Received 24 July 1996; revised 27 September 1996; accepted 1 October 1996
Abstract The effect of rat liver S9 on the mutagenicity of 10 nitrated polycyclic aromatic hydrocarbons Žnitro-PAHs. was evaluated with Salmonella typhimurium TA98NR using S9 from phenobarbital-, 3-methylcholanthrene ŽMC.-, b-naphthoflavone- and polychlorobiphenyl-treated and untreated rats. 2-Nitrofluorene Ž2-NFl., 2-nitrofluoren-9-one Ž2-NFlone., 2-nitrocarbazole Ž2-NCz., 3-NCz, 2-nitrodibenzothiophene Ž2-NDBT., 2-nitro-6 H-dibenzow b,d xpyran-6-one Ž2-NDBP. and 3-NDBP were metabolically activated by one or more of the S9 fractions, and the highest enhancement of the mutagenic potency of nitro-PAHs was observed with 3-MC-induced S9. Only in the case of 3-NFlone was the mutagenicity in strain TA98NR decreased by the addition of S9, regardless of S9 induction. 2-NDBP was most efficiently activated among nitro-PAHs tested by all S9 fractions used. The cytosolic fraction of S9 accounted for more of the activation of 2-NDBP than the microsomal fraction. NADH and NADPH were the most effective electron donors on the activation of 2-NDBP by S9. 2-NDBP was also metabolically activated by NADH plus commercial preparations of xanthine oxidase. These activations of 2-NDBP were inhibited by allopurinol, indicating that cytosolic xanthine oxidase in rat liver S9 participates in the activation of 2-NDBP. The potency of 2- and 3-NDBP isomers as base-substitution mutagens was also enhanced by S9. In the presence of S9, both compounds showed the highest mutagenicity in strain TA7005 ŽC P G ™ A P T. followed by strains TA7004 ŽG P C ™ A P T., TA7006 ŽC P G ™ G P C. and TA7002 ŽT P A ™ A P T., and this mutation specificity was similar to that without S9, indicating that the mechanism of mutagenesis caused by NDBP isomers with S9 is similar to that without S9. Keywords: Metabolic activation; Salmonella typhimurium; Nitro-6 H-dibenbzow b,d xpyran-6-one; Nitro-polycyclic aromatic hydrocarbons
1. Introduction
) Corresponding author. Tel.: q81-75-595-4650; Fax: q81-75595-4769.
Nitrated polycyclic aromatic hydrocarbons ŽnitroPAHs. have become of enormous concern because of their genotoxicity, e.g., mutagenicity w1–3x and carcinogenicity w4,5x, and ubiquity in ambient air
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 Ž 9 6 . 0 0 1 3 8 - 3
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T. Watanabe et al.r Mutation Research 388 (1997) 67–78
w6–8x. The mutagenic nitro-PAHs represented by nitrofluorenes, nitrofluoranthenes and nitropyrenes are metabolically activated by the endogenous nitroreductase in Salmonella typhimurium tester strains w1,9–11x. We reported that nitrodibenzofuran isomers were efficiently activated by not only endogenous nitroreductase in S. typhimurium TA98 but also rat liver S9 w12x. Nitro-6 H-dibenzow b,d xpyran-6-one ŽNDBP. isomers, which are recently identified as ambient air pollutants w13,14x, are potent mutagens in S. typhimurium tester strains w15,16x, and the mutagenicity of NDBP was also enhanced by the addition of Aroclor-induced S9 w16x. In the presence of S9, 2-NDBP exhibited base-substitution activity that was greater than its frameshift activity, whereas 3-NDBP exhibited more frameshift than base-substitution activity w16x. Several nitro-PAHs which have the structure similar to nitrodibenzofuran and NDBP, such as nitrofluorene ŽNFl., nitrofluoren-9-one ŽNFlone., nitrocarbazole ŽNCz. and nitrodibenzothiophene ŽNDBT., were detected in ambient air and diesel exhaust extracts w17–19x. Since these nitro-PAHs are expected to be activated by mammalian enzymes like nitrodibenzofuran and NDBP, assessing the mutagenicity of these nitro-PAHs in the presence of mammalian S9 enzymes is of importance for the evaluation of these nitro-PAHs on the risk for human health. In this report, we describe the ability of rat liver S9 to activate 2- and 3-nitro-PAHs isomers including NFl, NFlone, NCz, NDBT and NDBP. The enzymes participating in the metabolic activation of 2-NDBP in rat liver S9 are characterized by subfractions of S9, i.e., cytosolic and microsomal fractions, and selected electron donors and nitroreductase inhibitors. Four DNA bases can result in six different type base-pair substitution mutations ŽG P C ™ C P G, G P C ™ T P A, A P T ™ T P A, A P T ™ C P G transversions and A P T ™ G P C, G P C ™ A P T transitions.. Determining the pattern of six possible base substitutions induced by mutagen would be helpful to understand the mechanism of mutagenesis. Mutagenicity of 2- and 3-NDBP isomers were assayed by a set of six Salmonella typhimurium tester strains ŽTA7001 to TA7006., each of which reverts by only one specific base-pair substitution out of the six possible changes w20x, with and without S9 in order to evaluate the effect of the rat liver S9 metabolic
activation on the base substitution induction by NDBP isomers.
2. Materials and methods 2.1. Chemicals 2-NFl, 4-nitroquinoline-N-oxide Ž4-NQO., methyl methanesulfonate ŽMMS., N-methyl-N X-nitro-Nnitrosoguanidine ŽMNNG., b-naphthoflavone ŽbNF., hypoxanthine ŽHX., menadione ŽME. and allopurinol ŽAL. were purchased from Nacalai Tesque ŽKyoto, Japan.. 2-NFlone, 2-acetylaminofluorene
Fig. 1. Structure and CAS registry number of 2- and 3- nitrofluorene ŽNFI., nitrofluoren-9-one ŽNFlone., nitrocarbazole ŽNCz., nitrodibenzothiophene ŽNDBT. and nitrodibenzopyranone ŽNDBP. isomers tested.
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
ŽAAF., 1-methylnicotinamide ŽMN. and 2-hydroxypyrimidine ŽHP. were obtained from Tokyo Kasei ŽTokyo, Japan.. 2-NDBP, 3-NDBP and 3-NFlone were purchased from Aldrich Chemical Co. ŽMilwaukee, WI.. 3-Methylcholanthrene Ž3-MC., phenobarbital ŽPB., polychlorobiphenyl penta ŽPCB, KC-500., dicumarol ŽDI., piperonyl butoxide ŽPI. and dimethyl sulfoxide ŽDMSO. were obtained from Wako Pure Chemicals ŽOsaka, Japan.. Glucose 6phosphate ŽG-6-P., glucose-6-phosphate dehydrogenase ŽG-6-PDH., nicotinamide-adenine dinucleotide, reduced form ŽNADH. and nicotinamide-adenine dinucleotide phosphate, reduced form ŽNADPH. were purchased from Oriental Yeast ŽTokyo, Japan.. Xanthine oxidase ŽXOD, Grade III, EC 1.2.3.2. was obtained from Sigma Chemical Co. ŽSt. Louis, MO.. 3-NFl was gift from Dr. Miyata, National Institute of Health Sciences. 2-NCz and 3-NCz were synthesized according to Mendenhall and Smithw21x and Eikhman et al.w22x, respectively. 2-NDBT and 3-nitrodibenzothiophene5-oxide were synthesized by the method described by Cullinane et al.w23x. 3-Nitrodibenzothiophene-5oxide was reduced to 3-NDBT by hydrobromic acid w24x. Chemical structures were confirmed by 1 HNMR, mass spectrum and melting point measurement. The melting points of authentic 2-NCz, 3-NCz, 2-NDBT and 3-NDBT were 175 ; 1788C, 211 ;
69
2138C, 188.5 ; 189.58C and 156 ; 1578C, respectively ŽReference: 2-NCz 174 ; 175.58C w25x, 3-NCz 212 ; 2138C w26x, 2-NDBT 1868C w23x, 3-NDBT 153 ; 1548C w24x.. High-performance liquid chromatography analysis indicated the purities of NCz and NDBT isomers greater than 99% as judged by monitoring of the eluate at 254 nm. 2.2. Mutagenicity assay The structures, abbreviations and CAS registry numbers of the compounds tested are shown in Fig. 1. The genotypic characteristics of S. typhimurium strains are listed in Table 1. S. typhimurium strains TA98 and TA100 were kindly provided by Dr. B.N. Ames, University of California, Berkeley. CA. Strains TA98NR and TA98r1,8-DNP6 were kindly provided by Dr. H.S. Rosenkranz, University of Pittsburgh, PA. Strains TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006 were provided by Xenometrix Inc., Boulder, CO. The mutagenicity assay was performed as described by Maron and Ames w27x. All samples tested were dissolved in DMSO. Mixtures of cell suspension, sample solution and 0.1 M sodium phosphate buffer or exogenous enzyme sources, e.g., S9 mix, were incubated at 378C for 20 min with shaking before pouring on the plates. Each tester strain was checked routinely to
Table 1 Genotypes of the Salmonella typhimurium TA strains used Strain a
TA98 TA100
a
TA7001 b
Mutation
Cell wall
Repair
Plasmid
hisD3052 hisG46
rfa rfa
D uÕrB D uÕrB
pKM101 pKM101
hisG1775
rfa
D uÕrB
pKM101
Reversion event
Asp y 153™Gly y 153 ŽGAT .
TA7002
b
hisC9138
rfa
D uÕrB
pKM101
b
hisG9074
rfa
D uÕrB
pKM101
TA7004
b
hisG9133
rfa
D uÕrB
pKM101
TA7005
b
hisG9130
rfa
D uÕrB
pKM101
TA7006
b
hisC9070
rfa
D uÕrB
pKM101
a b
Maron and Ames w27x. Gee et al. w20x.
C P G ™ A P T transversion
ŽGAG .
Arg y 163™Gly y 163 ŽCGA .
G P C ™ A P T transition
ŽGAG .
Ala y 169™Glu y 169 ŽGCG .
