Mutation Research, 245 (1990) 277-285 Elsevier
277
MUTLET 0431
Mutagenicity of mononitrodihydrobenzo[a]pyrenes Peter P. Fu, Hyewook Jung, Linda S. Von Tungeln and Robert H. Heflich National Centerfor ToxicologicalResearch, Jefferson, AR 72079 (U.S.A.) (Received 7 May 1990) (Revision received 30 July 1990) (Accepted 31 July 1990)
Keywords: Dihydronitrobenzo[a]pyrenes; Bacterial mutagenicity; Nitro-PAHs; Nitrobenzo[a]pyrene
1-, 3-, and 6-nitrobenzo[a]pyrene (Nitro-BaP) are genotoxic environmental contaminants (Jager, 1978; Wang et al., 1978; Pitts et al., 1982; Gibson, 1983; Paputa-Peck et al., 1983; Schuetzle, 1983) and are formed in model atmospheres (Pitts et al., 1978). These three isomers are nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) derived from the carcinogenic and mutagenic PAH benzo[a]pyrene and, thus have been viewed as model compounds for the study of nitro-PAH carcinogenesis and mutagenesis (Beland et al., 1985; Chou et al., 1985, 1986; Tokiwa and Ohnishi, 1986; Fu et al., 1988; Fu, 1990). Previous studies indicate that 1and 3-nitro-BaP are potent mutagens in the Salmonella typhimurium reversion assay with and without exogenous $9 activation, while 6-nitro-
Correspondence: Dr. Peter P. Fu, National Center for Toxicological Research, Jefferson, AR 72079 (U.S.A.). Abbreviations: 7,8-dihydro- 1-NBaP, 1-nitro-7,8-dihydrobenzo[a]pyrene [other nitrodihydrobenzo[a]pyrenes are similarly abbreviated]; HPLC, high-performance liquid chromatography; 1-nitro-BaP, 1-nitrobenzo[a]pyrene; 3-nitro-BaP, 3-nitrobenzo[a]pyrene; 6-nitro-BaP, 6-nitrobenzo[a]pyrene; rtitroPAHs, nitro-polycyclic aromatic hydrocarbons; 1-nitroso-BaP, 1-nitrosobenzo[a]pyrene; TCPO, 3,3,3-trichloropropylene 1,2-oxide.
BaP is a mutagen only in the presence of $9 activation (Pitts et al., 1978, 1982, 1984; Tokiwa et al., 1981; Campbell et al., 1981; Fu et al., 1982a; Chou et al., 1983, 1985; 1986; Lofroth et al., 1984; Hass et al., 1986; Heflich et al., 1989). Nitroreduction is an important metabolic activation pathway for 1and 3-nitro-BaP, as evidenced by results showing that the direct-acting mutagenicity of these compounds is lower in TA98NR, which is deficient in a major nitroreductase component (Bryant et al., 1984; Pitts et al., 1984; Chou et al., 1985, 1986; Hass et al., 1986) than in TA98 and that their immediate nitro-reduced intermediates, 1- and 3-nitroso-BaP, exhibit much higher direct-acting rnutagenicity (Thornton-Manning et al., 1989; Heflich et al., 1989). 6-Nitroso-BaP, the immediate aitroreduction product of 6-nitro-BaP, also exhibits strong direct-acting mutagenic activity (Fu et al., 1988; Heflich et al., 1989). Recent studies indicate that hepatic microsomal metabolism of 1and 3-NBaP generates trans-7,8- and 9,10-dihydrodiols which are potent mutagens in S. typhimurium TA98 with and without $9 activation (Chou et al., 1985, 1986) (Table 2). The mutagenicity of these four trans-dihydrodiols assayed in the presence of $9 activation enzymes suggests that the bay-region trans-dihydrodiol epoxides are the metabolites
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
278
NO2 NO2 9,10-Dihydro-l-NBaP
9,10-Dihydro-3-NBaP
9,10-Dihydro-6-NBaP
NO2 7,8-Dihydro-l-NBaP
7,8-Dihydro-3-NBaP
7,8-Dinydro-6-N Ba P
Fig. 1. Structures and abbreviations of the dihydronitrobenzo[a]pyrenes used for the mutagenicitystudies. responsible for the mutation induction (Chou et al., 1985, 1986). In this study, we have determined the mutagenicity of 7,8- and 9,10-dihydro-NBaPs in S. typhimurium TA98, TA98NR, TA98/1,8-DNP6 and TA100 and compared these mutagenicities with those of the NBaP dihydrodiol analogs. The compounds studied include: 7,8-dihydro-l-NBaP, 7,8-dihydro-3-NBaP, 7,8-dihydro-6-NBaP, 9,10-dihydro-3-NBaP, 9.10-dihydro-3-NBaP and 9,10-dihydro-6-NBaP (Fig. 1). These compounds can potentially be metabolized by $9 activation enzymes to the corresponding epoxides by cytochrome P-450 (Conney, 1982). Based on studies of the mutagenicity of BaP diolepoxides and the corresponding tetrahydro-BaP oxides (Conney, 1982), these NBaP tetrahydro-epoxy compounds may alkylate DNA and produce mutations, as do the NBaP diol-epoxide analogs. Materials and methods
Chemicals Benzo[a]pyrene (BaP), 1-nitropyrene and 1,6-dinitropyrene were purchased from Aldrich Chemical Company, Milwaukee, WI. 7-Hydroxyl-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 7-hydr oxy - 3 - nitro - 7,8,9,10- tetrahydrobenzo[a] pyrene and 7-hydroxy-6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene were prepared as described previously (Von Tungeln and Fu, 1989). Acid-catalyzed de-
hydration of these compounds by p-toluenesulfonic acid in benzene under reflux gave 1-nitro9,10-H2-BaP, 3-nitro-9,10-H2-BaP and 6-nitro9,10-H2-BaP, respectively, in high yield. 1-Nitro7,8-H2-BaP, 3-nitro-7,8-H2-BaP and 6-nitro-7,8H2-BaP were similarly prepared from the corresponding nitro-10-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which were prepared from nitration of 10-keto-7,8,9,10-tetrahydrobenzo[a]pyrene followed by reduction of the nitrated products with sodium borohydride in methanol and tetrahydrofuran (unpublished results). 10-Keto-7,8,9,10tetrahydrobenzo[a]pyrene was prepared by oxidation of 7,8,9,10-tetrahydrobenzo[a]pyrene with 2,3-dichloro-5,6-dicyano- 1,4-benzoquinone and formic acid in dioxan (Fu et al., 1982b). The structures of all the synthesized compounds were confirmed by analysis of their UV-visible absorption, and their mass and proton nuclear magnetic resonance spectral data; their purities were >99% based on HPLC analysis.
