Xenobiotica the fate of foreign compounds in biological systems
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Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen F. A. Beland, B. A. Smith, J. R. Thornton-Manning & R. H. Heflich To cite this article: F. A. Beland, B. A. Smith, J. R. Thornton-Manning & R. H. Heflich (1992) Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen, Xenobiotica, 22:9-10, 1121-1133, DOI: 10.3109/00498259209051866 To link to this article: http://dx.doi.org/10.3109/00498259209051866
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Date: 05 May 2016, At: 22:22
XENOBIOTICA,
1992, VOL. 22,
NOS
9/10, 1121-1133
Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen F. A. BELAND?$*, B. A. S M I T H t , J. R. T H O R N T O N - M A N N I N G t § and R. H. H E F L I C H t
t National
Center for Toxicological Research, Jefferson, AR 72079, USA
t University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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Received 20 December 1990; accepted 17 April 1992 1. The mutagenicity of 1-nitropyrene metabolites in Chinese hamster ovary (CHO) 1cells, in the absence of rat liver S9, decreased in the order 6-hydroxy-1-nitropyrene~ nitropyrene 9,10-oxide> 1-nitropyrene 4,s-oxide 3-hydroxy-1 -nitropyrene 8hydroxy-1-nitropyrene > 1-nitropyrene. The order of mutagenicity with rat liver S9 was 1nitropyrene 4,s-oxide -6-hydroxy-1 -nitropyrene 1-nitropyrene 9,lO-oxide > 3hydroxy-1 -nitropyrene 1-nitropyrene > 8-hydroxy-1 -nitropyrene. 2. 1-Nitropyrene 4,s-oxide reacted with calf thymus DNA to give one or several closely related adducts. The same adducts were detected in CHO cells incubated with 1nitropyrene 4,s-oxide. Inclusion of a nitroreductase, xanthine oxidase, in the incubations with calf thymus DNA resulted in the formation of an additional adduct identified as N (deoxyguanosin-8-yl)-1 -aminopyrene (dG-C8-AP).
-
-
-
-
3. 1-Nitropyrene 9,lO-oxide reacted with calf thymus DNA to give an adduct pattern similar to that observed with 1-nitropyrene 4,s-oxide. Incubation of 1-nitropyrene 9,lOoxide with C H O cells resulted in the formation of the same adducts along with dG-C8-AP. 4. dG-C8-AP and N-(deoxyguanosin-8-y1)-l-amino-x-nitropyrene(x = 3, 6 or 8; dGC8-ANP) were detected in injection site DNA from Sprague-Dawley rats treated with 1nitropyrene. In mammary gland DNA, dG-C8-AP and an unidentified adduct were found. dG-C8-ANP was the only DNA adduct detected in the livers of newborn CD-1 mice and the lungs of A/J mice dosed with 1-nitropyrene.
1. Introduction Nitropyrenes, in particular I-nitropyrene and 1,3-, 1,6-, and 1,8-dinitropyrene (figure l), are widespread environmental pollutants that have been detected in photocopier toners, airborne particulates, coal fly-ash, and diesel engine exhaust (reviewed in IARC 1989). All four of these nitropyrenes are usually detected in environmental samples, with 1-nitropyrene typically being present in much greater concentrations than the dinitropyrenes. T h e dinitropyrenes, however, are extremely potent bacterial mutagens, which was the property that led to their initial discovery. As a result, the dinitropyrenes and 1-nitropyrene contribute nearly equally to the observed bacterial mutagenicity of most samples. I n addition to being mutagenic, nitropyrenes are tumourigens. 1-Nitropyrene, for example, induces tumours at the injection site and in the mammary gland in rats (Hirose et al. 1984, El-Bayoumy et al. 1988 b), in the lungs of adult mice (El-Bayoumy et al. 1984), and in the livers of newborn mice (Wislocki et al. 1986). T h e dinitropyrenes, in particular 1,6- and 1,8dinitropyrene, appear to be considerably more tumourigenic than 1-nitropyrene
*Address correspondence to: Frederick A. Beland, HFT-110, NCTR, Jefferson, AR 72079, USA. $Current address: U.S. EPA, HERL, MD-74, Research Triangle Park, N C 27711, USA. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
F. A. Beland et al.
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BNO*
OzN
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1-Nitropyrene
1,3-Dmi:ropyrens
N(0eoryguanorin-8-yljl-aminopyrene
Figure 1 .
1.B-Dinnropyrens
N(Deory uanosinS-yl)-l-arnlno gnitropyrene
Structures of 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene,and 1,FJ-dinitropyrene and metabolic pathways leading to identified DNA adducts.
From Beland and Kadlubar (1990).