T P A ™ G P C transversion
ŽGGT .
Gly y 169™Glu y 169 ŽGGG .
T P A ™ A P T transversion
ŽAAA .
Val y 153™Gly y 153 ŽGTT .
A P T ™ G P C transition
ŽGGT .
Ile y 217™Lys y 217 ŽATA .
TA7003
Detected mutation
ŽGGA .
C P G ™ G P C transversion
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T. Watanabe et al.r Mutation Research 388 (1997) 67–78
confirm their features for optimal response to known mutagens as follows: 4-NQO Ž0.5 or 1 mgrplate., MMS Ž650 mgrplate., MNNG Ž4 mgr plate. and AAF Ž5 mgrplate.. All experiments were performed in duplicate or triplicate with at least three doses and in at least three times. The slope of dose-response Žrevertantsrmg. was calculated by least-squares linear regression from the linear portion of the dose-response curve. Male Sprague-Dawley rats were treated with PB, 3-MC, b-NF or PCB, and liver S9, cytosol and microsomes were prepared as described before w12x. Liver S9, cytosol, microsomes and commercial preparations of XOD were used as exogenous enzyme source. NADPH system consisted of MgCl 2 Ž8 mmol., KCl Ž33 mmol., G-6-P Ž5 mmol., G-6-PDH Žfrom yeast; 0.5 unit., NADPH Ž4 mmol., sodium phosphate buffer Ž100 mmol, pH 7.4. and water per 1 ml of exogenous enzyme mix. NADPH system was used for the mutagenicity assay unless otherwise noted. NADH, HX, MN and HP systems contained MgCl 2 Ž8 mmol., KCl Ž33 mmol., sodium phosphate buffer Ž100 mmol, pH 7.4., water and an electron donor Ž4 mmol., i.e., NADH, HX, MN or HP, for exogenous enzyme mix Ž1 ml..
3. Results and discussion 3.1. Mutagenicity of 2- and 3-nitro-PAHs in S. typhimurium TA98, TA98 r 1,8-DNP6 and TA98NR without S9 All of nitro-PAHs tested were mutagenic in strain TA98 without S9, and the activity decreased in strains TA98NR and TA98r1,8-DNP6 , which are the ‘classical’ nitroreductase- and the O-acetyltransferase-deficient derivatives of strain TA98, respectively w9,28–30x ŽTable 2.. The mutagenic potency of the tested compounds was estimated by a slope of dose-response Žrevertantsrmg.. In the absence of S9, mutagenic potency of nitro-PAHs in strain TA98NR was 3 ; 35% and 17 ; 109% of that in strains TA98 and TA98r1,8-DNP6 , respectively. This suggests that nitroso andror hydroxylamino derivatives from these nitro-PAHs metabolized by nitroreductase in strain TA98 are mutagenic, and that nitroreduction is a significant step in the metabolic activation of most
nitro-PAHs as compared with an esterification step. The order of the mutagenic potency between 2- and 3-nitro-PAH isomers in strain TA98 were as follows: 2-NFl ) 3-NFl; 2-NFlone- 3-NFlone; 2-NCz) 3NCz; 2-NDBT- 3-NDBT and 2-NDBP- 3-NDBP. This order was the same as that observed in strain TA98NR without S9, except for NDBT isomers, indicating that isomer selectivity of the endogenous enzymes participating in the activation of nitro-PAHs in strain TA98NR are also similar to that of the ‘classical’ nitroreductase. 3.2. Metabolic actiÕation of 2- and 3-nitro-PAHs by S9 Mutagenic potency of 7 out of 10 nitro-PAHs tested, i.e., 2-NFl, 2-NFlone, 2-NCz, 3-NCz, 2NDBT, 2-NDBP and 3-NDBP, in strain TA98NR was increased by the addition of 3-MC-induced S9 ŽTable 2.. When these nitro-PAHs were assayed by strain TA98NR using NADPH system instead of S9 mix, they showed no significant changes in the mutagenicity compared with that in strain TA98NR without S9 Ždata not shown.. Ratios of the mutagenic potency, i.e., slope ratios, of nitro-PAHs in strain TA98NR with S9 to without S9 are shown in Table 2. The slope ratio of 2-NDBP was extremely higher than that of the other nitro-PAHs tested. All of 2-nitro-PAHs tested were more efficiently activated by S9 than 3-nitro isomers, while 3-NFlone, 3-NDBT and 3-NDBP are more effectively activated by bacterial enzymes than the 2-nitro isomers. These results suggest that most of the nitro-PAHs tested are activated by S9 and that the isomer selectivity of mammalian enzymes taking part in the activation of nitro-PAHs was not the same as that of bacterial enzymes. The effects of S9 concentration on the mutagenicity of nitro-PAHs were assayed using 3MC-induced S9 Ždata not shown.. The activity of nitro-PAHs was increased with the concentration of S9, and their increase reached plateau when the S9 concentration was more than 15% Žfor NDBP. or 10% Žfor the other nitro-PAHs. in S9 mix. 3.3. Effect of S9 induction on the mutagenicity of 2- and 3-nitro-PAHs In order to determine the influence of different induction of S9 enzymes, we compared the S9 frac-
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
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Table 2 Mutagenicity of nitrofluorene, nitrofluorenone, nitrocarbazole, nitrodibenzothiophene and nitrodibenzopyranone isomers in S. typhimurium TA98, TA98NR and TA98r1,8-DNP6 with and without S9 Sample
2-NFl
Dose Žmgrplate.
b
0.3 1 3 10 Slope Žrev.rmg. 3-NFl b 1 3 10 30 Slope Žrev.rmg. 2-NFlone b 0.1 0.3 1 3 Slope Žrev.rmg. 3-NFlone b 0.01 0.03 0.1 0.3 1 3 Slope Žrev.rmg. 2-NCz b 1 3 10 30 Slope Žrev.rmg. 3-NCz b 3 10 30 Slope Žrev.rmg. d 2-NDBT b 0.1 0.2 0.5 1 Slope Žrev.rmg. 3-NDBT b 0.05 0.1 0.2 0.5 1 Slope Žrev.rmg. 2-NDBP c 0.3 1 3 10 Slope Žrev.rmg. 3-NDBP c 1 3 10 Slope Žrev.rmg.
d
d
d
d
d
d
d
d
d
yS9
qS9
a
TA98
TA98NR
TA98r1,8-DNP6
TA98NR
63 " 12 210 " 34 638 " 117 – 212 39 " 5 150 " 5 350 " 39 1071 " 268 35 121 " 23 332 " 53 1373 " 64 – 1386 33 " 6 145 " 16 384 " 23 1035 " 218 – – 3420 45 " 1 166 " 12 465 " 69 1419 " 207 47 19 " 7 51 " 14 76 " 33 2 128 " 11 236 " 64 525 " 153 1210 " 280 1191 88 " 43 204 " 73 335 " 88 1001 " 224 – 1997 22 " 6 69 " 11 220 " 14 593 " 9 58 103 " 1 423 " 47 1290 " 24 129
–
28 " 13 93 " 5 266 " 6 827 " 45 82 16 " 1 70 " 11 199 " 22 605 " 60 20 46 " 12 125 " 24 402 " 44 1123 " 123 372 – – 46 " 12 197 " 27 620 " 82 2153 " 99 721 – 84 " 1 289 " 38 837 " 11 27 1"3 22 " 4 27 " 24 0.9 36 " 8 100 " 26 212 " 15 449 " 26 380 – 36 " 1 67 " 10 174 " 38 400 " 128 458 – 22 " 3 44 " 16 128 " 9 12 53 " 7 189 " 34 603 " 75 60
39 " 22 109 " 9 358 " 10 1147 " 35 114 – 44 " 4 105 " 8 244 " 12 7 – 104 " 24 333 " 60 1036 " 176 345 – – – 62 " 18 225 " 38 702 " 23 235 – 64 " 16 228 " 39 650 " 121 21 8"2 27 " 6 46 " 3 1 35 " 12 105 " 12 353 " 55 1000 " 117 1005 12 " 1 24 " 6 43 " 4 67 " 11 188 " 6 226 101 " 16 292 " 25 855 " 17 – 282 97 " 21 308 " 7 960 " 51 95
47 " 1 139 " 25 437 " 57 43 8"6 34 " 3 98 " 18 225 " 54 7 – 59 " 12 198 " 9 574 " 48 191 – – – 109 " 16 349 " 32 1143 " 177 382 15 " 2 51 " 10 149 " 24 410 " 35 13 2"1 7"3 22 " 15 0.7 43 " 18 86 " 12 194 " 68 417 " 73 413 17 " 5 32 " 1 59 " 25 133 " 18 292 " 1 287 1"3 6"2 9"1 29 " 6 2 48 " 6 102 " 8 317 " 32 30
Slope ratio in TA98NRŽqS9ryS9.