Mutagenicity assays Reversion to prototrophy was measured using Salmonella typhimurium histidine auxotrophic strains TA100, TA98, TA98NR and TA98/1,8DNP6 using methods outlined by Maron and Ames (1983). The post-mitochondrial supernatant fraction ($9) was prepared from a liver homogenate of Aroclor 1254-pretreated male Sprague-Dawley rats, and used at a concentration of 50 #l/plate. Experiments were also carried out with the epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO), added at a concentration of 930 /zg/plate to the assays with $9. The variability in assays conducted in triplicate was generally < _+20°70. All test chemicals were assayed on at least two separate occasions with similar results. Revertants/#g/plate were calculated from linear regression lines fitted to the increasing portion of dose-response curves. Results
The mutagenic activities of 7,8-dihydro- 1-NBaP, 7,8-dihydro-3-NBaP, 7,8-dihydro-6-NBaP, 9,10-
279
dihydro- 1-NBaP, 9,10-dihydro-3-NBaP and 9,10dihydro-6-NBaP toward Salmonella typhimurium
TA98, TA98NR, TA98/I,8-DNP6 and TA100 were determined in the absence and presence of rat-
TABLE 1 REVERSION
INDUCTION
Salmonella typhimurium Compound
BY 1-, 3- A N D
6-NITRO-DIHYDROBENZO[a]PYRENE
AND
STANDARD
MUTAGENS
STRAINS TA98, TA98NR, TA98/1,8-DNP6 AND TA100 /~g/plate
$9
Revertants/plate TA98
Solvent control
TA98NR
TA98/I,8-DNP6
TAI00
-
23 ±
5
17 ±
3
7 ±
1
127 ±
5
+
26 ±
1
28 ±
8
19 ±
4
140 ±
2
3311 + 220
1-Nitropyrene
1.0
-
1233 ±
83
258 ±
33
297 ±
42
1,6-Dinitropyrene
0.0025
-
1412 ± 250
1610 ±
46
292 ±
25
330 ±
63
Benzo[a]pyrene (BaP)
5.0
+
528 ± 114
570 ±
31
1016 +
89
9,10-Dihydro-l-NBaP
1.0
-
71 +
6
+
1195 +
73
3
202 +
161 +
28
179 +
2
19
390 ±
61
3.3
+
10.0
+
9,10-Dihydro-3-NBaP
1.0
+
3.3
+
5.0
+
9,10-Dihydro-6-NBaP
2.0 5.0 10.0
0.33 1.0
0.33 1.0 3.3
1.0 3.3
49
212 ±
33
120 ±
757 ±
157 _+
-
-
-
2273 ± 204
7 25
258 ___ 17
497 ± 163
1327 ± 211
103 ±
14
212 ±
14
200 ±
32
92 ±
15
420 + 228
327 +
28
45
247 +
3
350 ±
56
247 ±
25
2493 + 247
1162 ±
25
232 +
6
682 ±
78
357 +
3
453 +
18
280 ±
33
1672 ± 270
453 +
89
1033 ±
78
395 ±
78
3561 ± 720
+
122 ±
10
40
±
2
945 +
78
-
116 +
6
+
398 +
8
253 ±
47
-
50 ±
487 +
5
-
8
32
6
-
312 ±
1184 ± 138
38 ±
1351 ± 203 552 ±
81
3254 + 395
32 ±
7
-
43 ±
5
121 _+
11
164 ±
19
-
-
747 +_
28 ±
-
-
69
128
870 ±
96
±
8
765 ±
85
4
31 ±
7
157 +
6
170 +__ 31
28 ±
8
174 +
10
109 +_
6
72 +
5
210 ±
12
445 ±
60
34 +
3
255 ±
51
182 ±
8
108 +
7
243 ±
46
1197 ± 175
99 ±
17
425 ±
46
-
89 +
10
70 ±
12
48 ±
7
179 +
19
+
167 +
9
25 ±
19
44 ±
5
244 +
25
-
147 +
15
135 ±
20
88 ±
21
207 +
17
+
505 ±
26
635 ± 100
104 ±
17
327 ±
28
370 +
27
8
+
7,8-Dihydro-6-NBaP
348 +
5482 + 385
31 ±
+ 7,8-Dihydro-3-NBaP
2490 + 540
-
+ 3.3
87
+
+ 7,8-Dihydro-l-NBaP
305 _+
1925 ± 269
21
137 ±
14
247 +
2413 ± 210
327 ±
388 +
94
850 +_ 65
-
18
4
-
-
119
+
23 +
10
-
-
143 ±
13
-
20 +
3
-
-
126 +
23
+
26 ±
4
-
-
168 ±
11
+
+
11
IN
280
liver $9 activation enzymes. The mutagenicities of these compounds and the positive standards, 1-nitropyrene, 1,6-dinitropyrene and BaP are given
TABLE
2
REVERTANTS/•g/PLATE MUTAGENS
PRODUCED
BY
1-,
I N Salmonella t y p h i m u r i u m S T R A I N S
Compound
$9
1 - N i t r o - B a P
3-Nitro-BaP
6-Nitro-BaP
9,10-Dihydro-l-NBaP
9,10-Dihydro-3-NBaP
3-NBaP
tert.-9,10-c~hydrodiol c
9,10-Dihydro-6-NBaP
7,8-Dihydro-l-NBaP
I-NBaP
tert.-9,10-dihydrodiol a
7,8-Dihydro-3-NBaP
3-NBaP
tert.-7,8-dihydrodiol c
7,8-Dihydro-6-NBaP
AND
6-NITRO-DIHYDROBENZO[a]PYRENE TA98/1,8-DNP6
AND
AND
TA100
Revertants//zg/plate TA98NR
TA98/I,8-DNP6
TA100
-
2828 a
640 a
2997 a
94 b
+
1414 a
767 a
47 a
101 b
-
3185 c
327 c
4286 c
96 b
+
1418 c
882 c
51 c
141 b
-
9,10-dihydro-1NBaP (516) > 9,10-dihydro-6-NBaP (98) > 7,8-dihydro-6-NBaP ( < 1). 7,8-Dihydro- 1-NBaP, 9,10-dihydro-l-NBaP and 9,10-dihydro-3-NBaP exhibited lower mutagenic activity in S. typhimurium TA98NR than in TA98, and in turn much lower activities were observed in tester strain TA98/1,8-DNP6 than in TA98NR. 9,10-Dihydro-6-NBaP exhibited similar mutagenic potency in these three tester strains, while 7,8-dihydro-3NBaP had nearly equal activity in both TA98 and TA98NR and much lower activity in TA98/1,8DNP6. With the exception of 7,8-dihydro-6-NBaP
which was a very weak mutagen in both tester strains, the dihydro-NBaPs exhibited much lower mutagenic activity in S. typhimurium TA100 than in TA98, both in the presence and absence of $9 activation enzymes. In all the cases, their S9-mediated mutagenic activity was higher than their direct-acting mutagenicity. Among the six dihydroNBaPs tested, 7,8-dihydro-6-NBaP was the only structural isomer essentially inactive in both S. typhimurium strains TA98 and TA100 with and without $9 activation enzymes. 9,10-Dihydro-6NBaP was also inactive in both strains without $9 activation, and exhibited weak mutagenic activity when assayed in the presence of $9 enzymes. Table 3 summarizes the effects of the epoxide hydrolase inhibitor TCPO on the S9-mediated mutagenic activities of the six dihydro-NBaPs toward S. typhimurium TA98 and TA100. Although TCPO increased the mutagenic potency
TABLE 3 R E V E R S I O N I N D U C T I O N BY 1-, 3- A N D 6 - N I T R O D I H Y DROBENZO[a]PYRENE IN Salmonella typhimurium S T R A I N S TA98 A N D T A 1 0 0 W I T H $9 A C T I V A T I O N E N ZYMES IN THE PRESENCE AND ABSENCE OF EPOXIDE HYDROLASE INHIBITOR, 3,3,3-TRICHLOROPROPYLENE 1,2-OXIDE (TCPO) a Compound
$9
TCPO
Revertants/t~g/plate TA98
Solvent control
28 25
TA100
+ +
+
138 200
9 , 1 0 - D i h y d r o - 1-NBaP +
-
516
122
+
+
1377
2736
9,10-Dihydro-3-NBaP +
+
1938
3539
9,10-Dihydro-6-NBaP +
+
169
2915
7 , 8 - D i h y d r o - 1-NBaP
+
-
962
85
+
+
3978
6261
7,8-Dihydro-3-NBaP
+ +
+
584 2175
212 12878
7,8-Dihydro-6-NBaP
+ +
+
3 16
- 9 2398
a E x p e r i m e n t a l details described in M a t e r i a l s a n d M e t h o d s .