and, when tested in the same animal model, show the same organ specificity as 1-nitropyrene (Nesnow et al. 1984, Ohgaki et al. 1984, 1985, Tokiwa et al. 1984, Takayama et al. 1985, Maeda et al. 1986, Wislocki et al. 1986, Otofuji et al. 1987, King 1988). T h e conversion of 1-nitropyrene to a mutagen in bacteria is thought to involve nitroreduction to a reactive N-hydroxy arylamine intermediate. Howard et al. (1983), for example, demonstrated that the major adduct formed in DNA of S. typhimurium treated with 1-nitropyrene was N-(deoxyguanosin-8-y1)-1aminopyrene (dG-C8-AP), an adduct consistent with the metabolic conversion of 1nitropyrene to N-hydroxy-1 -aminopyrene (figure 1). Dinitropyrenes are also activated through nitroreduction. In the case of 1,6-dinitropyrene the major adduct is N-(deoxyguanosin-8-y1)-1-amino-6-nitropyrene (DjuriC et al. 1988; figure l ) , while 1,8-dinitropyrene gives N-(deoxyguanosin-8-y1)-1-amino-8-nitropyrene (Heflich et al. 1985 a, Andrews et al. 1986; figure 1). T h e same adducts are found in S. typhimurium treated with 1,6- and 1,8-dinitropyrene (Heflich et al. 1985 a, Andrews et al. 1986, DjuriC et al. 1986 b). T h e mutagenic efficiencies of dG-C8-AP and N-(deoxyguanosin-8-y1)-1amino-8-nitropyrene have been compared in S. typhimurium TA1538 and appear to be similar (Heflich et al. 1985a, DjuriC et al. 1986b). Therefore, the extreme mutagenicity of the dinitropyrenes is not due to a unique adduct but appears to be due to their efficient metabolism to a DNA-binding intermediate. This seems to be a
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result of the N-hydroxy amino intermediates of the dinitropyrenes (e.g. N-hydroxy1-amino-8-nitropyrene; figure 1) but not 1 -nitropyrene serving as substrates for transacetylases that catalyse the formation of reactive N-acetoxy derivatives (e.g. Nacetoxy- 1-amino-8-nitropyrene). 1-Nitropyrene is at best a very weak direct-acting mutagen in most mammalian cell lines; however, mutations can be induced by the inclusion of mammalian enzymes, such as the 9000 x g supernatant of a rat liver homogenate (S9; Li and Dutcher 1983, Heflich et al. 1990). T h e mammalian enzyme-mediated metabolism of 1-nitropyrene in vitro is complex and a number of products result from reduction and/or oxidation (reviewed in Beland and Kadlubar 1990, Smith et al. 1990 b; figure 2). Either of these pathways, or a combination of the two, could be involved in the activation of 1 -nitropyrene by mammalian enzymes to mutagenic derivatives. Metabolites indicative of nitroreduction, ring oxidation, and nitroreduction of ringoxidized products have been detected in vivo (reviewed in Beland and Kadlubar 1990, Smith et al. 1990 b). All of these pathways are involved in the activation of 1nitropyrene to DNA-binding species in microsomal incubations (DjuriC et al. 1986 a), which indicates that they may be important to tumourigenic activity in vivo. An adduct consistent with nitroreduction has been found in vivo along with, in some instances, other adducts (Stanton et al. 1985, Hashimoto and Shudo 1985, Mitchell
HO.
@
HO.'
0
@ 00,
H
6';" '-OH
0
Figure 2. 1 -Nitropyrene metabolites detected in in vitro incubations. From Beland et al. (1985).
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F . A. Beland et al.
1988, El-Bayoumy et al. 1988a, Roy et al. 1989). However, it is not known if the treatment protocols used in these adduct studies will induce tumours. In order to gain insight into the pathways by which 1-nitropyrene is activated in vivo, we have performed a series of interrelated in vitro and in vivo experiments. Specifically, we determined the mutagenicities of ring-oxidized metabolites of 1nitropyrene in Chinese hamster ovary (CHO) cells and compared the results to those obtained from incubating C H O cells with 1-nitropyrene in the presence of rat liver S9 preparations. Since these data indicated that 1-nitropyrene 4,s-oxide and 1nitropyrene 9,lO-oxide played an important role in the mutagenic response, we examined the DNA adducts formed by these oxides following reactions with calf thymus DNA, both in the presence and absence of a nitroreductase, xanthine oxidase, and compared these adducts with those responsible for the mutagenicity of the oxides in CHO cells. Finally, we treated Sprague-Dawley rats, CD-1 mice, and A/J mice with I-nitropyrene according to three regimens reported to induce tumours (El-Bayoumy et al. 1984, Hirose et al. 1984, Wislocki et al. 1986) and compared the DNA adducts present in the target tissues with those obtained in the C H O cells.
Materials and methods Materials 1-Nitropyrene (lot number CSL-86-100-20; 99.85-9998% 1-nitropyrene, 0.15-0.02% 1,3-, 1,6- and l,S-dinitropyrene, by mass spectral analyses) was purchased from Chemsyn Science Laboratories, Lenexa, KS, USA. 1-Nitropyrene 4,s-oxide and 1-nitropyrene 9,lO-oxide (Fifer et al. 1986), and 3-, 6and 8-hydroxy-I-nitropyrene (El-Bayoumy and Hecht 1983) were synthesized as described in the indicated references. [y-"P]ATP was synthesized from carrier-free [3zP]phosphate (ICN Biomedicals, Inc., Irvine, CA, USA) (Gupta et al. 1982). DNA modified with dG-C8-AP was prepared by reacting N-hydroxy-1 -amin~[~H]pyrene with calf thymus DNA to give a level of modification of 8.3 adducts per 1 x lo3 nucleotides (Heflich et al. 1985 b). DNA modified with N-(deoxyguanosin-8-yI)-l-amino-6-nitropyrene was synthesized by reacting N-hydroxy-l-amin0-6-nitro[~H]pyrenewith pS189 DNA to give a modification level of 4 8 adducts per 1 x lo3 nucleotides (Djurii: et al. 1988). Rat liver S9 was prepared as described in Heflich et al. (1990). Mutagenicity assays Mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were measured by quantifying resistance to 6-thioguanine. Assays conducted in the presence and absence of S9 activation were performed as described in Heflich et al. (1990). Reaction of 1-nitropyrene 4,5-oxide and I-nitropyrene 9,IO-oxide with D N A Calf thymus DNA was incubated with the oxides in the presence and absence of xanthine oxidase and hypoxanthine as outlined in DjuriC et al. (1986a). Treatment of animals Animals were treated with 1-nitropyrene and DNA was isolated from target tissues as described in Smith et 01. (1990b). 32P-postlabelling of adducts 32P-postlabellingof DNA adducts was performed using modifications of the n-butanol enhancement procedure described by Gupta (1985). DNA digests were treated with nuclease P1 essentially as described by Gupta and Earley (1988) as modified by Smith et al. (1990 b). Adducts were treated with hydrazine as indicated in Smith et al. (1990 b). DNA adducts were separated by the contact-transfer method of Lu et al. (1986). Development in the first direction ( D l ) was with 0.65 M sodium phosphate, pH 6.0, or 0 9 M sodium phosphate, pH 6.8. Solvents used for adduct separation in D3 and D4 (on individual 10 x lOcm Merck PEI-cellulose t.1.c. plates) were 3.6 M lithium formate, 8.5 M urea, pH 3.5, and 1.2 M lithium chloride, 0.5 M Tric-HCI, 8.0M urea, pH 8.0,respectively. An additional development in 0.9 M sodium phosphate, pH 6.8, was performed in the direction of D4 onto a 3-cm Whatman 1 wick (Lu et al. 1986). Adducts were located by autoradiography, as described by Gupta et 01. (1982).