2.7
1.0
1.8
0.6
1.8
1.4
2.4
0.8
141.0
3.2
72
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
tion from untreated and PB-, 3-MC-, b-NF- and PCB-pretreated rats for the activation of 2- and 3-nitro-PAHs using strain TA98NR ŽTable 3.. All the S9 fractions tested enhanced the mutagenicity of several nitro-PAHs, and the extent of enhancement by S9 varied with nitro-PAHs and S9 inducers. Five out of 8 nitro-PAHs, 2-NFl, 2-NFlone, 2-NCz, 3-NCz and 2-NDBP, showed the highest enhancement of the mutagenic activity in the presence of 3-MC-induced S9. 3-NDBP was activated by 3-MC-induced S9 in the same level as that using S9 from rats treated with PB and b-NF. 2-NDBP and 3-NDBP were activated by all of the S9 tested. On the contrary, mutagenicity of 3-NFlone decreased by the addition of S9 regardless of the enzymes induction. 3.4. Mutagenicity of 2-NDBP with cytosol or microsomes Since 2-NDBP was most effectively activated by 3-MC-induced S9 among the nitro-PAHs tested, the characterization of the enzymes participating in the 2-NDBP activation was performed using 3-MC-induced S9. Subfractions of 3-MC-induced S9, i.e., cytosol and microsomes, were compared for their ability to activate 2-NDBP ŽFig. 2.. Both cytosolic and microsomal fractions activated 2-NDBP. However, the mutagenicity of 2-NDBP with cytosol was seven times higher than that with microsomes, indicating that the enzymes participating in the activation of 2-NDBP were mainly present in cytosolic fraction. 3.5. Effect of different electron donors and nitroreductase inhibitor on the mutagenicity of 2-NDBP Knowing that cytosolic xanthine oxidase ŽXOD., aldehyde oxidase ŽAO. and NADŽP.H-quinone ox-
ido-reductase ŽNQOR. participate in the nitroreduction of some nitroarenes w31–34x, we evaluated the involvement of these enzymes in the activation of 2-NDBP using their electron donors and inhibitors. As shown in Fig. 2, 2-NDBP was remarkably activated in the presence of S9 mix including NADPH or NADH, which were electron donors for NQORmediated nitroreduction w34x and NADH was an electron donor for XOD w35x. Mutagenic potency of 2-NDBP was slightly enhanced by the addition of hypoxanthine ŽHX.-S9 mix, while HX was also an electron donor for XOD-mediated nitroreduction w31x. Swaminathan et al.w35x reported that 4-nitrobiphenyl was activated through nitroreduction by NADH-XOD mix in aerobic conditions. The XOD-mediated nitroreduction of 1-nitropyrene and 3-nitrofluoranthene using HX as an electron donor was inhibited by oxygen w31x. These reports suggest that XOD could catalyze nitroreduction of nitro-PAHs; however, the efficiency is depend on an electron donor. The difference of activation of 2-NDBP by HX-S9 mix and NADH-S9 mix would be attributed to this property of XOD. S9 mix including 1-methylnicotinamide ŽMN. and 2-hydroxypyrimidine ŽHP., electron donors for AO w32,33x, also enhanced the mutagenicity of 2-NDBP, but the extent of enhancement was much lower than that by NADŽP.H-S9 mix. The enhancement of the mutagenicity of 2-NDBP was not observed by the addition of the electron donors alone Ždata not shown.. These results demonstrated that XOD and NQOR would participate in activating 2-NDBP. This assumption is also supported by the result that the mutagenicity of 2-NDBP dependent on the activation by S9 mix was remarkably inhibited by AL, XOD inhibitor ŽFig. 2.. In the presence of 0.1 mM of AL, mutagenic activity of 2-NDBP was 16% of that without inhibitor. Since Hajos and Winston w34x reported that DI, NQOR inhibitor, depressed XOD catalyzed nitroreduction of dinitropy-
Notes to Table 2: Results are presented as the mean net Hisq revertantsrplate "SD Žn s 6.. Spontaneous revertantsrplate: TA98 yS9 30 " 10; TA98NR yS9 21 " 6, qS9Ž10%. 28 " 9, Ž15%. 31 " 6; TA98r1,8-DNP6 yS9 17 " 3. a S9 was prepared from the rats pretreated with 3-MC. b S9 mix was composed of NADPH system and 10%Žvrv. of S9. c S9 mix was composed of NADPH system and 15%Žvrv. of S9. d The slope of dose-response Žrevertantsrmg. was calculated by least-squares linear regression from the linear portion of the dose-response curve.
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
rene isomers by 50%, the decrease in the mutagenicity of 2-NDBP by DI might be due to not only the inhibition of NQOR but also the inhibition of XOD. ME, well-known AO inhibitor, also suppressed the activation of 2-NDBP by S9 to 60% of that without inhibitor. Since naphthoquinones, such as ME, are the best electron acceptors in the NQOR-mediated oxidation of NADŽP.H w36x, the depression of muta-
73
genic potency of 2-NDBP observed in the presence of ME would be attributed to the inhibitory effect of ME on the electron transfer from NADPH to 2-NDBP catalyzed by NQOR. The suppressive effect of PI, inhibitor of microsomal cytochrome P-450 system, on the activation of 2-NDBP by S9 was the weakest among the nitroreductase inhibitors used. This supports the presumption that the enzymes taking part in
Table 3 Effect of S9 inducer on the mutagenicity of nitrofluorene, nitrofluorenone, nitrocarbazole and nitrodibenzopyranone isomers in S. typhimurium TA98NR Sample
Dose
yS9
Žmgrplate. 2-NFl
c
1 3 10 Slope Žrev.rmg. 3-NFl c 3 10 30 Slope Žrev.rmg. 2-NFlone c 0.3 1 3 Slope Žrev.rmg. 3-NFlone c 0.3 1 3 Slope Žrev.rmg. 2-NCz c 3 10 30 Slope Žrev.rmg. e 3-NCz c 3 10 30 Slope Žrev.rmg. 2-NDBP d 0.3 1 3 Slope Žrev.rmg. 3-NDBP d 1 3 10 Slope Žrev.rmg.
e
e
e
e
e
e
e
qS9 None
47 " 1 139 " 25 437 " 57 43 34 " 3 98 " 18 225 " 54 7 59 " 12 198 " 9 574 " 48 191 109 " 16 349 " 32 1143 " 177 382 51 " 10 149 " 24 410 " 35 13 2"1 7"3 22 " 15 0.7 1"3 6"2 9"1 2 48 " 6 102 " 8 317 " 32 30
a
45 " 8 144 " 1 465 " 50 46 22 " 8 70 " 11 123 " 1 3 12 " 2 84 " 3 251 " 94 85 55 " 29 224 " 12 542 " 54 179 65 " 6 189 " 35 528 " 43 17 3"1 13 " 5 20 " 8 0.6 51 " 11 130 " 8 403 " 36 132 129 " 3 257 " 49 774 " 133 75
PB
b
51 " 7 147 " 7 458 " 3 45 20 " 3 79 " 17 146 " 17 4 11 " 6 34 " 8 101 " 6 33 38 " 9 144 " 17 428 " 24 143 65 " 1 200 " 38 617 " 65 20 6"4 12 " 6 31 " 5 0.9 27 " 5 78 " 12 243 " 33 80 121 " 21 331 " 43 995 " 166 98
3-MC
b
109 " 9 358 " 10 1147 " 35 114 44 " 4 105 " 8 244 " 12 7 104 " 24 333 " 60 1036 " 176 345 62 " 18 225 " 38 702 " 23 235 64 " 16 228 " 39 650 " 121 21 8"2 27 " 6 46 " 3 1 101 " 16 292 " 25 855 " 17 282 97 " 21 308 " 7 960 " 51 95
b-NF
b
104 " 54 366 " 4 965 " 17 95 31 " 5 100 " 16 186 " 19 5 63 " 1 185 " 4 538 " 66 177 63 " 1 211 " 32 634 " 41 211 67 " 16 224 " 54 637 " 20 21 7"6 24 " 5 31 " 1 0.9 65 " 9 206 " 16 608 " 78 202 152 " 17 306 " 23 958 " 129 93
PCB
b
110 " 51 231 " 66 651 " 191 62 21 " 8 64 " 13 162 " 16 5 68 " 13 211 " 13 648 " 43 215 39 " 9 138 " 29 431 " 88 144 30 " 1 102 " 16 351 " 10 11 8"2 25 " 3 39 " 21 1 37 " 1 126 " 17 390 " 30 130 141 " 1 341 " 10 909 " 75 88
Results are presented as the mean net hisq revertantsrplate "SD Ž n s 6.. Spontaneous revertantsrplate: yS9 21 " 6; qS9 None 31 " 11, PB 28 " 7, 3-MC 31 " 6, b-NF 32 " 5, PCB 35 " 7. a S9 was prepared from unpretreated rats. b S9 was prepared from the rats pretreated with PB, 3-MC, b-NF and PCB. c S9 mix was composed of NADPH system and 10% Žvrv. of S9. d S9 mix was composed of NADPH system and 15% Žvrv. of S9. e The slope of dose-response Žrevertantsrmg. was calculated by least-squares linear regression from the linear portion of the dose-response curve.
74
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
the activation of 2-NDBP were mainly present in cytosol. 3.6. Metabolic actiÕation of 2-NDBP by XOD
Fig. 2. Mutagenicity of 2-NDBP in S. typhimurium TA98NR with S9, S9-subfractions, electron donors and nitroreductase inhibitors. NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; NADH, nicotinamide adenine dinucleotide, reduced form; HX, hypoxanthine; MN, 1-methylnicotinamide; HP, 2-hydroxypyrimidine; AL, allopurinol; DI, dicumarol; ME, menadione; PI, piperonyl butoxide.
The results described above suggest that cytosolic XOD participates in the 2-NDBP activation by S9. Our previous result that rat liver XOD is significantly induced by the pretreatment with 3-MC w12x also supports this assumption. In order to investigate the ability of XOD to transform 2-NDBP to active metabolites, mutagenicity of 2-NDBP was tested in the presence of commercial preparations of XOD ŽFig. 3.. As shown in Fig. 3, 2-NDBP was activated by XOD mix including NADH as an electron donor, and mutagenic potency of 2-NDBP was dependent on XOD concentration. AL obviously inhibited the metabolic activation of 2-NDBP by NADH-XOD mix. 2-NDBP was also activated by NADPH-XOD mix, which included NADPH as an electron donor. However, the mutagenicity of 2-NDBP in the presence of NADPH-XOD Ž20 mU. mix was much lower than that with NADH-XOD Ž20 mU. mix. These results support the presumption that cytosolic XOD activates 2-NDBP, and suggest the existence of the enzymes using NADPH as an electron donor to transform 2-NDBP to active metabolites in S9 besides XOD.
Fig. 3. Mutagenicity of 2-NDBP in S. typhimurium TA98NR with XOD. 4 mmol of NADH or NADPH was used per 1 ml of XOD mix. 0.1 mM of allopurinol ŽAL. was used with 20 mU of XOD and NADPH.