282 of the compounds in both TA98 and TA100, the increases were much greater in TA100 (22-74-fold) than in TA98 (3-4-fold). The order or mutagenic potency tested in TA98 with TCPO was: 7,8-dihydro-l-NBaP (3978 revertants/#g/plate) > 7,8-dihydro-3-NBaP (2175) > 9,10-dihydro-3-NBaP (1938) > 9,10-dihydro-l-NBaP (1377) > 9,10dihydro-6-NBaP (169) > 7,8-dihydro-6-NBaP (16). The order or mutagenic potency tested in TA100 with TCPO was: 7,8-dihydro-3-NBaP (12878 revertants/#g/plate) > 7,8-dihydro-1NBaP (6261) > 9,10-dihydro-3-NBaP (3959) > 9,10-dihydro-l-NBaP (2736) > 9,10-dihydro-6NBaP (2915) > 7,8-dihydro-6-NBaP (2398). When assayed at a dose of 1.0 #g/plate, both 7,8-dihydro-l-NBaP and 7,8-3-NBaP exhibited cytotoxicity (loss of the bacterial lawn) in the presence of TCPO. Discussion
The direct-acting mutagenicity of the six dihydro-NBaP compounds was much lower than that observed with $9 activation. The mutagenicity of 7,8-dihydro-3-NBaP and 9,10-dihydro-3-NBaP in all strains of TA98 was similar, indicating that the major component of the bacterial nitroreductase(s) deficient in TA98NR (Bryant et al., 1984) was not involved in the activation and that the transesterificase deficient in TA98/1,8-DNP6 (McCoy et al., 1983) was not required for activation. On the other hand, 7,8-dihydro-l-NBaP exhibited lower mutagenicity in both TA98NR and TA98/1,8-DNP6, indicating the involvement of both the major nitroreductase and transesterificase in the metabolic activation of this compound. These findings indicate that metabolic activation of these compounds involves different mechanisms as well as different metabolizing enzymes. Both 7,8-dihydro-6-NBaP and 9,10-dihydro-6-NBaP, which have their nitro substituents perpendicular to the aromatic moiety, exhibited very weak or no direct-acting mutagenicity in both TA98 and TA100. These results are in agreement with the hypothesis we previously proposed that nitroPAHs with a perpendicular orientation are very
weak direct-acting mutagens in Salmonella (Fu et al., 1985, 1986, 1988, 1989; Fu, 1990). As is the case for the parent compounds, 7,8-dihydro-BaP and 9,10-dihydro-BaP, all six dihydro-NBaPs bear an olefinic aromatic double bond and can be facilely metabolized to the corresponding epoxides by cytochrome P-450 isozymes contained in the $9 mix (Conney, 1982). With $9, all four of the dihydro-l-NBaP and dihydro-3-NBaP isomers were potent mutagens in TA98, producing from 516 to 962 revertants/ #g/plate, and their mutagenicity was much lower in TA98NR and TA98/1,8-DNP6. These results were similar to those found for the 1- and 3-NBaP dihydrodiols (Chou et al., 1985, 1986; Table 3), and indicate that after ring-oxidation of the dihydro derivatives, nitroreduction followed by esterification were required in order to exert maximum mutagenicity. However, it is important to consider that $9 contains epoxide hydrolase which can readily catalyze the hydrolysis of NBaP-tetrahydro-epoxides to the corresponding NBaP-tetrahydro-diols. Indeed, with the incorporation of the epoxide hydrolase inhibitor, TCPO, to the incubation system, much higher mutagenic activities were observed for all six dihydro-NBaP. These results are inconsistent with the mutagenicity assays of 7,8-dihydro-BaP and 9,10-dihydro-BaP tested with $9 in the presence and absence of TCPO (Conney, 1982). With TCPO, the much greater increase of mutagenicity in Salmonella typhimurium TA100 than in TA98 suggests that the metabolites responsible for induction of mutation in TA98 and TA100 may be different. In most cases, mutation in TA98 is frameshift-type while mutation in TA100 is base-pair substitution type (Hartman et al., 1986). It is also known that arylamines which mainly bind to DNA through reactions at the nitrogen atoms are generally more mutagenic in TA98 than in TA100, and that the mutagenicity of PAHs which form DNA adducts via the epoxy ring is usually greater in TA100 than in TA98. Thus, gathering together all these observations, it may be that the mutagenic activity of dihydro-NBaPs in TA98 with $9 and with or without TCPO is largely due to nitroreduction of the nitro substituent of the
283
NR
H\ /
HNOH
NO NR
DNA N
DNA ~
- $9
• NO 2
DNA ~ , 2 + $9
2
0N,
]
MUTATION
~R
HNOH
H~N/DNA /
EH
DNA.~ ~
NO2 HO
/
HNOH
OH :
NR
OH -
)
~
H DNAH
OH
DNA N I
Fig. 2. Proposed metabolicactivation pathways of 9,10-dihydro-l-NBaPin S. typhimurium TA98 and TA100. NR, $9, and EH designate bacterial nitroreductases,9000 g supernatantof liver homogenates,and epoxidehydrolase,respectively.