Metabolic activation of 1 -nitropyrene
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Results Mutagenicity in CHO cells of 1 -nitropyrene and oxidized 1 -nitropyrene metabolites Mutagenicity assays were conducted with C H O cells exposed to 1-nitropyrene, 3-, 6- and 8-hydroxy-l-nitropyrene,1-nitropyrene 4,S-oxide) and 1-nitropyrene 9,lO-oxide (see figure 2 for structures) both in the presence and the absence of S9 activation. Data are summarized in table 1. In the absence of S9, 6-hydroxy-lnitropyrene and 1-nitropyrene 9,lO-oxide were the most mutagenic compounds. 3and 8-Hydroxy-1 -nitropyrene and 1-nitropyrene 4,s-oxide were weaker mutagens, while 1-nitropyrene was essentially non-mutagenic. T h e order of mutagenic potency with S9 was 1-nitropyrene 4,s-oxide 6-hydroxy-1 -nitropyrene 1-nitropyrene 9,lO-oxide > 3-hydroxy-1 -nitropyrene 1-nitropyrene > 8-hydroxy-1 -nitropyrene. S9 significantly decreased the mutagenicity of 6-hydroxy-1 -nitropyrene.
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N
N
Reaction of 1 -nitropyrene 4,5-oxide and 1 -nitropyrene 9,lO-oxide with D N A 32P-Postlabelling analysis of the 1-nitropyrene 4,s-oxide-modified calf thymus DNA indicated either one, or possibly several closely related, adducts (figure 3A). DNA reacted with 1-nitropyrene 4,s-oxide in the presence of xanthine oxidase and hypoxanthine also had these adducts as well as an additional adduct having migration characteristics identical to the bis-phosphate of dG-C8-AP (figure 3B). Calf thymus DNA was also reacted with 1-nitropyrene 9,lO-oxide and analysed by 32Ppostlabelling (figure 4A). Two adducts were observed that migrated in the vicinity of the 'direct-reaction' adducts obtained from 1-nitropyrene 4,s-oxide. A similar adduct pattern was obtained when calf thymus DNA was incubated with 1nitropyrene 9,lO-oxide in the presence of xanthine oxidase and hypoxanthine (figure 4B).
Table 1.
Summary of the mutagenicity of 1-nitropyrene and oxidized 1-nitropyrene metabolites in CHO cells. Minus S9 Compound 1-Nitropyrene 3-Hydroxy-1-nitropyrene 6-Hydroxy-1-nitropyrene 8-Hydroxy-1-nitropyrene 1-Nitropyrene 4,S-oxide 1-Nitropyrene 9,lO-oxide
Mutation frequency" 1.4f0.7 5.0 f 1.5"
52.2 k60'*d.' 4 0 f 1.2 6.7 3.1 286k 5.7'~~
Plus s 9 Nb
Mutation frequency
12 8 19 7 10 9
3.4f 1.0 4 5 1.0 13.3+2.6'*' 0.4 f2.4 18.9 k7.9 15.0+4.7'
*
Nb 8 5 8
7 8 9
a Data are from Heflich et al. (1990). Mutation frequencies (mutants per 1 x lo6 clonable cells per lOnmol compound per ml) were calculated from the increasing portions of dose-response curves. The spontaneous mutations ( 4 k 2, - S9, n = 21; 5 f 2, S9, n = 20) have been subtracted from each assay. Data are presented as the meanf SEM. Statistical comparisons were made by Student's t-test. Differences were considered significant when p < 0.05. Number of determinations. 'Significantly different from 1-nitropyrene. Significantly greater than in the presence of S9. Significantly greater than 3- and 8-hydroxy-1-nitropyrene. Significantly greater than 1-nitropyrene 4,s-oxide.
+
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Figure 3. 3zP-Postlabellingautoradiograms of adducts obtained from: A, calf thymus DNA reacted with 1-nitropyrene 4,s-oxide; B, calf thymus DNA reacted with 1-nitropyrene 4,s-oxide in the presence of xanthine oxidase and hypoxanthine; C, DNA from CHO cells incubated with 1nitropyrene 4,s-oxide. Data are from Smith et al. (1990a)
Figure 4. 32P-Postlabellingautoradiograms of adducts obtained from: A, calf thymus DNA reacted with 1-nitropyrene 9,lO-oxide; B, calf thyus DNA reacted with 1-nitropyrene 9,lO-oxide in the presence of xanthine oxidase and hypoxanthine; C, DNA from CHO cells incubated with 1nitropyrene 9,lO-oxide. Data are from Smith et al. (1990a).