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
3.7. Base-pair substitution mutation induced by 2- and 3-NDBP isomers with and without S9
75
pendently, although the numbers of revertants did not reach 2-fold increase over back ground even at the highest dose. 3-NDBP was mutagenic in strains TA7002, TA7004, TA7005 and TA7006 without S9. In the presence of S9, both compounds were mutagenic in strains TA7002, TA7004, TA7005 and TA7006. These are the strains responded to both isomers without S9, except for TA7006 to 2-NDBP. The slopes Žrevertantsr10 mg. of the dose-response curves in these 4 strains increased by the addition of S9 ŽFig. 4.. Whether the activation enzymes are endogenous or exogenous, both compounds showed the highest mutagenicity in strain TA7005 ŽC P G ™ A P T. followed by TA7004 ŽG P C ™ A P T., TA7006 ŽC P G ™ G P C. and TA7002 ŽT P A ™ A P T.. The
Table 4 shows the mutagenicity of 2- and 3-NDBP isomers in base-pair substitution mutation with and without S9 mix. Both 2-NDBP and 3-NDBP were mutagenic in strain TA100 and the potency was enhanced by 3-MC-induced S9. Enhancement of the mutagenicity of 2-NDBP by S9 was higher than that of 3-NDBP, consistent with these compounds acting as frameshift mutagens. In the absence of S9, 2NDBP was negative in strains TA7001, TA7003 and TA7006, and was judged equivocal in its effect in strains TA7002, TA7004 and TA7005. The revertant colonies in the latter three strains increased dose-de-
Table 4 Mutagenicity of 2-NDBP and 3-NDBP in S. typhimurium TA100, TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006 with and without S9 Sample
yS9 rq S9
Dose Žmgrplate.
TA100
TA7001
2-NDBP
yS9
0 2 5 10 20 0 0.5 1 2 5 10 20
127 " 22 201 " 21 273 " 30 359 " 39 497 " 74 116 " 20 169 " 12 216 " 38 292 " 4 552 " 65 1182 " 88 –
2"1 3"2 2"1 2"2 2"1 3"2 – – 4"2 3"2 4"2 2"1
0 2 5 10 20 50 0 1 2 5 10 20 50
115 " 12 153 " 23 218 " 27 343 " 35 520 " 131 1148 " 58 116 " 20 169 " 3 196 " 10 347 " 42 627 " 22 1036 " 170 –
2"1 – 2"2 4"2 2"2 2"1 4"1 – – 4"2 4"3 3"3 4"1
qS9
3-NDBP
a
yS9
qS9
a
TA7002 2"2 3"1 4"3 4"2 5"2 3"2 – – 13 " 7 21 " 9 35 " 13 65 " 5 3"1 – 7"3 12 " 5 15 " 3 18 " 2 5"5 – – 9"7 12 " 9 23 " 7 "39 " 2
TA7003
TA7004
TA7005
TA7006
1 " 0.5 1 " 0.6 1 " 0.4 1 " 0.4 1 " 0.4 1"1 – – 2"1 2"1 3"2 2"2
28 " 7 25 " 3 33 " 13 34 " 14 40 " 14 37 " 9 – – 54 " 10 75 " 18 111 " 29 154 " 20
19 " 5 21 " 4 31 " 10 33 " 8 35 " 7 25 " 6 – – 76 " 19 131 " 47 233 " 79 270 " 99
8"1 7"3 8"3 10 " 4 8"5 5"3 – – 15 " 3 19 " 7 24 " 10 33 " 6
2"2 – 2"1 1"1 2"2 2"1 1 " 0.4 – – 1 " 0.9 1 " 0.4 3"1 2"1
30 " 4 – 34 " 1 42 " 7 64 " 16 110 " 9 37 " 9 – – 93 " 16 104 " 12 178 " 2 320 " 19
16 " 5 – 32 " 4 54 " 13 119 " 33 236 " 110 22 " 2 – – 90 " 16 136 " 23 317 " 49 451 " 65
3"1 – 9"1 10 " 3 18 " 8 37 " 1 2"1 – – 18 " 1 27 " 3 41 " 16 50 " 7
Results are presented as the mean Hisq revertantsrplate "SD Ž n s 6.. S9 mix was composed of NADPH system and 15% Žvrv. of S9 which was prepared from rats pretreated with 3-MC.
a
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T. Watanabe et al.r Mutation Research 388 (1997) 67–78
ent urban air would be ubiquitously polluted with 2-NDBP. Additional research and exposure monitoring is necessary before the human exposure and cancer risk of 2-NDBP can be evaluated.
Acknowledgements We are grateful to Dr. Naoki Miyata, National Institute of Health Sciences, for the generous gifts of 3-NFl. We are grateful to Prof. Bruce N. Ames, University of California, who kindly supplied Salmonella typhimurium strains TA98 and TA100, and to Prof. Herbert S. Rosenkranz, Chairman, University of Pittsburgh, who kindly supplied S. typhimurium strains TA98NR and TA98r1,8-DNP6 . We thank Xenometrix Inc. for providing S. typhimurium strains TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006. We thank Ms. Satomi Shirakawa, Ms. Yuka Miyoshi, Ms. Tamiko Kintou and Ms. Kazuko Maegawa for their technical assistance in this investigation. Fig. 4. Mutagenic potency of 2-NDBPŽA. and 3-NDBPŽB. with and without S9 mix in a set of six S. typhimurium tester strains ŽTA7001 to TA7006.. Mutagenic potency was calculated by least-squares linear regression from the linear portion of the dose-response curve, and presented as a number of revertants per 10 mg of each compound. I, without S9 mix; B, with S9 mix.
result that NDBP isomers showed the similar mutation specificities with and without S9 ŽFig. 4. indicate that the mechanism of mutagenesis caused by NDBP isomers with S9 is similar to that without S9, that is, S9 enzymes transform NDBP isomers to the same active metabolites as those formed by bacterial enzymes. C P G ™ A P T transversion, which was mainly induced by NDBP isomers, was found in an activated c-Ki-ras gene of a fibrosarcoma induced by 1,8-dinitropyrene, and is considered responsible for the activation of c-Ki-ras w37x. The fact that 2-NDBP was efficiently activated by mammalian enzymes described above implies that 2-NDBP might be hazardous to human health. 2-NDBP was quantitated in air particulate extract in 1992 for the first time w13x, and it was found that 2-NDBP is not only emitted from diesel engines but also formed through atmospheric transformation w14,38x, suggesting that ambi-
References w1x Tokiwa, H. and Y. Ohnishi Ž1986. Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol., 17, 23–60. w2x Rodriguez, H. and G.L. Murison Ž1990. Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells, Mutation Res., 240, 73–81. w3x Shane, B.S., G.L. Squadrito, D.F. Church and W.A. Pryor Ž1991. Comparative mutagenicity of nitrofluoranthenes in Salmonella typhimurium TA98, TA98NR, and TA98r1,8DNP6 , Environ. Mol. Mutagen., 17, 130–138. w4x Tokiwa, H., R. Nakagawa, K. Horikawa and A. Ohkubo Ž1987. The nature of the mutagenicity and carcinogenicity of nitrated, aromatic compounds in the environment, Environ. Health Perspect., 73, 191–199. w5x Horikawa, K., N. Sera, T. Otofuji, K. Murakami, H. Tokiwa, M. Iwagawa, K. Izumi and H. Otsuka Ž1991. Pulmonary carcinogenicity of 3,9- and 3,7-dinitrofluoranthene, 3-nitrofluoranthene and benzow axpyrene in F344 rats, Carcinogenesis, 12, 1003–1007. w6x Arey, J., B. Zielinska, W.P. Harger, R. Atkinson and A.M. Winer Ž1988. The contribution of nitrofluoranthenes and nitropyrenes to the mutagenic activity of ambient particulate organic matter collected in southern California, Mutation Res., 207, 45–51. w7x Nishioka, M.G., C.C. Howard, D.A. Contos, L.M. Ball and J.
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w20x Gee, P., D.M. Maron and B.N. Ames Ž1994. Detection and classification of mutagens: A set of base-specific Salmonella tester strains, Proc. Natl. Acad. Sci. USA, 91, 11606–11610. w21x Mendenhall, G.D. and P.A.S. Smith Ž1973. 2-Nitrocarbazole, Org. Synth., 5, 829–833. w22x Eikhman, R.K., V.O. Lukashevich and E.A. Silaeva Ž1939. Production of nitro and amino derivatives of carbazole, Org. Chem. Ind., 6, 93–95. w23x Cullinane, N.M., C.G. Davies and G.I. Davies Ž1936. Substitution derivatives of diphenylene sulphide and diphenylenesulphone, J. Chem. Soc., 1435–1437. w24x Brown, R.K., N.A. Nelson and J.C. Wood Ž1952. Nitro and amino derivatives of dibenzothiophene, J. Am. Chem. Soc., 74, 1165–1167. w25x Smith, P.A.S. and B.B. Brown Ž1951. The reaction of aryl azides with hydrogen halides, J. Am. Chem. Soc., 73, 2438– 2441. w26x Smith, P.A.S. and B.B. Brown Ž1951. The synthesis of hetero- cyclic compounds from aryl azides. I. Bromo and nitro carbazoles, J. Am. Chem. Soc., 73, 2435–2437. w27x Maron, D.M. and B.N. Ames Ž1983. Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173–215. w28x Rosenkranz H.S. and R. Mermelstein Ž1983. Mutagenicity and genotoxicity of nitroarenes: all nitro-containing chemicals were not created equal, Mutation Res., 114, 217–267. w29x Quilliam, M.A., F. Messier, C. Lu, P.A. Andrews, B.E. McCarry and D.R. McCalla Ž1982. The metabolism of nitrosubstituted polycyclic aromatic hydrocarbons in Salmonella typhimurium, in: Proceedings, International Conference on Polycyclic Aromatic Hydrocarbons, Vol. 5, Battelle, Columbus OH, pp. 667– 672. w30x McCoy, E.C., G.D. McCoy and H.S. Rosenkranz Ž1982. Esterification of arylhydroxylamines: evidence for a specific gene product in mutagenesis, Biochem. Biophys. Res. Commun., 108, 1362–1367. w31x Bauer, S.L. and P.C. Howard Ž1990. The kinetics of 1-nitropyrene and 3-nitrofluoranthene metabolism using bovine liver xanthine oxidase, Cancer Lett., 54, 37–42. w32x Tatsumi, K, S. Kitamura and N. Narai Ž1986. Reductive metabolism of aromatic nitro compounds including carcinogens by rabbit liver preparations, Cancer Res., 46, 1089– 1093. w33x Bauer, S.L. and P.C. Howard Ž1991. Kinetics and cofactor requirements for the nitroreductive metabolism of 1-nitropyrene and 3-nitrofluoranthene by rabbit liver aldehyde oxidase, Carcinogenesis, 9, 1545–1549. w34x Hajos, A.K.D. and G.W. Winston Ž1991. Dinitropyrene nitroreductase activity of purified NADŽP.H-quinone oxidoreductase: role in rat liver cytosol and induction by Aroclor1254 pretreatment, Carcinogenesis, 12, 697–702. w35x Swaminathan, S. and J.F. Hatcher Ž1986. Xanthine oxidasemediated mutagenicity of the bladder carcinogen 4-nitrobiphenyl, Mutation Res., 172, 37–45. w36x Ernster, L. Ž1967. DT-diaphorase, Methods Enzymol., 10, 309– 317.