NBaP-tetrahydro-epoxide compounds. On the other hand, the mutagenic activity of these compounds in TA100, especially with TCPO, appears to be mainly through the interaction of the epoxy ring with DNA. Fig. 2, using 7,8-dihydro-l-NBaP as an example, summarizes the four metabolic activation pathways leading to mutation induction proposed above. Without exogenous $9 activation, nitroreduction, either with or without the involvement of acetylation, is an important activation pathway (shown as pathway 1 in Fig. 2). This pathway is expected to be more important in TA98 than in TA100, as evidenced by the much higher direct-acting mutagenicities observed in this strain
(see Table 1). In contrast, when assayed with $9 and TCPO, the markedly higher mutagenic activity of the tested dihydro compounds in TA100 compared with TA98 suggests that DNA adducts are being formed by reaction with ring carbons of epoxide metabolites stabilized by the presence of the epoxide hydrolase inhibitor TCPO (shown as activation pathway 2 in Fig. 2). When assayed with $9 and without TCPO, both activation pathways 3 and 4 could be involved, although pathway 4 is probable more important than pathway 3 since the tetrahydro-epoxide should be facilely hydrolyzed to the corresponding tetrahydro-trans-diol. Some dihydro nitro compounds activated by pathway 4 require acetylation in order to exert the maximum
284 mutagenicity and
these
display relatively low
m u t a g e n i c responses in T A 9 8 / I , 8 - D N P 6 (Tables 1 a n d 2), Since b o t h a c t i v a t i o n pathways 3 a n d 4 involve n i t r o r e d u c t i o n a n d D N A - a d d u c t f o r m a t i o n t h r o u g h reaction with a m i n o nitrogens, comp o u n d s activated by these pathways should produce higher m u t a g e n i c i t y in TA98 t h a n in TA100. M o s t o f the n i t r o - P A H s a n d a r y l a m i n e s so far studied are derived from n o n - m u t a g e n i c P A H s , such as biphenyl, n a p h t h a l e n e , a n t h r a c e n e , pyrene a n d fluorene. T h u s , m e t a b o l i s m o f these comp o u n d s t h r o u g h r i n g - o x i d a t i o n generally results in detoxification. O n the other h a n d , n i t r o r e d u c t i o n of nitro-PAHs and N-oxidation of arylamines generates N - h y d r o x y a m i n o - P A H s which b i n d with D N A a n d induce m u t a t i o n s largely via a frameshift m e c h a n i s m ( R o s e n k r a n z a n d Mermelstein, 1983). This a c c o u n t s for the general p h e n o m e n o n that the m u t a g e n i c i t y o f b o t h n i t r o - P A H s a n d a r y l a m i n e s is greater in TA98 t h a n in TA100. However, the m u t a g e n i c i t y results of the six d i h y d r o - N B a P s assayed with $9 a n d T C P O clearly indicate that these c o m p o u n d s exhibit m u c h higher m u t a g e n i c i t y in T A 1 0 0 t h a n in TA98. T h u s , these findings suggest that r i n g - o x i d a t i o n can be a n i m p o r t a n t p a t h w a y for the metabolic a c t i v a t i o n o f nitroP A H s to m u t a g e n s , a n d are also in a g r e e m e n t with the previous findings that r i n g - o x i d a t i o n o f 1- a n d 3 - N B a P to the c o r r e s p o n d i n g diol epoxides is a p o t e n t i a l activation p a t h w a y ( C h o u et al., 1985, 1986; T h o r n t o n - M a n n i n g et al., 1988).
References Beland, F.A., R.H. Heflich, P.C. Howard and P.P. Fu (1985) The in vitro metabolic activation of nitro polycyclicaromatic hydrocarbons, in: R.G. Harvey (Ed.), Polycyclic Hydrocarbons and Carcinogenesis, ACS Symp. Ser. 283, American Chemical Society, Washington, DC, pp. 371-396. Bryant, D.W., D.R. McCalla, P. Lultschik, M.A. Quilliam and B.E. McCary (1984) Metabolism of 1,8-dinitropyrene by Salmonella typhimurium, Chem.-Biol. Interact., 49, 351-368. Campbell, J., G.C. Crumplin, J.V. Garner, R.C. Garner, C.N. Martin and A. Rutter (1981) Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagens and stimulators of DNA repair synthesis in cultured human cells, Carcinogenesis, 2, 559-565.
Chou, M.W., F.E. Evans, S.K. Yang and P.P. Fu (1983) Evidence for a 2,3-epoxide as an intermediate in the microsomal metabolism of 6-nitrobenzo[a]pyrene, Carcinogenesis, 4, 699-702. Chou, M.W., R.H. Heflich and P.P. Fu (1985) Multiple metabolic pathways for the mutagenic activation of 3-nitrobenzo[a]pyrene, Carcinogenesis, 6, 1235-1238. Chou, M.W., R.H. Heflich and P.P. Fu (1986) Metabolism of l-nitrobenzo[a]pyrene by rat liver microsomes to potent mutagenic metabolites, Carcinogenesis, 7, 1837-1844. Conney, A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G.H.A. Clowes Memorial Lecture, Cancer Res., 42, 4875-4917. Fu, P.P. (1990) Metabolic activation of nitro-polycyclic aromatic hydrocarbons, Drug Metab. Rev., in press. Fu, P.P., M.W. Chou, S.K. Yang, F.A. Beland, F.F. Kadlubar, D.A. Casciano, R.H. Heflich and F.E. Evans (1982a) Metabolism of the mutagenic environmental pollutant, 6-nitrobenzo[a]pyrene; metabolic activation via ring oxidation, Biochem. Biophys. Res. Commun., 103, 1037-1043. Fu, P.P., J.D. Clark and A.Y. Huang (1982b) Synthesis of 10-oxo-7,8,9,10-tetrahydrobenzo[alpyrene, J. Chem. Res.(S), 121. Fu, P.P., M.W. Chou, D.W. Miller, G.L. White, R.H. Heflich and F.A. Beland 0985) The orientation of the nitrosubstituent predicts the direct-acting bacterial mutagenicity of nitrated polycyclicaromatic hydrocarbons, Mutation Res., 143, 173-181. Fu, P.P., R.H. Heflich, L.S. Von Tungeln, D.T.C. Yang, E.K. Fifer and F.A. Beland (1986) Effect of the nitro group conformation on the rat liver microsomal metabolism and bacterial mutagenicity of 2- and 9-nitroanthracene, Carcinogenesis, 7, 1819-1927. Fu, P.P., M.W. Chou and F.A. Beland (1988) Effects of nitro substitution on the in vitro metabolic activation of polycyclic aromatic hydrocarbons, in: S.K. Yang and B.D. Silverman (Eds.), Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships, CRC Press, Boca Raton, FL, pp. 37-65. Fu, P.P., Y.-C. Ni, Y.M. Zhang, R.H. Heflich, Y.K. Wang and J.S. Lai (1989) Effect of the orientation of nitro substituent on the bacterial mutagenicity of dinitrobenzo[e]pyrenes, Mutation Res., 225, 121-125. Gibson, T.L. (1983) Sources of direct-acting nitroarene mutagens in airborne particulate matter, Mutation Res., 122, 115-121. Hartman, P.E., B.N. Ames, J.R. Roth, W.M. Barnes and D.E. Levin (1986) Target sequences for mutagenesis in Salmonella histidine-requiring mutants, Environ. Mutagen., 8,631-641. Hass, B.S., R.H. Heflich, M.W. Chou, G.L. White, P.P. Fu and D.A. Casciano (1986) Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrene in Salmonella typhimurium strains, Mutation Res., 171, 123-129. Heflich, R.H., L.E. Unruh, J.R. Thornton-Manning, L.S. Von