D N A adduct analysis in CHO cells exposed to I-nitropyrene 4,5-oxide and 1nitropyrene 9,lO-oxide In C H O cells incubated with 1-nitropyrene 4,5-oxideY the major adducts detected corresponded with those observed by the direct reaction of the oxide with DNA (figure 3C). C H O cells exposed to 1-nitropyrene 9,lO-oxide also had adducts that migrated in the vicinity of the ‘direct-reaction’ adducts; in addition, there appeared to be a small amount of the bis-phosphate of dG-CS-AP (figure 4C). At a 10 PM dose, 1-nitropyrene 9,lO-oxide produced approximately four times as many mutations as 1-nitropyrene 4,s-oxide (table 2). Likewise, 1-nitropyrene 9,lO-oxide bound to the C H O cell DNA approximately four-fold more than 1-nitropyrene 4,soxide. D N A adduct analysis in animals treated with 1 -nitropyrene 32P-Postlabelling was used for the analysis of DNA adduct formation in tissues that have been demonstrated to be targets for tumourigenesis by I-nitropyrene. I n female Sprague-Dawley rats, two adducts were apparent in DNA extracted from the
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subcutaneous injection site (figure 5A). One of these was tentatively identified as dGC8-AP by comparison of its chromatographic behaviour with the adduct standard, dG-C8-AP, that was analysed in parallel (figure 6A). T h e second adduct migrated slightly below dG-Cg-AP. This less polar adduct appeared to be a dinitropyrene adduct, N-(deoxyguanosin-8-y1)-1-amino-x-nitropyrene (x = 3, 6 or 8; dG-C8ANP), based on the similarity of its chromatographic behaviour with that of the standard, N-(deoxyguanosin-8-y1)-1-amino-6-nitropyrene(figure 6D). Neither adduct was apparent in injection site DNA isolated from animals that received the vehicle only (figure 5B). In mammary gland DNA from the 1-nitropyrene-treated Sprague-Dawley rats, two adducts were found (figure SC) that were not observed in control animals (figure SD). T h e minor adduct migrated to the same position as the standard, dG-C8-AP, while the major adduct was more polar. Both liver DNA from newborn CD-1 mice (figure 5E) and lung DNA from A/J mice (figure 5G) contained a single major adduct that migrated to the same position as the standard, dG-C8ANP (figure 6D). This adduct was not detected in liver (figure 5F) or lung (figure 5H) DNA from control mice. T h e identification of the D N A adducts was facilitated by the fact that the two standards, dG-Cg-AP and dG-C8-ANP, responded differently following exposure to hydrazine in the presence of palladium on carbon or incubation with nuclease P1 (figure 6). When the dG-C8-AP standard was treated with hydrazine and palladium on carbon, the adduct was recovered in 76% yield (figure 6B), whereas < 1% of the dG-C8-ANP standard was recovered after a similar treatment (figure 6E). In contrast, incubation of the dG-C8-AP standard with nuclease P1 caused a 97% decrease in the intensity of this adduct (figure 6C), while the intensity of dG-C8ANP was decreased by < 20% (figure 6F). T h e in vivo adducts identified as dG-C8AP and dG-C8-ANP behaved in a manner identical to the standards upon treatment with nuclease P1 and hydrazine (not shown). T h e concentration of the DNA adducts in the target tissues was quantified through comparisons to the D N A standards modified with dG-C8-AP and dG-C8ANP. As shown in table 3, dG-C8-ANP was formed to approximately an eight-fold greater extent than dG-C8-AP in injection site D N A of Sprague-Dawley rats, while the unknown adduct in the mammary gland DNA was estimated to be present at 10 times the amount of dG-C8-AP.
Discussion In this study we have used both in vitro mutagenesis and in vivo D N A binding assays to obtain a better understanding of the pathways by which I-nitropyrene is metabolized to a mammalian-cell mutagen and carcinogen. Initially, C H O cells were incubated with a series of 1-nitropyrene metabolites (figure 2). Of these, 6-hydroxy1-nitropyrene, 1-nitropyrene 9,10-oxide, and 1-nitropyrene 4,S-oxide were the strongest mutagens in C H O cells. These mutagenic responses were produced both in the presence and absence of S9 activation (table l), which indicates that these compounds are the metabolites primarily responsible for the S9-mediated mutagenicity of 1-nitropyrene in CHO cells. T h e direct-acting mutagenicity of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,lO-oxide could be due to DNA binding either through the ring epoxide or via an N-hydroxy arylamine intermediate produced by nitroreduction. Both routes of DNA binding have been demonstrated with these compounds in cell-free incub-
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Figure 5. Autoradiograms of 32P-postlabelled adducts in wiwo in the DNA of target tissues for 1 nitropyrene tumourigenesis. Injection site DNA from Sprague-Dawley rats treated with 1-nitropyrene (A) or solvent alone (B). Mammary gland DNA from Sprague-Dawley rats treated with 1-nitropyrene (C) or solvent alone (D). Liver DNA from CD-1 mice treated with 1-nitropyrene (E) or solvent alone (F). Lung DNA from A/J mice treated with I-nitropyrene (G) or solvent alone (H). Data are from Smith et al.
(1990b).
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Figure 6. Autoradiograms of 32P-postlabelled DNA adduct standards dG-C8-AP (A-C) and dG-C8ANP (D-F), before (A, D) and after treatment with hydrazine (B, E) or nuclease P1 (C, F). Data are from Smith et al. (1990b).
Table 2.
Mutations and DNA adducts produced in CHO cells by 1-nitropyrene 4,s-oxide and 1nitropyrene 9,lO-oxide. Compound
Mutants per 1 x lo6 clonable C H O cells’
DNA adduct concentration (adducts/106 nucleotides)b
6.7 k 3.1 28.6 & 5.7
3.8 147
1-Nitropyrene 4,s-oxide ( 1 0 ~ ~ ) 1-Nitropyrene 9,lO-oxide ( 1 0 ~ ~ )
Data are from table 1. b T h e concentration of DNA adducts was determined by 3zP-postlabelling through comparison to a standard modified with dG-C8-AP at a level of 0.5 adducts per 1 x lo6 nucleotides. Data are from Smith et al. (1990a). a
Table 3.
Concentration of DNA adducts formed in target tissues of animals treated with 1-nitropyrene. DNA adduct concentration (adducts/lO’ nuc1eotides)a
Species
Tissue
Sprague-Dawley rats
Injection site Mammary gland Liver Lung
CD-1 mice A/J mice
dG-C8-AP 0.8 k 0.4 0-2k0 0 6
dG-C8-ANP
Unknown
6.2 0.9 1.8 0.4b 7.0k3.1 7.9 f 2.9
a Data are from Smith et al.(1990 b). Adduct levels were quantified by comparison to DNA standards modified with dG-C8-AP and dG-C8-ANP. Results are reported as the meankstandard deviation of individual determinations from 4 to 8 animals. Quantified through comparison to DNA standards modified with dG-C8-ANP.