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w37x Tahira, T., K. Hayashi, M. Ochiai, N. Tsuchida, M. Nagao and T. Sugimura Ž1986. Structure of the c-Ki-ras gene in a rat fibrosarcoma induced by 1,8-dinitropyrene, Mol. Cell. Biol., 1349–1351.
w38x Helmig, D., J. Arey, W.P. Harger and R. Atkinson and J. Lopez-Cancio Ž1992. Formation of mutagenic nitrodibenzopyranones and their occurrence in ambient air, Environ. Sci. Technol., 26, 622–624.
Metabolic activation of 2- and 3-nitrodibenzopyranone isomers and related compounds by rat liver S9 and the effect of S9 on the mutational specificity of nitrodibenzopyranones Tetsushi Watanabe a,) , Hideaki Kaji a , Mari Takashima a , Terue Kasai a , Joellen Lewtas b, Teruhisa Hirayama a a
b
Kyoto Pharmaceutical UniÕersity, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607, Japan National Health and EnÕironmental Effects Research Laboratory, U.S. EnÕironmental Protection Agency, Research, Triangle Park, NC 27711, USA Received 24 July 1996; revised 27 September 1996; accepted 1 October 1996
Abstract The effect of rat liver S9 on the mutagenicity of 10 nitrated polycyclic aromatic hydrocarbons Žnitro-PAHs. was evaluated with Salmonella typhimurium TA98NR using S9 from phenobarbital-, 3-methylcholanthrene ŽMC.-, b-naphthoflavone- and polychlorobiphenyl-treated and untreated rats. 2-Nitrofluorene Ž2-NFl., 2-nitrofluoren-9-one Ž2-NFlone., 2-nitrocarbazole Ž2-NCz., 3-NCz, 2-nitrodibenzothiophene Ž2-NDBT., 2-nitro-6 H-dibenzow b,d xpyran-6-one Ž2-NDBP. and 3-NDBP were metabolically activated by one or more of the S9 fractions, and the highest enhancement of the mutagenic potency of nitro-PAHs was observed with 3-MC-induced S9. Only in the case of 3-NFlone was the mutagenicity in strain TA98NR decreased by the addition of S9, regardless of S9 induction. 2-NDBP was most efficiently activated among nitro-PAHs tested by all S9 fractions used. The cytosolic fraction of S9 accounted for more of the activation of 2-NDBP than the microsomal fraction. NADH and NADPH were the most effective electron donors on the activation of 2-NDBP by S9. 2-NDBP was also metabolically activated by NADH plus commercial preparations of xanthine oxidase. These activations of 2-NDBP were inhibited by allopurinol, indicating that cytosolic xanthine oxidase in rat liver S9 participates in the activation of 2-NDBP. The potency of 2- and 3-NDBP isomers as base-substitution mutagens was also enhanced by S9. In the presence of S9, both compounds showed the highest mutagenicity in strain TA7005 ŽC P G ™ A P T. followed by strains TA7004 ŽG P C ™ A P T., TA7006 ŽC P G ™ G P C. and TA7002 ŽT P A ™ A P T., and this mutation specificity was similar to that without S9, indicating that the mechanism of mutagenesis caused by NDBP isomers with S9 is similar to that without S9. Keywords: Metabolic activation; Salmonella typhimurium; Nitro-6 H-dibenbzow b,d xpyran-6-one; Nitro-polycyclic aromatic hydrocarbons
1. Introduction
) Corresponding author. Tel.: q81-75-595-4650; Fax: q81-75595-4769.
Nitrated polycyclic aromatic hydrocarbons ŽnitroPAHs. have become of enormous concern because of their genotoxicity, e.g., mutagenicity w1–3x and carcinogenicity w4,5x, and ubiquity in ambient air
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 2 1 8 Ž 9 6 . 0 0 1 3 8 - 3
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T. Watanabe et al.r Mutation Research 388 (1997) 67–78
w6–8x. The mutagenic nitro-PAHs represented by nitrofluorenes, nitrofluoranthenes and nitropyrenes are metabolically activated by the endogenous nitroreductase in Salmonella typhimurium tester strains w1,9–11x. We reported that nitrodibenzofuran isomers were efficiently activated by not only endogenous nitroreductase in S. typhimurium TA98 but also rat liver S9 w12x. Nitro-6 H-dibenzow b,d xpyran-6-one ŽNDBP. isomers, which are recently identified as ambient air pollutants w13,14x, are potent mutagens in S. typhimurium tester strains w15,16x, and the mutagenicity of NDBP was also enhanced by the addition of Aroclor-induced S9 w16x. In the presence of S9, 2-NDBP exhibited base-substitution activity that was greater than its frameshift activity, whereas 3-NDBP exhibited more frameshift than base-substitution activity w16x. Several nitro-PAHs which have the structure similar to nitrodibenzofuran and NDBP, such as nitrofluorene ŽNFl., nitrofluoren-9-one ŽNFlone., nitrocarbazole ŽNCz. and nitrodibenzothiophene ŽNDBT., were detected in ambient air and diesel exhaust extracts w17–19x. Since these nitro-PAHs are expected to be activated by mammalian enzymes like nitrodibenzofuran and NDBP, assessing the mutagenicity of these nitro-PAHs in the presence of mammalian S9 enzymes is of importance for the evaluation of these nitro-PAHs on the risk for human health. In this report, we describe the ability of rat liver S9 to activate 2- and 3-nitro-PAHs isomers including NFl, NFlone, NCz, NDBT and NDBP. The enzymes participating in the metabolic activation of 2-NDBP in rat liver S9 are characterized by subfractions of S9, i.e., cytosolic and microsomal fractions, and selected electron donors and nitroreductase inhibitors. Four DNA bases can result in six different type base-pair substitution mutations ŽG P C ™ C P G, G P C ™ T P A, A P T ™ T P A, A P T ™ C P G transversions and A P T ™ G P C, G P C ™ A P T transitions.. Determining the pattern of six possible base substitutions induced by mutagen would be helpful to understand the mechanism of mutagenesis. Mutagenicity of 2- and 3-NDBP isomers were assayed by a set of six Salmonella typhimurium tester strains ŽTA7001 to TA7006., each of which reverts by only one specific base-pair substitution out of the six possible changes w20x, with and without S9 in order to evaluate the effect of the rat liver S9 metabolic
activation on the base substitution induction by NDBP isomers.
2. Materials and methods 2.1. Chemicals 2-NFl, 4-nitroquinoline-N-oxide Ž4-NQO., methyl methanesulfonate ŽMMS., N-methyl-N X-nitro-Nnitrosoguanidine ŽMNNG., b-naphthoflavone ŽbNF., hypoxanthine ŽHX., menadione ŽME. and allopurinol ŽAL. were purchased from Nacalai Tesque ŽKyoto, Japan.. 2-NFlone, 2-acetylaminofluorene
Fig. 1. Structure and CAS registry number of 2- and 3- nitrofluorene ŽNFI., nitrofluoren-9-one ŽNFlone., nitrocarbazole ŽNCz., nitrodibenzothiophene ŽNDBT. and nitrodibenzopyranone ŽNDBP. isomers tested.
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
ŽAAF., 1-methylnicotinamide ŽMN. and 2-hydroxypyrimidine ŽHP. were obtained from Tokyo Kasei ŽTokyo, Japan.. 2-NDBP, 3-NDBP and 3-NFlone were purchased from Aldrich Chemical Co. ŽMilwaukee, WI.. 3-Methylcholanthrene Ž3-MC., phenobarbital ŽPB., polychlorobiphenyl penta ŽPCB, KC-500., dicumarol ŽDI., piperonyl butoxide ŽPI. and dimethyl sulfoxide ŽDMSO. were obtained from Wako Pure Chemicals ŽOsaka, Japan.. Glucose 6phosphate ŽG-6-P., glucose-6-phosphate dehydrogenase ŽG-6-PDH., nicotinamide-adenine dinucleotide, reduced form ŽNADH. and nicotinamide-adenine dinucleotide phosphate, reduced form ŽNADPH. were purchased from Oriental Yeast ŽTokyo, Japan.. Xanthine oxidase ŽXOD, Grade III, EC 1.2.3.2. was obtained from Sigma Chemical Co. ŽSt. Louis, MO.. 3-NFl was gift from Dr. Miyata, National Institute of Health Sciences. 2-NCz and 3-NCz were synthesized according to Mendenhall and Smithw21x and Eikhman et al.w22x, respectively. 2-NDBT and 3-nitrodibenzothiophene5-oxide were synthesized by the method described by Cullinane et al.w23x. 3-Nitrodibenzothiophene-5oxide was reduced to 3-NDBT by hydrobromic acid w24x. Chemical structures were confirmed by 1 HNMR, mass spectrum and melting point measurement. The melting points of authentic 2-NCz, 3-NCz, 2-NDBT and 3-NDBT were 175 ; 1788C, 211 ;
69
2138C, 188.5 ; 189.58C and 156 ; 1578C, respectively ŽReference: 2-NCz 174 ; 175.58C w25x, 3-NCz 212 ; 2138C w26x, 2-NDBT 1868C w23x, 3-NDBT 153 ; 1548C w24x.. High-performance liquid chromatography analysis indicated the purities of NCz and NDBT isomers greater than 99% as judged by monitoring of the eluate at 254 nm. 2.2. Mutagenicity assay The structures, abbreviations and CAS registry numbers of the compounds tested are shown in Fig. 1. The genotypic characteristics of S. typhimurium strains are listed in Table 1. S. typhimurium strains TA98 and TA100 were kindly provided by Dr. B.N. Ames, University of California, Berkeley. CA. Strains TA98NR and TA98r1,8-DNP6 were kindly provided by Dr. H.S. Rosenkranz, University of Pittsburgh, PA. Strains TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006 were provided by Xenometrix Inc., Boulder, CO. The mutagenicity assay was performed as described by Maron and Ames w27x. All samples tested were dissolved in DMSO. Mixtures of cell suspension, sample solution and 0.1 M sodium phosphate buffer or exogenous enzyme sources, e.g., S9 mix, were incubated at 378C for 20 min with shaking before pouring on the plates. Each tester strain was checked routinely to
Table 1 Genotypes of the Salmonella typhimurium TA strains used Strain a
TA98 TA100
a
TA7001 b
Mutation
Cell wall
Repair
Plasmid
hisD3052 hisG46
rfa rfa
D uÕrB D uÕrB
pKM101 pKM101
hisG1775
rfa
D uÕrB
pKM101
Reversion event
Asp y 153™Gly y 153 ŽGAT .