285 Tungeln and P.P. Fu (1989) Mutagenicity of 1-, 3- and 6-nitrosobenzo[a]pyrene in Salmonella typhimurium and Chinese hamster ovary ceils, Mutation Res., 225, 157-163. Jager, J. (1978) Detection and characterization of nitro derivatives of some polycyclic aromatic hydrocarbons by fluorescence quenching after thin-layer chromatography: application to air pollution analysis, J. Chromatogr., 152, 575-578. Lofroth, G., R. Toftgard, L. Nilsson, E. Agurell and J.-A. Gustafsson (1984) Short-term bioassays of nitro derivatives of benzo[a]pyrene and perylene, Carcinogenesis, 5,925-930. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCoy, E.C., M. Anders and H.S. Rosenkranz (1983) The basis of the insensitivity of Salmonella typhimurium strain TA98/1,8-DNP6 to the mutagenic action of nitroarenes, Mutation Res., 121, 17-23. Paputa-Peck, M.C., R.S. Marano, D. Schuetzle, T.L. Riley, C.V. Hampton, T.J. Prater, L.M. Skewes, T.E. Jensen, P.H. Ruehle, L.C. Bosch and W.P. Duncan (1983) Determination of nitrated polynuclear aromatic hydrocarbons in particulate extracts by capillary column gas chromatography with nitrogen selective detection, Anal. Chem., 55, 1946-1954. Pitts Jr., J.N., K.A. Van Cauwenberghe, D. Grosjean, J.P. Schmid, D.R. Fits, W.L. Belser Jr., G.B. Knudson and P.M. Hynds (1978) Atmospheric reactions of polycyclic aromatic hydrocarbons: facile formation of mutagenic nitro derivatives, Science, 202, 515-519. Pitts Jr., J.N., D.M. Lokensgard, W. Harger, T.S. Fisher, V. Mejia, J.J. Schuler, G.M. Scorziell and Y.A. Katzenstein (1982) Mutagens in diesel exhaust particulate, identification and direct activities of 6-nitrobenzo[a]pyrene, 9-nitroanthracene, 1-nitropyrene and 5H-phenanthro[4,5-bed]pyran-5-one, Mutation Res., 103,241-249.
Pitts Jr., J.N., B. Zielinska and W.P. Harger (1984) Isomeric mononitrobenzo[a]pyrenes: synthesis, identification and mutagenic activities, Mutation Res., 140, 81-85. Rosenkranz, H.S., and R. Mermelstein (1983) Mutagenicity and genotoxicity of nitroarenes, All nitro-containing chemicals were not created equal, Mutation Res., 114, 217-267. Schuetzle, D. (1983) Sampling of vehicle emissions for chemical analysis and biological testing, Environ. Health Perspect., 47, 65-80. Thornton-Manning, J.R., M.W. Chou, P.P. Fu and R.H. Heflich (1988) Microsomal activation of 1- and 3-nitrobenzo[a]pyrene to mutagens in Chinese hamster ovary cells, Mutagenesis, 3, 233-237. Thornton-Manning, J.R., W.L. Campbell, B.S. Hass, J.J. Chen, P.P. Fu, C.E. Cerniglia and R.H. Heflich (1989) Role of nitroreduction in the synergistic mutational response induced by mixtures of 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium, Environ. Mol. Mutagen., 13, 203-210. Tokiwa, H., and H. Ohnishi (1986) Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment, CRC Crit. Rev. Toxicol., 17, 23-60. Tokiwa, H., R. Nakagawa and Y. Ohnishi (1981) Mutagenic assay of aromatic nitro compounds with Salmonella typhimurium, Mutation Res., 91, 321-325. Von Tungeln, L.S., and P.P. Fu (1989) High performance liquid chromatographic separation of ring-oxidized metabolites of nitro-polycyclic aromatic hydrocarbons, J. Chromatogr., 461,315-326. Wang, Y.Y., S.M., Rappaport, R.F. Sawyer, R.E. Talcott and E.T. Wei (1978) Direct-acting mutagen in automobile exhaust, Cancer Lett., 5, 39-47. Communicated by H.S. Rosenkranz
277
MUTLET 0431
Mutagenicity of mononitrodihydrobenzo[a]pyrenes Peter P. Fu, Hyewook Jung, Linda S. Von Tungeln and Robert H. Heflich National Centerfor ToxicologicalResearch, Jefferson, AR 72079 (U.S.A.) (Received 7 May 1990) (Revision received 30 July 1990) (Accepted 31 July 1990)
Keywords: Dihydronitrobenzo[a]pyrenes; Bacterial mutagenicity; Nitro-PAHs; Nitrobenzo[a]pyrene
1-, 3-, and 6-nitrobenzo[a]pyrene (Nitro-BaP) are genotoxic environmental contaminants (Jager, 1978; Wang et al., 1978; Pitts et al., 1982; Gibson, 1983; Paputa-Peck et al., 1983; Schuetzle, 1983) and are formed in model atmospheres (Pitts et al., 1978). These three isomers are nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) derived from the carcinogenic and mutagenic PAH benzo[a]pyrene and, thus have been viewed as model compounds for the study of nitro-PAH carcinogenesis and mutagenesis (Beland et al., 1985; Chou et al., 1985, 1986; Tokiwa and Ohnishi, 1986; Fu et al., 1988; Fu, 1990). Previous studies indicate that 1and 3-nitro-BaP are potent mutagens in the Salmonella typhimurium reversion assay with and without exogenous $9 activation, while 6-nitro-
Correspondence: Dr. Peter P. Fu, National Center for Toxicological Research, Jefferson, AR 72079 (U.S.A.). Abbreviations: 7,8-dihydro- 1-NBaP, 1-nitro-7,8-dihydrobenzo[a]pyrene [other nitrodihydrobenzo[a]pyrenes are similarly abbreviated]; HPLC, high-performance liquid chromatography; 1-nitro-BaP, 1-nitrobenzo[a]pyrene; 3-nitro-BaP, 3-nitrobenzo[a]pyrene; 6-nitro-BaP, 6-nitrobenzo[a]pyrene; rtitroPAHs, nitro-polycyclic aromatic hydrocarbons; 1-nitroso-BaP, 1-nitrosobenzo[a]pyrene; TCPO, 3,3,3-trichloropropylene 1,2-oxide.