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ations utilizing mammalian nitroreductases and isolated D N A (DjuriC et al. 1986 a). T h e presence of the mammalian nitroreductase, xanthine oxidase, and hypoxanthine had no obvious effect on the adduct pattern produced by 1-nitropyrene 9,lO-oxide (figure 4), but an additional adduct with properties consistent with dG-C8-AP was observed in reactions conducted with 1-nitropyrene 4,s-oxide (figure 3). It appears that dG-C8-AP was produced from the reduction of 1-nitropyrene 4,s-oxide to 1nitropyrene followed by nitroreduction to N-hydroxy-1 -aminopyrene. Sugiura et al. (1980) have shown that xanthine oxidase and hypoxanthine will reduce arene oxides, while we (Howard et al. 1983) have demonstrated that the same system will catalyse the reduction of 1-nitropyrene to N-hydroxy-1 -aminopyrene, which will react with DNA to give dG-C8-AP. I-Nitropyrene 4,s-oxide produced a DNA adduct profile in C H O cells (figure 3C) similar to that found by reacting this compound directly with DNA (figure 3A). T h e adduct profile produced in CHO cells by 1-nitropyrene 9,lO-oxide (figure 4C) also included a small amount ( 5%) of an adduct that migrated in the vicinity of dGC8-AP. This adduct was not seen when the 9,lO-oxide was incubated with calf thymus DNA either in the presence or absence of xanthine oxidase and hypoxanthine. 1-Nitropyrene 9,lO-oxide also bound to C H O cell DNA to a much greater extent and was more mutagenic than 1-nitropyrene 4,S-oxide, while binding of the two oxides to calf thymus DNA was nearly equal (DjuriC et al. 1986a). T h e differential activities of these compounds in C H O cells could reflect the fact that 1nitropyrene 4,s-oxide is more readily detoxified than 1-nitropyrene 9,lO-oxide through epoxide hydrase-catalysed hydration (DjuriC et al. 1987). T h e results obtained with the CHO cells indicated that ring-oxidized metabolites could be responsible for the mutations induced in mammalian cells by 1nitropyrene; therefore, we ascertained if adducts derived from these metabolites were present in target tissues for 1-nitropyrene-induced tumours. In the injection site DNA from Sprague-Dawley rats, two major adducts were observed by 32Ppostlabelling (figure 5A). Based upon their chromatographic behaviour and their sensitivity to nuclease P1 and hydrazine relative to adduct standards, these were identified as adducts derived from the nitroreduction of 1-nitropyrene and a dinitropyrene. Two adducts also were detected in mammary gland DNA from Sprague-Dawley rats (figure SC). One of these had chromatographic characteristics and hydrazine and nuclease P1 sensitivities similar to dG-C8-AP. T h e identity of the other adduct is unknown, but it may result from ring oxidation. In the other two animal models, newborn CD-1 mice and A/J mice, the only DNA adduct detected in liver and lung, respectively, had characteristics consistent with dG-C8-ANP (figures 5E and 5G). Although the major adducts detected in CD-1 and A/J mice and at the injection site in Sprague-Dawley rats resulted from the nitroreduction of dinitropyrenes (table 3), mass spectral analyses of the I-nitropyrene used in these experiments indicated very low levels of dinitropyrene contamination. This indicates that dinitropyrenes are activated very efficiently to electrophilic metabolites, to an extent far better than 1-nitropyrene. There appear to be a number of reasons for this. First, as noted previously, the major metabolic pathways for 1-nitropyrene in vivo are through ring oxidation. Although ring-oxidized metabolites have been reported to bind to DNA (DjuriC et al. 1986 a, Smith et al. 1990a), the data we obtained in the present study indicate that, with the possible exception of the mammary gland in Sprague-Dawley rats, this does not occur to a large extent in target tissues for 1-
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nitropyrene tumourigenesis. In contrast, the only metabolic pathways that have been reported for dinitropyrenes are through nitroreduction (DjuriC et al. 1985, 1986c, 1988, Heflich et al. 1986), which presumably results in the formation of reactive N-hydroxy arylamine intermediates. Second, 1-nitropyrene undergoes nitroreduction primarily via a one-electron route, while the initial reduction of 1,6and 1-8-dinitropyrene is mainly by a two-electron pathway (Eddy et al. 1986). In addition, the one-electron reduction product of 1-nitropyrene, a radical anion, is quite sensitive to oxygen and readily undergoes redox cycling to regenerate 1nitropyrene (DjuriC et al. 1986 c). This will result in decreased concentrations of the electrophile, N-hydroxy-1-aminopyrene. Third, the N-hydroxy arylamine metabolites of dinitropyrenes are converted by 0-acetyltransferases to N-acetoxy arylamines (figure l ) , with a further increase in reactivity. For example, in in vitro studies with rat liver cytosol, 0-acetylation of N-hydroxy-1 -amino-3-, 6-, or 8nitropyrene increased the extent of D N A binding by 2W0-fold (DjuriC et al. 1985). This does not occur with the reduced 1-nitropyrene metabolite, N-hydroxy-laminopyrene, for reasons that are presently unknown. In summary, mutagenesis assays using C H O cells indicate that ring-oxidized metabolites of 1-nitropyrene can contribute to the mutagenic response of this compound in mammalian cells. However, the presence of dG-C8-AP in the DNA from the injection site and mammary glands of Sprague-Dawley rats indicates that the metabolic pathway responsible for activation of 1-nitropyrene to a tumourigen in these tissues is nitroreduction. An unidentified adduct present in mammary gland DNA indicates that pathways other than nitroreduction are also involved in the metabolic activation of 1-nitropyrene in this tissue. Nevertheless, when the mammary gland data are compared with the CHO cell results, it appears that this unidentified pathway does not involve 1-nitropyrene 4,s-oxide or 1-nitropyrene 9,10-oxide. Finally, the fact that only dinitropyrene-derived adducts were found in DNA from the livers of CD-1 mice and the lungs of A/J mice raises the question as to whether or not the previously reported tumour formation in these tissues can be attributed to 1-nitropyrene.
Acknowledgements We thank Nancy F. Fullerton for technical assistance, M. Matilde Marques for providing the 1-nitropyrene-modified DNA, Marcia Mah for preparing the 1,6dinitropyrene-modified DNA, Glenn Talaska for giving advice on 32P-postlabelling assays, Walter A. Korfmacher for conducting the mass spectral analyses, and Cindy Hartwick for helping prepare this manuscript. Research in this article was conducted, in part, under contract to the Health Effects Institute (HEI), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-812059) and automotive manufacturers. T h e contents of this article do not necessarily reflect the views of the HEI, nor do they necessarily reflect the policies of the EPA, or automotive manufacturers.
References ANDREWS, P. J., QUILLIAM, M. A,, MCCARRY, B. E., BRYANT, D. W., and MCCALLA, D. R., 1986, Identification of the DNA adduct formed by metabolism of 1,8-dinitropyrene in Salmonella typhimurium. Carcinogenesis, 7 , 105-1 10. BELAND, F. A,, and KADLUBAR, F. F., 1990, Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons. In Handbook of Experimental Pharmacology, Vol. 94/I, edited by C. S. Cooper and P. L. Grover (Berlin: Springer-Verlag), pp. 267-325.