TA7002
b
hisC9138
rfa
D uÕrB
pKM101
b
hisG9074
rfa
D uÕrB
pKM101
TA7004
b
hisG9133
rfa
D uÕrB
pKM101
TA7005
b
hisG9130
rfa
D uÕrB
pKM101
TA7006
b
hisC9070
rfa
D uÕrB
pKM101
a b
Maron and Ames w27x. Gee et al. w20x.
C P G ™ A P T transversion
ŽGAG .
Arg y 163™Gly y 163 ŽCGA .
G P C ™ A P T transition
ŽGAG .
Ala y 169™Glu y 169 ŽGCG .
T P A ™ G P C transversion
ŽGGT .
Gly y 169™Glu y 169 ŽGGG .
T P A ™ A P T transversion
ŽAAA .
Val y 153™Gly y 153 ŽGTT .
A P T ™ G P C transition
ŽGGT .
Ile y 217™Lys y 217 ŽATA .
TA7003
Detected mutation
ŽGGA .
C P G ™ G P C transversion
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T. Watanabe et al.r Mutation Research 388 (1997) 67–78
confirm their features for optimal response to known mutagens as follows: 4-NQO Ž0.5 or 1 mgrplate., MMS Ž650 mgrplate., MNNG Ž4 mgr plate. and AAF Ž5 mgrplate.. All experiments were performed in duplicate or triplicate with at least three doses and in at least three times. The slope of dose-response Žrevertantsrmg. was calculated by least-squares linear regression from the linear portion of the dose-response curve. Male Sprague-Dawley rats were treated with PB, 3-MC, b-NF or PCB, and liver S9, cytosol and microsomes were prepared as described before w12x. Liver S9, cytosol, microsomes and commercial preparations of XOD were used as exogenous enzyme source. NADPH system consisted of MgCl 2 Ž8 mmol., KCl Ž33 mmol., G-6-P Ž5 mmol., G-6-PDH Žfrom yeast; 0.5 unit., NADPH Ž4 mmol., sodium phosphate buffer Ž100 mmol, pH 7.4. and water per 1 ml of exogenous enzyme mix. NADPH system was used for the mutagenicity assay unless otherwise noted. NADH, HX, MN and HP systems contained MgCl 2 Ž8 mmol., KCl Ž33 mmol., sodium phosphate buffer Ž100 mmol, pH 7.4., water and an electron donor Ž4 mmol., i.e., NADH, HX, MN or HP, for exogenous enzyme mix Ž1 ml..
3. Results and discussion 3.1. Mutagenicity of 2- and 3-nitro-PAHs in S. typhimurium TA98, TA98 r 1,8-DNP6 and TA98NR without S9 All of nitro-PAHs tested were mutagenic in strain TA98 without S9, and the activity decreased in strains TA98NR and TA98r1,8-DNP6 , which are the ‘classical’ nitroreductase- and the O-acetyltransferase-deficient derivatives of strain TA98, respectively w9,28–30x ŽTable 2.. The mutagenic potency of the tested compounds was estimated by a slope of dose-response Žrevertantsrmg.. In the absence of S9, mutagenic potency of nitro-PAHs in strain TA98NR was 3 ; 35% and 17 ; 109% of that in strains TA98 and TA98r1,8-DNP6 , respectively. This suggests that nitroso andror hydroxylamino derivatives from these nitro-PAHs metabolized by nitroreductase in strain TA98 are mutagenic, and that nitroreduction is a significant step in the metabolic activation of most
nitro-PAHs as compared with an esterification step. The order of the mutagenic potency between 2- and 3-nitro-PAH isomers in strain TA98 were as follows: 2-NFl ) 3-NFl; 2-NFlone- 3-NFlone; 2-NCz) 3NCz; 2-NDBT- 3-NDBT and 2-NDBP- 3-NDBP. This order was the same as that observed in strain TA98NR without S9, except for NDBT isomers, indicating that isomer selectivity of the endogenous enzymes participating in the activation of nitro-PAHs in strain TA98NR are also similar to that of the ‘classical’ nitroreductase. 3.2. Metabolic actiÕation of 2- and 3-nitro-PAHs by S9 Mutagenic potency of 7 out of 10 nitro-PAHs tested, i.e., 2-NFl, 2-NFlone, 2-NCz, 3-NCz, 2NDBT, 2-NDBP and 3-NDBP, in strain TA98NR was increased by the addition of 3-MC-induced S9 ŽTable 2.. When these nitro-PAHs were assayed by strain TA98NR using NADPH system instead of S9 mix, they showed no significant changes in the mutagenicity compared with that in strain TA98NR without S9 Ždata not shown.. Ratios of the mutagenic potency, i.e., slope ratios, of nitro-PAHs in strain TA98NR with S9 to without S9 are shown in Table 2. The slope ratio of 2-NDBP was extremely higher than that of the other nitro-PAHs tested. All of 2-nitro-PAHs tested were more efficiently activated by S9 than 3-nitro isomers, while 3-NFlone, 3-NDBT and 3-NDBP are more effectively activated by bacterial enzymes than the 2-nitro isomers. These results suggest that most of the nitro-PAHs tested are activated by S9 and that the isomer selectivity of mammalian enzymes taking part in the activation of nitro-PAHs was not the same as that of bacterial enzymes. The effects of S9 concentration on the mutagenicity of nitro-PAHs were assayed using 3MC-induced S9 Ždata not shown.. The activity of nitro-PAHs was increased with the concentration of S9, and their increase reached plateau when the S9 concentration was more than 15% Žfor NDBP. or 10% Žfor the other nitro-PAHs. in S9 mix. 3.3. Effect of S9 induction on the mutagenicity of 2- and 3-nitro-PAHs In order to determine the influence of different induction of S9 enzymes, we compared the S9 frac-
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
71
Table 2 Mutagenicity of nitrofluorene, nitrofluorenone, nitrocarbazole, nitrodibenzothiophene and nitrodibenzopyranone isomers in S. typhimurium TA98, TA98NR and TA98r1,8-DNP6 with and without S9 Sample
2-NFl
Dose Žmgrplate.
b
0.3 1 3 10 Slope Žrev.rmg. 3-NFl b 1 3 10 30 Slope Žrev.rmg. 2-NFlone b 0.1 0.3 1 3 Slope Žrev.rmg. 3-NFlone b 0.01 0.03 0.1 0.3 1 3 Slope Žrev.rmg. 2-NCz b 1 3 10 30 Slope Žrev.rmg. 3-NCz b 3 10 30 Slope Žrev.rmg. d 2-NDBT b 0.1 0.2 0.5 1 Slope Žrev.rmg. 3-NDBT b 0.05 0.1 0.2 0.5 1 Slope Žrev.rmg. 2-NDBP c 0.3 1 3 10 Slope Žrev.rmg. 3-NDBP c 1 3 10 Slope Žrev.rmg.
d
d
d
d
d
d
d
d
d
yS9
qS9
a
TA98
TA98NR
TA98r1,8-DNP6
TA98NR
63 " 12 210 " 34 638 " 117 – 212 39 " 5 150 " 5 350 " 39 1071 " 268 35 121 " 23 332 " 53 1373 " 64 – 1386 33 " 6 145 " 16 384 " 23 1035 " 218 – – 3420 45 " 1 166 " 12 465 " 69 1419 " 207 47 19 " 7 51 " 14 76 " 33 2 128 " 11 236 " 64 525 " 153 1210 " 280 1191 88 " 43 204 " 73 335 " 88 1001 " 224 – 1997 22 " 6 69 " 11 220 " 14 593 " 9 58 103 " 1 423 " 47 1290 " 24 129
–
28 " 13 93 " 5 266 " 6 827 " 45 82 16 " 1 70 " 11 199 " 22 605 " 60 20 46 " 12 125 " 24 402 " 44 1123 " 123 372 – – 46 " 12 197 " 27 620 " 82 2153 " 99 721 – 84 " 1 289 " 38 837 " 11 27 1"3 22 " 4 27 " 24 0.9 36 " 8 100 " 26 212 " 15 449 " 26 380 – 36 " 1 67 " 10 174 " 38 400 " 128 458 – 22 " 3 44 " 16 128 " 9 12 53 " 7 189 " 34 603 " 75 60
39 " 22 109 " 9 358 " 10 1147 " 35 114 – 44 " 4 105 " 8 244 " 12 7 – 104 " 24 333 " 60 1036 " 176 345 – – – 62 " 18 225 " 38 702 " 23 235 – 64 " 16 228 " 39 650 " 121 21 8"2 27 " 6 46 " 3 1 35 " 12 105 " 12 353 " 55 1000 " 117 1005 12 " 1 24 " 6 43 " 4 67 " 11 188 " 6 226 101 " 16 292 " 25 855 " 17 – 282 97 " 21 308 " 7 960 " 51 95
47 " 1 139 " 25 437 " 57 43 8"6 34 " 3 98 " 18 225 " 54 7 – 59 " 12 198 " 9 574 " 48 191 – – – 109 " 16 349 " 32 1143 " 177 382 15 " 2 51 " 10 149 " 24 410 " 35 13 2"1 7"3 22 " 15 0.7 43 " 18 86 " 12 194 " 68 417 " 73 413 17 " 5 32 " 1 59 " 25 133 " 18 292 " 1 287 1"3 6"2 9"1 29 " 6 2 48 " 6 102 " 8 317 " 32 30
Slope ratio in TA98NRŽqS9ryS9.