BaP is a mutagen only in the presence of $9 activation (Pitts et al., 1978, 1982, 1984; Tokiwa et al., 1981; Campbell et al., 1981; Fu et al., 1982a; Chou et al., 1983, 1985; 1986; Lofroth et al., 1984; Hass et al., 1986; Heflich et al., 1989). Nitroreduction is an important metabolic activation pathway for 1and 3-nitro-BaP, as evidenced by results showing that the direct-acting mutagenicity of these compounds is lower in TA98NR, which is deficient in a major nitroreductase component (Bryant et al., 1984; Pitts et al., 1984; Chou et al., 1985, 1986; Hass et al., 1986) than in TA98 and that their immediate nitro-reduced intermediates, 1- and 3-nitroso-BaP, exhibit much higher direct-acting rnutagenicity (Thornton-Manning et al., 1989; Heflich et al., 1989). 6-Nitroso-BaP, the immediate aitroreduction product of 6-nitro-BaP, also exhibits strong direct-acting mutagenic activity (Fu et al., 1988; Heflich et al., 1989). Recent studies indicate that hepatic microsomal metabolism of 1and 3-NBaP generates trans-7,8- and 9,10-dihydrodiols which are potent mutagens in S. typhimurium TA98 with and without $9 activation (Chou et al., 1985, 1986) (Table 2). The mutagenicity of these four trans-dihydrodiols assayed in the presence of $9 activation enzymes suggests that the bay-region trans-dihydrodiol epoxides are the metabolites
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
278
NO2 NO2 9,10-Dihydro-l-NBaP
9,10-Dihydro-3-NBaP
9,10-Dihydro-6-NBaP
NO2 7,8-Dihydro-l-NBaP
7,8-Dihydro-3-NBaP
7,8-Dinydro-6-N Ba P
Fig. 1. Structures and abbreviations of the dihydronitrobenzo[a]pyrenes used for the mutagenicitystudies. responsible for the mutation induction (Chou et al., 1985, 1986). In this study, we have determined the mutagenicity of 7,8- and 9,10-dihydro-NBaPs in S. typhimurium TA98, TA98NR, TA98/1,8-DNP6 and TA100 and compared these mutagenicities with those of the NBaP dihydrodiol analogs. The compounds studied include: 7,8-dihydro-l-NBaP, 7,8-dihydro-3-NBaP, 7,8-dihydro-6-NBaP, 9,10-dihydro-3-NBaP, 9.10-dihydro-3-NBaP and 9,10-dihydro-6-NBaP (Fig. 1). These compounds can potentially be metabolized by $9 activation enzymes to the corresponding epoxides by cytochrome P-450 (Conney, 1982). Based on studies of the mutagenicity of BaP diolepoxides and the corresponding tetrahydro-BaP oxides (Conney, 1982), these NBaP tetrahydro-epoxy compounds may alkylate DNA and produce mutations, as do the NBaP diol-epoxide analogs. Materials and methods
Chemicals Benzo[a]pyrene (BaP), 1-nitropyrene and 1,6-dinitropyrene were purchased from Aldrich Chemical Company, Milwaukee, WI. 7-Hydroxyl-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, 7-hydr oxy - 3 - nitro - 7,8,9,10- tetrahydrobenzo[a] pyrene and 7-hydroxy-6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene were prepared as described previously (Von Tungeln and Fu, 1989). Acid-catalyzed de-
hydration of these compounds by p-toluenesulfonic acid in benzene under reflux gave 1-nitro9,10-H2-BaP, 3-nitro-9,10-H2-BaP and 6-nitro9,10-H2-BaP, respectively, in high yield. 1-Nitro7,8-H2-BaP, 3-nitro-7,8-H2-BaP and 6-nitro-7,8H2-BaP were similarly prepared from the corresponding nitro-10-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which were prepared from nitration of 10-keto-7,8,9,10-tetrahydrobenzo[a]pyrene followed by reduction of the nitrated products with sodium borohydride in methanol and tetrahydrofuran (unpublished results). 10-Keto-7,8,9,10tetrahydrobenzo[a]pyrene was prepared by oxidation of 7,8,9,10-tetrahydrobenzo[a]pyrene with 2,3-dichloro-5,6-dicyano- 1,4-benzoquinone and formic acid in dioxan (Fu et al., 1982b). The structures of all the synthesized compounds were confirmed by analysis of their UV-visible absorption, and their mass and proton nuclear magnetic resonance spectral data; their purities were >99% based on HPLC analysis.
Mutagenicity assays Reversion to prototrophy was measured using Salmonella typhimurium histidine auxotrophic strains TA100, TA98, TA98NR and TA98/1,8DNP6 using methods outlined by Maron and Ames (1983). The post-mitochondrial supernatant fraction ($9) was prepared from a liver homogenate of Aroclor 1254-pretreated male Sprague-Dawley rats, and used at a concentration of 50 #l/plate. Experiments were also carried out with the epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO), added at a concentration of 930 /zg/plate to the assays with $9. The variability in assays conducted in triplicate was generally < _+20°70. All test chemicals were assayed on at least two separate occasions with similar results. Revertants/#g/plate were calculated from linear regression lines fitted to the increasing portion of dose-response curves. Results
The mutagenic activities of 7,8-dihydro- 1-NBaP, 7,8-dihydro-3-NBaP, 7,8-dihydro-6-NBaP, 9,10-
279