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BELAND, F. A., HEFLICH, R. H., HOWARD, P. C., and Fu, P. P., 1985, The in vitro metabolic activation of nitro polycyclic aromatic hydrocarbons. In Polycyclic Hydrocarbons and Carcinogenesis, ACS Symposium Series, No. 283, edited by R. G. Harvey (Washington, DC: American Chemical Society), pp. 371-396. DJURIC,Z., COLES, B., FIFER,E. K., KETTERER, B., and BELAND, F. A,, 1987, I n wiwo and in witro formation of glutathione conjugates from the K-region epoxides of 1-nitropyrene. Carcinogenesis, 8, 17811786. DJURIC, Z., FIFER,E. K., and BELAND, F. A., 1985,Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA. Carcinogenesis, 6, 941-944. DJURIC,Z., FIFER, E. K., HOWARD, P. C., and BELAND, F. A,, 1986 a, Oxidative microsomalmetabolism of 1-nitropyrene and DNA-binding of oxidized metabolites following nitroreduction. Carcinogenesis, 7, 1073-1079. DJURIC,Z., HEFLICH, R. H., FIFER, E. K., and BELAND, F. A,, 1986 b, Metabolic activationof mutagenic and tumorigenic dinitropyrenes. In Biochemical and Molecular Epidemiology of Cancer, edited by C. Harris (New York: Alan R. Liss), pp. 4 4 1 4 7 . D. W., HEFLICH, R. H., and BELAND, F. A,, 1986c, Aerobic and anaerobic reduction DJURIC,Z., POTTER, of nitrated pyrenes in vitro. Chemico-Biological Interactions, 59. 309-324. DJURIC,Z., FIFER,E. K., YAMAZOE, Y., and BELAND, F. A,, 1988, DNA binding by 1-nitropyreneand 1,6dinitropyrene in witro and in wiwo: effects of nitroreductase induction. Carcinogenesis, 9, 357-364. R., 1986, Dichotomy in the EDDY,E. P., McCoy, E. C., ROSENKRANZ, H. S., and MERMELSTEIN, mutagenicity and genotoxicity of nitropyrenes: apparent effect of the number of electrons involved in nitroreduction. Mutation Research, 161. 109-1 11. EL-BAYOUMY, K., and HECHT,S. S., 1983, Identification and mutagenicity of metabolites of 1nitropyrene formed by rat liver. Cancer Research, 43, 3132-3137. EL-BAYOUMY, K., HECHT,S. S., SACKL, T., and STONER, G. D., 1984, Tumorigenicity and metabolism of 1-nitropyrene in A/J mice. Carcinogenesis, 5 , 1449-1452. EL-BAYOUMY, K., SHIUE,G.-H., and HECHT,S. S., 1988a, Metabolism and DNA binding of 1 nitropyrene and 1-nitrosopyrene in newborn mice. Chemical Research in Toxicology, 1 , 243-247. EL-BAYOUMY, K., RIVENSON, A., JOHNSON, B., DIBELLO,J., LITTLE,P., and HECHT,S. S., 1988b, Comparative tumorigenicity of 1-nitropyrene, 1-nitrosopyrene, and 1-aminopyrene administered by gavage to Sprague-Dawley rats. Cancer Research, 48, 42564260. FIFER,E. K., HOWARD, P. C., HEFLICH, R.H., and BELAND, F. A., 1986, Synthesis and mutagenicity of 1nitropyrene 4,S-oxide and 1-nitropyrene 9,10-oxide, microsomal metabolites of 1-nitropyrene. Mutagenesis, 1, 433438. GUPTA,R. C., 1985, Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen : DNA adducts. Cancer Research, 45, 5656-5662. GUPTA,R. C., and EARLEY, K., 1988, 32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis, 9, 1687-1693. GUPTA,R. C., REDDY, M. V., and RANDERATH, K., 1982, 32P-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis, 3, 1081-1092. HASHIMOTO, Y., and SHUDO,K., 1985, Modification of nucleic acids with 1-nitropyrene in the rat: identification of the modified nucleic acid base. Japanese Journal of Cancer Research (Gann), 76, 253-256. HEFLICH, R. H., DJURIC,Z., FIFER, E. K., CERNIGLIA, C. E., and BELAND, F. A., 1986, Metabolism of dinitropyrenes to DNA-binding derivatives in witro and in wiwo. In Carcinogenic and Mutagenic Effects of Diesel Engine Exhaust, edited by N. Ishinishi, A. Koizumi, R. 0. McClellan and W. Stober (Amsterdam: Elsevier Science Publishers B.V. (Biomedical Division)), pp. 185-1 97. HEFLICH, R. H., FIFER,E. K., DJURIC,Z., and BELAND, F. A,, 1985a, DNA adduct formation and mutation induction by nitropyrenes in Salmonella and Chinese hamster ovary cells: relationships with nitroreduction and acetylation. Enwironmental Health Perspectiwes, 62, 135-143. R. H., HOWARD, P. C., and BELAND, F. A,, 1985b, 1-Nitrosopyrene: an intermediate in the HEFLICH, metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538. Mutation Research, 149, 25-32. HEFLICH, R. H., THORNTON-MANNING, J. R.,KINOUCHI, T., and BELAND, F. A,, 1990, Mutagenicity of oxidized microsomal metabolites of 1 -nitropyrene in Chinese hamster ovary cells. Mutagenesis, 5 , 151-157. HIROSE, M., LEE,M.-S., WANG,C. Y., and KING,C. M., 1984, Induction of rat mammary gland tumors by 1-nitropyrene, a recently recognized environmental mutagen. Cancer Research, 44.11 58-1 162. HOWARD, P. C., HEFLICH, R. H., EVANS, F. E., and BELAND, F. A,, 1983, Formation of DNA adducts in witro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1nitropyrene. Cancer Research, 43, 2052-2058. I A R C Monographs on the Ewaluation of Carcinogenic Risks to Humans: Diesel and Gasoline Engine Exhausts and Some Nitroarenes, Volume 46, 1989 (Lyon: IARC), pp. 1-458. KING,C. M., 1988, Metabolism and biological effects of nitropyrene and related compounds. Health Effects Institute Research Report Number 16 (Cambridge, Health Effects Institute), pp. 1-22.