2.7
1.0
1.8
0.6
1.8
1.4
2.4
0.8
141.0
3.2
72
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
tion from untreated and PB-, 3-MC-, b-NF- and PCB-pretreated rats for the activation of 2- and 3-nitro-PAHs using strain TA98NR ŽTable 3.. All the S9 fractions tested enhanced the mutagenicity of several nitro-PAHs, and the extent of enhancement by S9 varied with nitro-PAHs and S9 inducers. Five out of 8 nitro-PAHs, 2-NFl, 2-NFlone, 2-NCz, 3-NCz and 2-NDBP, showed the highest enhancement of the mutagenic activity in the presence of 3-MC-induced S9. 3-NDBP was activated by 3-MC-induced S9 in the same level as that using S9 from rats treated with PB and b-NF. 2-NDBP and 3-NDBP were activated by all of the S9 tested. On the contrary, mutagenicity of 3-NFlone decreased by the addition of S9 regardless of the enzymes induction. 3.4. Mutagenicity of 2-NDBP with cytosol or microsomes Since 2-NDBP was most effectively activated by 3-MC-induced S9 among the nitro-PAHs tested, the characterization of the enzymes participating in the 2-NDBP activation was performed using 3-MC-induced S9. Subfractions of 3-MC-induced S9, i.e., cytosol and microsomes, were compared for their ability to activate 2-NDBP ŽFig. 2.. Both cytosolic and microsomal fractions activated 2-NDBP. However, the mutagenicity of 2-NDBP with cytosol was seven times higher than that with microsomes, indicating that the enzymes participating in the activation of 2-NDBP were mainly present in cytosolic fraction. 3.5. Effect of different electron donors and nitroreductase inhibitor on the mutagenicity of 2-NDBP Knowing that cytosolic xanthine oxidase ŽXOD., aldehyde oxidase ŽAO. and NADŽP.H-quinone ox-
ido-reductase ŽNQOR. participate in the nitroreduction of some nitroarenes w31–34x, we evaluated the involvement of these enzymes in the activation of 2-NDBP using their electron donors and inhibitors. As shown in Fig. 2, 2-NDBP was remarkably activated in the presence of S9 mix including NADPH or NADH, which were electron donors for NQORmediated nitroreduction w34x and NADH was an electron donor for XOD w35x. Mutagenic potency of 2-NDBP was slightly enhanced by the addition of hypoxanthine ŽHX.-S9 mix, while HX was also an electron donor for XOD-mediated nitroreduction w31x. Swaminathan et al.w35x reported that 4-nitrobiphenyl was activated through nitroreduction by NADH-XOD mix in aerobic conditions. The XOD-mediated nitroreduction of 1-nitropyrene and 3-nitrofluoranthene using HX as an electron donor was inhibited by oxygen w31x. These reports suggest that XOD could catalyze nitroreduction of nitro-PAHs; however, the efficiency is depend on an electron donor. The difference of activation of 2-NDBP by HX-S9 mix and NADH-S9 mix would be attributed to this property of XOD. S9 mix including 1-methylnicotinamide ŽMN. and 2-hydroxypyrimidine ŽHP., electron donors for AO w32,33x, also enhanced the mutagenicity of 2-NDBP, but the extent of enhancement was much lower than that by NADŽP.H-S9 mix. The enhancement of the mutagenicity of 2-NDBP was not observed by the addition of the electron donors alone Ždata not shown.. These results demonstrated that XOD and NQOR would participate in activating 2-NDBP. This assumption is also supported by the result that the mutagenicity of 2-NDBP dependent on the activation by S9 mix was remarkably inhibited by AL, XOD inhibitor ŽFig. 2.. In the presence of 0.1 mM of AL, mutagenic activity of 2-NDBP was 16% of that without inhibitor. Since Hajos and Winston w34x reported that DI, NQOR inhibitor, depressed XOD catalyzed nitroreduction of dinitropy-
Notes to Table 2: Results are presented as the mean net Hisq revertantsrplate "SD Žn s 6.. Spontaneous revertantsrplate: TA98 yS9 30 " 10; TA98NR yS9 21 " 6, qS9Ž10%. 28 " 9, Ž15%. 31 " 6; TA98r1,8-DNP6 yS9 17 " 3. a S9 was prepared from the rats pretreated with 3-MC. b S9 mix was composed of NADPH system and 10%Žvrv. of S9. c S9 mix was composed of NADPH system and 15%Žvrv. of S9. d The slope of dose-response Žrevertantsrmg. was calculated by least-squares linear regression from the linear portion of the dose-response curve.
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
rene isomers by 50%, the decrease in the mutagenicity of 2-NDBP by DI might be due to not only the inhibition of NQOR but also the inhibition of XOD. ME, well-known AO inhibitor, also suppressed the activation of 2-NDBP by S9 to 60% of that without inhibitor. Since naphthoquinones, such as ME, are the best electron acceptors in the NQOR-mediated oxidation of NADŽP.H w36x, the depression of muta-
73
genic potency of 2-NDBP observed in the presence of ME would be attributed to the inhibitory effect of ME on the electron transfer from NADPH to 2-NDBP catalyzed by NQOR. The suppressive effect of PI, inhibitor of microsomal cytochrome P-450 system, on the activation of 2-NDBP by S9 was the weakest among the nitroreductase inhibitors used. This supports the presumption that the enzymes taking part in
Table 3 Effect of S9 inducer on the mutagenicity of nitrofluorene, nitrofluorenone, nitrocarbazole and nitrodibenzopyranone isomers in S. typhimurium TA98NR Sample
Dose
yS9
Žmgrplate. 2-NFl
c
1 3 10 Slope Žrev.rmg. 3-NFl c 3 10 30 Slope Žrev.rmg. 2-NFlone c 0.3 1 3 Slope Žrev.rmg. 3-NFlone c 0.3 1 3 Slope Žrev.rmg. 2-NCz c 3 10 30 Slope Žrev.rmg. e 3-NCz c 3 10 30 Slope Žrev.rmg. 2-NDBP d 0.3 1 3 Slope Žrev.rmg. 3-NDBP d 1 3 10 Slope Žrev.rmg.
e
e
e
e
e
e
e
qS9 None
47 " 1 139 " 25 437 " 57 43 34 " 3 98 " 18 225 " 54 7 59 " 12 198 " 9 574 " 48 191 109 " 16 349 " 32 1143 " 177 382 51 " 10 149 " 24 410 " 35 13 2"1 7"3 22 " 15 0.7 1"3 6"2 9"1 2 48 " 6 102 " 8 317 " 32 30
a
45 " 8 144 " 1 465 " 50 46 22 " 8 70 " 11 123 " 1 3 12 " 2 84 " 3 251 " 94 85 55 " 29 224 " 12 542 " 54 179 65 " 6 189 " 35 528 " 43 17 3"1 13 " 5 20 " 8 0.6 51 " 11 130 " 8 403 " 36 132 129 " 3 257 " 49 774 " 133 75
PB
b
51 " 7 147 " 7 458 " 3 45 20 " 3 79 " 17 146 " 17 4 11 " 6 34 " 8 101 " 6 33 38 " 9 144 " 17 428 " 24 143 65 " 1 200 " 38 617 " 65 20 6"4 12 " 6 31 " 5 0.9 27 " 5 78 " 12 243 " 33 80 121 " 21 331 " 43 995 " 166 98
3-MC
b
109 " 9 358 " 10 1147 " 35 114 44 " 4 105 " 8 244 " 12 7 104 " 24 333 " 60 1036 " 176 345 62 " 18 225 " 38 702 " 23 235 64 " 16 228 " 39 650 " 121 21 8"2 27 " 6 46 " 3 1 101 " 16 292 " 25 855 " 17 282 97 " 21 308 " 7 960 " 51 95
b-NF
b
104 " 54 366 " 4 965 " 17 95 31 " 5 100 " 16 186 " 19 5 63 " 1 185 " 4 538 " 66 177 63 " 1 211 " 32 634 " 41 211 67 " 16 224 " 54 637 " 20 21 7"6 24 " 5 31 " 1 0.9 65 " 9 206 " 16 608 " 78 202 152 " 17 306 " 23 958 " 129 93
PCB
b
110 " 51 231 " 66 651 " 191 62 21 " 8 64 " 13 162 " 16 5 68 " 13 211 " 13 648 " 43 215 39 " 9 138 " 29 431 " 88 144 30 " 1 102 " 16 351 " 10 11 8"2 25 " 3 39 " 21 1 37 " 1 126 " 17 390 " 30 130 141 " 1 341 " 10 909 " 75 88
Results are presented as the mean net hisq revertantsrplate "SD Ž n s 6.. Spontaneous revertantsrplate: yS9 21 " 6; qS9 None 31 " 11, PB 28 " 7, 3-MC 31 " 6, b-NF 32 " 5, PCB 35 " 7. a S9 was prepared from unpretreated rats. b S9 was prepared from the rats pretreated with PB, 3-MC, b-NF and PCB. c S9 mix was composed of NADPH system and 10% Žvrv. of S9. d S9 mix was composed of NADPH system and 15% Žvrv. of S9. e The slope of dose-response Žrevertantsrmg. was calculated by least-squares linear regression from the linear portion of the dose-response curve.