dihydro- 1-NBaP, 9,10-dihydro-3-NBaP and 9,10dihydro-6-NBaP toward Salmonella typhimurium
TA98, TA98NR, TA98/I,8-DNP6 and TA100 were determined in the absence and presence of rat-
TABLE 1 REVERSION
INDUCTION
Salmonella typhimurium Compound
BY 1-, 3- A N D
6-NITRO-DIHYDROBENZO[a]PYRENE
AND
STANDARD
MUTAGENS
STRAINS TA98, TA98NR, TA98/1,8-DNP6 AND TA100 /~g/plate
$9
Revertants/plate TA98
Solvent control
TA98NR
TA98/I,8-DNP6
TAI00
-
23 ±
5
17 ±
3
7 ±
1
127 ±
5
+
26 ±
1
28 ±
8
19 ±
4
140 ±
2
3311 + 220
1-Nitropyrene
1.0
-
1233 ±
83
258 ±
33
297 ±
42
1,6-Dinitropyrene
0.0025
-
1412 ± 250
1610 ±
46
292 ±
25
330 ±
63
Benzo[a]pyrene (BaP)
5.0
+
528 ± 114
570 ±
31
1016 +
89
9,10-Dihydro-l-NBaP
1.0
-
71 +
6
+
1195 +
73
3
202 +
161 +
28
179 +
2
19
390 ±
61
3.3
+
10.0
+
9,10-Dihydro-3-NBaP
1.0
+
3.3
+
5.0
+
9,10-Dihydro-6-NBaP
2.0 5.0 10.0
0.33 1.0
0.33 1.0 3.3
1.0 3.3
49
212 ±
33
120 ±
757 ±
157 _+
-
-
-
2273 ± 204
7 25
258 ___ 17
497 ± 163
1327 ± 211
103 ±
14
212 ±
14
200 ±
32
92 ±
15
420 + 228
327 +
28
45
247 +
3
350 ±
56
247 ±
25
2493 + 247
1162 ±
25
232 +
6
682 ±
78
357 +
3
453 +
18
280 ±
33
1672 ± 270
453 +
89
1033 ±
78
395 ±
78
3561 ± 720
+
122 ±
10
40
±
2
945 +
78
-
116 +
6
+
398 +
8
253 ±
47
-
50 ±
487 +
5
-
8
32
6
-
312 ±
1184 ± 138
38 ±
1351 ± 203 552 ±
81
3254 + 395
32 ±
7
-
43 ±
5
121 _+
11
164 ±
19
-
-
747 +_
28 ±
-
-
69
128
870 ±
96
±
8
765 ±
85
4
31 ±
7
157 +
6
170 +__ 31
28 ±
8
174 +
10
109 +_
6
72 +
5
210 ±
12
445 ±
60
34 +
3
255 ±
51
182 ±
8
108 +
7
243 ±
46
1197 ± 175
99 ±
17
425 ±
46
-
89 +
10
70 ±
12
48 ±
7
179 +
19
+
167 +
9
25 ±
19
44 ±
5
244 +
25
-
147 +
15
135 ±
20
88 ±
21
207 +
17
+
505 ±
26
635 ± 100
104 ±
17
327 ±
28
370 +
27
8
+
7,8-Dihydro-6-NBaP
348 +
5482 + 385
31 ±
+ 7,8-Dihydro-3-NBaP
2490 + 540
-
+ 3.3
87
+
+ 7,8-Dihydro-l-NBaP
305 _+
1925 ± 269
21
137 ±
14
247 +
2413 ± 210
327 ±
388 +
94
850 +_ 65
-
18
4
-
-
119
+
23 +
10
-
-
143 ±
13
-
20 +
3
-
-
126 +
23
+
26 ±
4
-
-
168 ±
11
+
+
11
IN
280
liver $9 activation enzymes. The mutagenicities of these compounds and the positive standards, 1-nitropyrene, 1,6-dinitropyrene and BaP are given
TABLE
2
REVERTANTS/•g/PLATE MUTAGENS
PRODUCED
BY
1-,
I N Salmonella t y p h i m u r i u m S T R A I N S
Compound
$9
1 - N i t r o - B a P
3-Nitro-BaP
6-Nitro-BaP
9,10-Dihydro-l-NBaP
9,10-Dihydro-3-NBaP
3-NBaP
tert.-9,10-c~hydrodiol c
9,10-Dihydro-6-NBaP
7,8-Dihydro-l-NBaP
I-NBaP
tert.-9,10-dihydrodiol a
7,8-Dihydro-3-NBaP
3-NBaP
tert.-7,8-dihydrodiol c
7,8-Dihydro-6-NBaP
AND
6-NITRO-DIHYDROBENZO[a]PYRENE TA98/1,8-DNP6
AND
AND
TA100
Revertants//zg/plate TA98NR
TA98/I,8-DNP6
TA100
-
2828 a
640 a
2997 a
94 b
+
1414 a
767 a
47 a
101 b
-
3185 c
327 c
4286 c
96 b
+
1418 c
882 c
51 c
141 b
-
9,10-dihydro-1NBaP (516) > 9,10-dihydro-6-NBaP (98) > 7,8-dihydro-6-NBaP ( < 1). 7,8-Dihydro- 1-NBaP, 9,10-dihydro-l-NBaP and 9,10-dihydro-3-NBaP exhibited lower mutagenic activity in S. typhimurium TA98NR than in TA98, and in turn much lower activities were observed in tester strain TA98/1,8-DNP6 than in TA98NR. 9,10-Dihydro-6-NBaP exhibited similar mutagenic potency in these three tester strains, while 7,8-dihydro-3NBaP had nearly equal activity in both TA98 and TA98NR and much lower activity in TA98/1,8DNP6. With the exception of 7,8-dihydro-6-NBaP
which was a very weak mutagen in both tester strains, the dihydro-NBaPs exhibited much lower mutagenic activity in S. typhimurium TA100 than in TA98, both in the presence and absence of $9 activation enzymes. In all the cases, their S9-mediated mutagenic activity was higher than their direct-acting mutagenicity. Among the six dihydroNBaPs tested, 7,8-dihydro-6-NBaP was the only structural isomer essentially inactive in both S. typhimurium strains TA98 and TA100 with and without $9 activation enzymes. 9,10-Dihydro-6NBaP was also inactive in both strains without $9 activation, and exhibited weak mutagenic activity when assayed in the presence of $9 enzymes. Table 3 summarizes the effects of the epoxide hydrolase inhibitor TCPO on the S9-mediated mutagenic activities of the six dihydro-NBaPs toward S. typhimurium TA98 and TA100. Although TCPO increased the mutagenic potency
TABLE 3 R E V E R S I O N I N D U C T I O N BY 1-, 3- A N D 6 - N I T R O D I H Y DROBENZO[a]PYRENE IN Salmonella typhimurium S T R A I N S TA98 A N D T A 1 0 0 W I T H $9 A C T I V A T I O N E N ZYMES IN THE PRESENCE AND ABSENCE OF EPOXIDE HYDROLASE INHIBITOR, 3,3,3-TRICHLOROPROPYLENE 1,2-OXIDE (TCPO) a Compound
$9
TCPO
Revertants/t~g/plate TA98
Solvent control
28 25
TA100
+ +
+
138 200
9 , 1 0 - D i h y d r o - 1-NBaP +
-
516
122
+
+
1377
2736
9,10-Dihydro-3-NBaP +
+
1938
3539
9,10-Dihydro-6-NBaP +
+
169
2915
7 , 8 - D i h y d r o - 1-NBaP
+
-
962
85
+
+
3978
6261
7,8-Dihydro-3-NBaP
+ +
+
584 2175
212 12878
7,8-Dihydro-6-NBaP
+ +
+
3 16
- 9 2398
a E x p e r i m e n t a l details described in M a t e r i a l s a n d M e t h o d s .