Downloaded by [RMIT University Library] at 22:22 05 May 2016
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LI,A. P., and DUTCHER, J. S., 1983, Mutagenicity of mono-, di- and tri-nitropyrenes in Chinese hamster ovary cells. Mutation Research, 119, 387-392. Lu, L.-J. W., DISHER, R. M., REDDY, M. V . , and RANDERATH, K., 1986, 32P-Postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene. Cancer Research, 46, 3046-3054. MAEDA, T., IZUMI,K., OTSUKA, H., MANABE, Y., KINOUCHI, T., and OHNISHI, Y., 1986, Induction of squamous cell carcinoma in the rat lung by 1,6-dinitropyrene. Journal of the National Cancer Institute, 76, 693-701. MITCHELL, C. E., 1988, Formation of DNA adducts in mouse tissues after intratracheal instillation of 1nitropyrene. Carcinogenesis, 9, 857-860. NESNOW, S., TRIPLETT, L. L., and SLAGA, T. J., 1984, Tumor initiating activities of 1-nitropyrene and its nitrated products in SENCAR mice. Cancer Letters, 23, 1-8. H., NEGISHI, C., WAKABAYASHI, K., KUSAMA, K., SATO,S., and SUCIMURA, T., 1984, Induction OHGAKI, of sarcomas in rats by subcutaneous injection of dinitropyrenes. Carcinogenesis, 5, 583-585. T . , 1985, Absence of OHGAKI, H., HASEGAWA, H., KATO,T., NEGISHI,C., SATO,S., and SUGIMURA, carcinogenicity of 1-nitropyrene, correction of previous results, and demonstration of carcinogenicity of 1,6-dinitropyrene in rats. Cancer Letters, 25, 239-245. OTOFUJI, T., HORIKAWA, K., MAEDA, T., SANO,N., IZUMI,K., OTSUKA, H., and TOKIWA, H., 1987, Tumorigenicity test of 1,3- and 1,8-dinitropyrene in BALB/c mice. Journal ofthe National Cancer Institute, 79, 185-188. ROY,A. K., EL-BAYOUMY, K., and HECHT,S. S., 1989, 32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female SpragueDawIey rats. Carcinogenesis, 10, 195-1 98. R. H., OHNISHI, Y., OHUCHIDA, A., KINOUCHI, T.,THORNTON-MANNING, J. R., SMITH,B. A,, HEFLICH, F. A., 1990a, DNA adduct formation by 1-nitropyrene 4 3 - and 9,lO-oxide. In and BELAND, Nitroarenes: Occurrence, Metabolism, and Biological Impact edited by P. C. Howard, S. S. Hecht, and F. A. Beland (New York: Plenum Press), pp. 181-187. SMITH,B. A., KORFMACHER, W. A,, and BELAND, F. A,, 1990 b, DNA adduct formation in target tissues of Sprague-Dawley rats, CD-1 mice and A/J mice following tumorigenic doses of 1-nitropyrene. Carcinogenesis, 11, 1705-1710. STANTON, C. A., CHOW,F. L., PHILLIPS, D. H., GROVER, P. L., GARNER, R. C., and MARTIN, C. N., 1985, Evidence for N-(deoxyguanosin-8-yI)-l-aminopyrene as a major DNA adduct in female rats treated with 1-nitropyrene. Carcinogenesis, 6, 535-538. SUCIURA, M., YAMAZOE, Y., KAMATAKI, T., and KATO,R., 1980, Reduction of epoxy derivatives of benzo(a)pyrene by microsomal cytochrome P-450. Cancer Research, 40,291CL2914. TAKAYAMA, S., ISHIKAWA, T., NAKAJIMA, H., and SATO,S., 1985, Lung carcinoma induction in Syrian golden hamsters by intratracheal instillation of 1,6-dinitropyrene. Japanese Journal of Cancer Research ( C a m ) , 76, 457461. S., OTSUKA, H., MANABE, Y., KINOUCHI, T., and TOKIWA, H., OTOFUJI, T., HORIKAWA, K., KITAMORI, OHNISHI, Y., 1984,1,6-Dinitropyrene: mutagenicity in Salmonella and carcinogenicity in BALB/c mice. Journal of the National Cancer Institute, 73, 1359-1363. E. S., Lu, A. Y. H., DOOLEY, K. L., Fu, P. P., HAN-HSU, H., BELAND, F. A., and WISLOCKI, P. G., BAGAN, KADLUBAR, F. F., 1986, Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis, 7, 1317-1 322.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen F. A. Beland, B. A. Smith, J. R. Thornton-Manning & R. H. Heflich To cite this article: F. A. Beland, B. A. Smith, J. R. Thornton-Manning & R. H. Heflich (1992) Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen, Xenobiotica, 22:9-10, 1121-1133, DOI: 10.3109/00498259209051866 To link to this article: http://dx.doi.org/10.3109/00498259209051866
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Date: 05 May 2016, At: 22:22
XENOBIOTICA,
1992, VOL. 22,
NOS
9/10, 1121-1133
Metabolic activation of 1-nitropyrene to a mammalian cell mutagen and a carcinogen F. A. BELAND?$*, B. A. S M I T H t , J. R. T H O R N T O N - M A N N I N G t § and R. H. H E F L I C H t
t National
Center for Toxicological Research, Jefferson, AR 72079, USA
t University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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Received 20 December 1990; accepted 17 April 1992 1. The mutagenicity of 1-nitropyrene metabolites in Chinese hamster ovary (CHO) 1cells, in the absence of rat liver S9, decreased in the order 6-hydroxy-1-nitropyrene~ nitropyrene 9,10-oxide> 1-nitropyrene 4,s-oxide 3-hydroxy-1 -nitropyrene 8hydroxy-1-nitropyrene > 1-nitropyrene. The order of mutagenicity with rat liver S9 was 1nitropyrene 4,s-oxide -6-hydroxy-1 -nitropyrene 1-nitropyrene 9,lO-oxide > 3hydroxy-1 -nitropyrene 1-nitropyrene > 8-hydroxy-1 -nitropyrene. 2. 1-Nitropyrene 4,s-oxide reacted with calf thymus DNA to give one or several closely related adducts. The same adducts were detected in CHO cells incubated with 1nitropyrene 4,s-oxide. Inclusion of a nitroreductase, xanthine oxidase, in the incubations with calf thymus DNA resulted in the formation of an additional adduct identified as N (deoxyguanosin-8-yl)-1 -aminopyrene (dG-C8-AP).