74
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
the activation of 2-NDBP were mainly present in cytosol. 3.6. Metabolic actiÕation of 2-NDBP by XOD
Fig. 2. Mutagenicity of 2-NDBP in S. typhimurium TA98NR with S9, S9-subfractions, electron donors and nitroreductase inhibitors. NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; NADH, nicotinamide adenine dinucleotide, reduced form; HX, hypoxanthine; MN, 1-methylnicotinamide; HP, 2-hydroxypyrimidine; AL, allopurinol; DI, dicumarol; ME, menadione; PI, piperonyl butoxide.
The results described above suggest that cytosolic XOD participates in the 2-NDBP activation by S9. Our previous result that rat liver XOD is significantly induced by the pretreatment with 3-MC w12x also supports this assumption. In order to investigate the ability of XOD to transform 2-NDBP to active metabolites, mutagenicity of 2-NDBP was tested in the presence of commercial preparations of XOD ŽFig. 3.. As shown in Fig. 3, 2-NDBP was activated by XOD mix including NADH as an electron donor, and mutagenic potency of 2-NDBP was dependent on XOD concentration. AL obviously inhibited the metabolic activation of 2-NDBP by NADH-XOD mix. 2-NDBP was also activated by NADPH-XOD mix, which included NADPH as an electron donor. However, the mutagenicity of 2-NDBP in the presence of NADPH-XOD Ž20 mU. mix was much lower than that with NADH-XOD Ž20 mU. mix. These results support the presumption that cytosolic XOD activates 2-NDBP, and suggest the existence of the enzymes using NADPH as an electron donor to transform 2-NDBP to active metabolites in S9 besides XOD.
Fig. 3. Mutagenicity of 2-NDBP in S. typhimurium TA98NR with XOD. 4 mmol of NADH or NADPH was used per 1 ml of XOD mix. 0.1 mM of allopurinol ŽAL. was used with 20 mU of XOD and NADPH.
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
3.7. Base-pair substitution mutation induced by 2- and 3-NDBP isomers with and without S9
75
pendently, although the numbers of revertants did not reach 2-fold increase over back ground even at the highest dose. 3-NDBP was mutagenic in strains TA7002, TA7004, TA7005 and TA7006 without S9. In the presence of S9, both compounds were mutagenic in strains TA7002, TA7004, TA7005 and TA7006. These are the strains responded to both isomers without S9, except for TA7006 to 2-NDBP. The slopes Žrevertantsr10 mg. of the dose-response curves in these 4 strains increased by the addition of S9 ŽFig. 4.. Whether the activation enzymes are endogenous or exogenous, both compounds showed the highest mutagenicity in strain TA7005 ŽC P G ™ A P T. followed by TA7004 ŽG P C ™ A P T., TA7006 ŽC P G ™ G P C. and TA7002 ŽT P A ™ A P T.. The
Table 4 shows the mutagenicity of 2- and 3-NDBP isomers in base-pair substitution mutation with and without S9 mix. Both 2-NDBP and 3-NDBP were mutagenic in strain TA100 and the potency was enhanced by 3-MC-induced S9. Enhancement of the mutagenicity of 2-NDBP by S9 was higher than that of 3-NDBP, consistent with these compounds acting as frameshift mutagens. In the absence of S9, 2NDBP was negative in strains TA7001, TA7003 and TA7006, and was judged equivocal in its effect in strains TA7002, TA7004 and TA7005. The revertant colonies in the latter three strains increased dose-de-
Table 4 Mutagenicity of 2-NDBP and 3-NDBP in S. typhimurium TA100, TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006 with and without S9 Sample
yS9 rq S9
Dose Žmgrplate.
TA100
TA7001
2-NDBP
yS9
0 2 5 10 20 0 0.5 1 2 5 10 20
127 " 22 201 " 21 273 " 30 359 " 39 497 " 74 116 " 20 169 " 12 216 " 38 292 " 4 552 " 65 1182 " 88 –
2"1 3"2 2"1 2"2 2"1 3"2 – – 4"2 3"2 4"2 2"1
0 2 5 10 20 50 0 1 2 5 10 20 50
115 " 12 153 " 23 218 " 27 343 " 35 520 " 131 1148 " 58 116 " 20 169 " 3 196 " 10 347 " 42 627 " 22 1036 " 170 –
2"1 – 2"2 4"2 2"2 2"1 4"1 – – 4"2 4"3 3"3 4"1
qS9
3-NDBP
a
yS9
qS9
a
TA7002 2"2 3"1 4"3 4"2 5"2 3"2 – – 13 " 7 21 " 9 35 " 13 65 " 5 3"1 – 7"3 12 " 5 15 " 3 18 " 2 5"5 – – 9"7 12 " 9 23 " 7 "39 " 2
TA7003
TA7004
TA7005
TA7006
1 " 0.5 1 " 0.6 1 " 0.4 1 " 0.4 1 " 0.4 1"1 – – 2"1 2"1 3"2 2"2
28 " 7 25 " 3 33 " 13 34 " 14 40 " 14 37 " 9 – – 54 " 10 75 " 18 111 " 29 154 " 20
19 " 5 21 " 4 31 " 10 33 " 8 35 " 7 25 " 6 – – 76 " 19 131 " 47 233 " 79 270 " 99
8"1 7"3 8"3 10 " 4 8"5 5"3 – – 15 " 3 19 " 7 24 " 10 33 " 6
2"2 – 2"1 1"1 2"2 2"1 1 " 0.4 – – 1 " 0.9 1 " 0.4 3"1 2"1
30 " 4 – 34 " 1 42 " 7 64 " 16 110 " 9 37 " 9 – – 93 " 16 104 " 12 178 " 2 320 " 19
16 " 5 – 32 " 4 54 " 13 119 " 33 236 " 110 22 " 2 – – 90 " 16 136 " 23 317 " 49 451 " 65
3"1 – 9"1 10 " 3 18 " 8 37 " 1 2"1 – – 18 " 1 27 " 3 41 " 16 50 " 7
Results are presented as the mean Hisq revertantsrplate "SD Ž n s 6.. S9 mix was composed of NADPH system and 15% Žvrv. of S9 which was prepared from rats pretreated with 3-MC.
a
76
T. Watanabe et al.r Mutation Research 388 (1997) 67–78
ent urban air would be ubiquitously polluted with 2-NDBP. Additional research and exposure monitoring is necessary before the human exposure and cancer risk of 2-NDBP can be evaluated.
Acknowledgements We are grateful to Dr. Naoki Miyata, National Institute of Health Sciences, for the generous gifts of 3-NFl. We are grateful to Prof. Bruce N. Ames, University of California, who kindly supplied Salmonella typhimurium strains TA98 and TA100, and to Prof. Herbert S. Rosenkranz, Chairman, University of Pittsburgh, who kindly supplied S. typhimurium strains TA98NR and TA98r1,8-DNP6 . We thank Xenometrix Inc. for providing S. typhimurium strains TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006. We thank Ms. Satomi Shirakawa, Ms. Yuka Miyoshi, Ms. Tamiko Kintou and Ms. Kazuko Maegawa for their technical assistance in this investigation. Fig. 4. Mutagenic potency of 2-NDBPŽA. and 3-NDBPŽB. with and without S9 mix in a set of six S. typhimurium tester strains ŽTA7001 to TA7006.. Mutagenic potency was calculated by least-squares linear regression from the linear portion of the dose-response curve, and presented as a number of revertants per 10 mg of each compound. I, without S9 mix; B, with S9 mix.
result that NDBP isomers showed the similar mutation specificities with and without S9 ŽFig. 4. indicate that the mechanism of mutagenesis caused by NDBP isomers with S9 is similar to that without S9, that is, S9 enzymes transform NDBP isomers to the same active metabolites as those formed by bacterial enzymes. C P G ™ A P T transversion, which was mainly induced by NDBP isomers, was found in an activated c-Ki-ras gene of a fibrosarcoma induced by 1,8-dinitropyrene, and is considered responsible for the activation of c-Ki-ras w37x. The fact that 2-NDBP was efficiently activated by mammalian enzymes described above implies that 2-NDBP might be hazardous to human health. 2-NDBP was quantitated in air particulate extract in 1992 for the first time w13x, and it was found that 2-NDBP is not only emitted from diesel engines but also formed through atmospheric transformation w14,38x, suggesting that ambi-
References w1x Tokiwa, H. and Y. Ohnishi Ž1986. Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol., 17, 23–60. w2x Rodriguez, H. and G.L. Murison Ž1990. Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells, Mutation Res., 240, 73–81. w3x Shane, B.S., G.L. Squadrito, D.F. Church and W.A. Pryor Ž1991. Comparative mutagenicity of nitrofluoranthenes in Salmonella typhimurium TA98, TA98NR, and TA98r1,8DNP6 , Environ. Mol. Mutagen., 17, 130–138. w4x Tokiwa, H., R. Nakagawa, K. Horikawa and A. Ohkubo Ž1987. The nature of the mutagenicity and carcinogenicity of nitrated, aromatic compounds in the environment, Environ. Health Perspect., 73, 191–199. w5x Horikawa, K., N. Sera, T. Otofuji, K. Murakami, H. Tokiwa, M. Iwagawa, K. Izumi and H. Otsuka Ž1991. Pulmonary carcinogenicity of 3,9- and 3,7-dinitrofluoranthene, 3-nitrofluoranthene and benzow axpyrene in F344 rats, Carcinogenesis, 12, 1003–1007. w6x Arey, J., B. Zielinska, W.P. Harger, R. Atkinson and A.M. Winer Ž1988. The contribution of nitrofluoranthenes and nitropyrenes to the mutagenic activity of ambient particulate organic matter collected in southern California, Mutation Res., 207, 45–51. w7x Nishioka, M.G., C.C. Howard, D.A. Contos, L.M. Ball and J.
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w8x
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
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