282 of the compounds in both TA98 and TA100, the increases were much greater in TA100 (22-74-fold) than in TA98 (3-4-fold). The order or mutagenic potency tested in TA98 with TCPO was: 7,8-dihydro-l-NBaP (3978 revertants/#g/plate) > 7,8-dihydro-3-NBaP (2175) > 9,10-dihydro-3-NBaP (1938) > 9,10-dihydro-l-NBaP (1377) > 9,10dihydro-6-NBaP (169) > 7,8-dihydro-6-NBaP (16). The order or mutagenic potency tested in TA100 with TCPO was: 7,8-dihydro-3-NBaP (12878 revertants/#g/plate) > 7,8-dihydro-1NBaP (6261) > 9,10-dihydro-3-NBaP (3959) > 9,10-dihydro-l-NBaP (2736) > 9,10-dihydro-6NBaP (2915) > 7,8-dihydro-6-NBaP (2398). When assayed at a dose of 1.0 #g/plate, both 7,8-dihydro-l-NBaP and 7,8-3-NBaP exhibited cytotoxicity (loss of the bacterial lawn) in the presence of TCPO. Discussion
The direct-acting mutagenicity of the six dihydro-NBaP compounds was much lower than that observed with $9 activation. The mutagenicity of 7,8-dihydro-3-NBaP and 9,10-dihydro-3-NBaP in all strains of TA98 was similar, indicating that the major component of the bacterial nitroreductase(s) deficient in TA98NR (Bryant et al., 1984) was not involved in the activation and that the transesterificase deficient in TA98/1,8-DNP6 (McCoy et al., 1983) was not required for activation. On the other hand, 7,8-dihydro-l-NBaP exhibited lower mutagenicity in both TA98NR and TA98/1,8-DNP6, indicating the involvement of both the major nitroreductase and transesterificase in the metabolic activation of this compound. These findings indicate that metabolic activation of these compounds involves different mechanisms as well as different metabolizing enzymes. Both 7,8-dihydro-6-NBaP and 9,10-dihydro-6-NBaP, which have their nitro substituents perpendicular to the aromatic moiety, exhibited very weak or no direct-acting mutagenicity in both TA98 and TA100. These results are in agreement with the hypothesis we previously proposed that nitroPAHs with a perpendicular orientation are very
weak direct-acting mutagens in Salmonella (Fu et al., 1985, 1986, 1988, 1989; Fu, 1990). As is the case for the parent compounds, 7,8-dihydro-BaP and 9,10-dihydro-BaP, all six dihydro-NBaPs bear an olefinic aromatic double bond and can be facilely metabolized to the corresponding epoxides by cytochrome P-450 isozymes contained in the $9 mix (Conney, 1982). With $9, all four of the dihydro-l-NBaP and dihydro-3-NBaP isomers were potent mutagens in TA98, producing from 516 to 962 revertants/ #g/plate, and their mutagenicity was much lower in TA98NR and TA98/1,8-DNP6. These results were similar to those found for the 1- and 3-NBaP dihydrodiols (Chou et al., 1985, 1986; Table 3), and indicate that after ring-oxidation of the dihydro derivatives, nitroreduction followed by esterification were required in order to exert maximum mutagenicity. However, it is important to consider that $9 contains epoxide hydrolase which can readily catalyze the hydrolysis of NBaP-tetrahydro-epoxides to the corresponding NBaP-tetrahydro-diols. Indeed, with the incorporation of the epoxide hydrolase inhibitor, TCPO, to the incubation system, much higher mutagenic activities were observed for all six dihydro-NBaP. These results are inconsistent with the mutagenicity assays of 7,8-dihydro-BaP and 9,10-dihydro-BaP tested with $9 in the presence and absence of TCPO (Conney, 1982). With TCPO, the much greater increase of mutagenicity in Salmonella typhimurium TA100 than in TA98 suggests that the metabolites responsible for induction of mutation in TA98 and TA100 may be different. In most cases, mutation in TA98 is frameshift-type while mutation in TA100 is base-pair substitution type (Hartman et al., 1986). It is also known that arylamines which mainly bind to DNA through reactions at the nitrogen atoms are generally more mutagenic in TA98 than in TA100, and that the mutagenicity of PAHs which form DNA adducts via the epoxy ring is usually greater in TA100 than in TA98. Thus, gathering together all these observations, it may be that the mutagenic activity of dihydro-NBaPs in TA98 with $9 and with or without TCPO is largely due to nitroreduction of the nitro substituent of the
283
NR
H\ /
HNOH
NO NR
DNA N
DNA ~
- $9
• NO 2
DNA ~ , 2 + $9
2
0N,
]
MUTATION
~R
HNOH
H~N/DNA /
EH
DNA.~ ~
NO2 HO
/
HNOH
OH :
NR
OH -
)
~
H DNAH
OH
DNA N I
Fig. 2. Proposed metabolicactivation pathways of 9,10-dihydro-l-NBaPin S. typhimurium TA98 and TA100. NR, $9, and EH designate bacterial nitroreductases,9000 g supernatantof liver homogenates,and epoxidehydrolase,respectively.
NBaP-tetrahydro-epoxide compounds. On the other hand, the mutagenic activity of these compounds in TA100, especially with TCPO, appears to be mainly through the interaction of the epoxy ring with DNA. Fig. 2, using 7,8-dihydro-l-NBaP as an example, summarizes the four metabolic activation pathways leading to mutation induction proposed above. Without exogenous $9 activation, nitroreduction, either with or without the involvement of acetylation, is an important activation pathway (shown as pathway 1 in Fig. 2). This pathway is expected to be more important in TA98 than in TA100, as evidenced by the much higher direct-acting mutagenicities observed in this strain
(see Table 1). In contrast, when assayed with $9 and TCPO, the markedly higher mutagenic activity of the tested dihydro compounds in TA100 compared with TA98 suggests that DNA adducts are being formed by reaction with ring carbons of epoxide metabolites stabilized by the presence of the epoxide hydrolase inhibitor TCPO (shown as activation pathway 2 in Fig. 2). When assayed with $9 and without TCPO, both activation pathways 3 and 4 could be involved, although pathway 4 is probable more important than pathway 3 since the tetrahydro-epoxide should be facilely hydrolyzed to the corresponding tetrahydro-trans-diol. Some dihydro nitro compounds activated by pathway 4 require acetylation in order to exert the maximum
284 mutagenicity and
these
display relatively low
m u t a g e n i c responses in T A 9 8 / I , 8 - D N P 6 (Tables 1 a n d 2), Since b o t h a c t i v a t i o n pathways 3 a n d 4 involve n i t r o r e d u c t i o n a n d D N A - a d d u c t f o r m a t i o n t h r o u g h reaction with a m i n o nitrogens, comp o u n d s activated by these pathways should produce higher m u t a g e n i c i t y in TA98 t h a n in TA100. M o s t o f the n i t r o - P A H s a n d a r y l a m i n e s so far studied are derived from n o n - m u t a g e n i c P A H s , such as biphenyl, n a p h t h a l e n e , a n t h r a c e n e , pyrene a n d fluorene. T h u s , m e t a b o l i s m o f these comp o u n d s t h r o u g h r i n g - o x i d a t i o n generally results in detoxification. O n the other h a n d , n i t r o r e d u c t i o n of nitro-PAHs and N-oxidation of arylamines generates N - h y d r o x y a m i n o - P A H s which b i n d with D N A a n d induce m u t a t i o n s largely via a frameshift m e c h a n i s m ( R o s e n k r a n z a n d Mermelstein, 1983). This a c c o u n t s for the general p h e n o m e n o n that the m u t a g e n i c i t y o f b o t h n i t r o - P A H s a n d a r y l a m i n e s is greater in TA98 t h a n in TA100. However, the m u t a g e n i c i t y results of the six d i h y d r o - N B a P s assayed with $9 a n d T C P O clearly indicate that these c o m p o u n d s exhibit m u c h higher m u t a g e n i c i t y in T A 1 0 0 t h a n in TA98. T h u s , these findings suggest that r i n g - o x i d a t i o n can be a n i m p o r t a n t p a t h w a y for the metabolic a c t i v a t i o n o f nitroP A H s to m u t a g e n s , a n d are also in a g r e e m e n t with the previous findings that r i n g - o x i d a t i o n o f 1- a n d 3 - N B a P to the c o r r e s p o n d i n g diol epoxides is a p o t e n t i a l activation p a t h w a y ( C h o u et al., 1985, 1986; T h o r n t o n - M a n n i n g et al., 1988).
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