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-
-
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3. 1-Nitropyrene 9,lO-oxide reacted with calf thymus DNA to give an adduct pattern similar to that observed with 1-nitropyrene 4,s-oxide. Incubation of 1-nitropyrene 9,lOoxide with C H O cells resulted in the formation of the same adducts along with dG-C8-AP. 4. dG-C8-AP and N-(deoxyguanosin-8-y1)-l-amino-x-nitropyrene(x = 3, 6 or 8; dGC8-ANP) were detected in injection site DNA from Sprague-Dawley rats treated with 1nitropyrene. In mammary gland DNA, dG-C8-AP and an unidentified adduct were found. dG-C8-ANP was the only DNA adduct detected in the livers of newborn CD-1 mice and the lungs of A/J mice dosed with 1-nitropyrene.
1. Introduction Nitropyrenes, in particular I-nitropyrene and 1,3-, 1,6-, and 1,8-dinitropyrene (figure l), are widespread environmental pollutants that have been detected in photocopier toners, airborne particulates, coal fly-ash, and diesel engine exhaust (reviewed in IARC 1989). All four of these nitropyrenes are usually detected in environmental samples, with 1-nitropyrene typically being present in much greater concentrations than the dinitropyrenes. T h e dinitropyrenes, however, are extremely potent bacterial mutagens, which was the property that led to their initial discovery. As a result, the dinitropyrenes and 1-nitropyrene contribute nearly equally to the observed bacterial mutagenicity of most samples. I n addition to being mutagenic, nitropyrenes are tumourigens. 1-Nitropyrene, for example, induces tumours at the injection site and in the mammary gland in rats (Hirose et al. 1984, El-Bayoumy et al. 1988 b), in the lungs of adult mice (El-Bayoumy et al. 1984), and in the livers of newborn mice (Wislocki et al. 1986). T h e dinitropyrenes, in particular 1,6- and 1,8dinitropyrene, appear to be considerably more tumourigenic than 1-nitropyrene
*Address correspondence to: Frederick A. Beland, HFT-110, NCTR, Jefferson, AR 72079, USA. $Current address: U.S. EPA, HERL, MD-74, Research Triangle Park, N C 27711, USA. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.
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BNO*
OzN
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1-Nitropyrene
1,3-Dmi:ropyrens
N(0eoryguanorin-8-yljl-aminopyrene
Figure 1 .
1.B-Dinnropyrens
N(Deory uanosinS-yl)-l-arnlno gnitropyrene
Structures of 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene,and 1,FJ-dinitropyrene and metabolic pathways leading to identified DNA adducts.
From Beland and Kadlubar (1990).
and, when tested in the same animal model, show the same organ specificity as 1-nitropyrene (Nesnow et al. 1984, Ohgaki et al. 1984, 1985, Tokiwa et al. 1984, Takayama et al. 1985, Maeda et al. 1986, Wislocki et al. 1986, Otofuji et al. 1987, King 1988). T h e conversion of 1-nitropyrene to a mutagen in bacteria is thought to involve nitroreduction to a reactive N-hydroxy arylamine intermediate. Howard et al. (1983), for example, demonstrated that the major adduct formed in DNA of S. typhimurium treated with 1-nitropyrene was N-(deoxyguanosin-8-y1)-1aminopyrene (dG-C8-AP), an adduct consistent with the metabolic conversion of 1nitropyrene to N-hydroxy-1 -aminopyrene (figure 1). Dinitropyrenes are also activated through nitroreduction. In the case of 1,6-dinitropyrene the major adduct is N-(deoxyguanosin-8-y1)-1-amino-6-nitropyrene (DjuriC et al. 1988; figure l ) , while 1,8-dinitropyrene gives N-(deoxyguanosin-8-y1)-1-amino-8-nitropyrene (Heflich et al. 1985 a, Andrews et al. 1986; figure 1). T h e same adducts are found in S. typhimurium treated with 1,6- and 1,8-dinitropyrene (Heflich et al. 1985 a, Andrews et al. 1986, DjuriC et al. 1986 b). T h e mutagenic efficiencies of dG-C8-AP and N-(deoxyguanosin-8-y1)-1amino-8-nitropyrene have been compared in S. typhimurium TA1538 and appear to be similar (Heflich et al. 1985a, DjuriC et al. 1986b). Therefore, the extreme mutagenicity of the dinitropyrenes is not due to a unique adduct but appears to be due to their efficient metabolism to a DNA-binding intermediate. This seems to be a
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Metabolic activation of 1 -nitropyrene
result of the N-hydroxy amino intermediates of the dinitropyrenes (e.g. N-hydroxy1-amino-8-nitropyrene; figure 1) but not 1 -nitropyrene serving as substrates for transacetylases that catalyse the formation of reactive N-acetoxy derivatives (e.g. Nacetoxy- 1-amino-8-nitropyrene). 1-Nitropyrene is at best a very weak direct-acting mutagen in most mammalian cell lines; however, mutations can be induced by the inclusion of mammalian enzymes, such as the 9000 x g supernatant of a rat liver homogenate (S9; Li and Dutcher 1983, Heflich et al. 1990). T h e mammalian enzyme-mediated metabolism of 1-nitropyrene in vitro is complex and a number of products result from reduction and/or oxidation (reviewed in Beland and Kadlubar 1990, Smith et al. 1990 b; figure 2). Either of these pathways, or a combination of the two, could be involved in the activation of 1 -nitropyrene by mammalian enzymes to mutagenic derivatives. Metabolites indicative of nitroreduction, ring oxidation, and nitroreduction of ringoxidized products have been detected in vivo (reviewed in Beland and Kadlubar 1990, Smith et al. 1990 b). All of these pathways are involved in the activation of 1nitropyrene to DNA-binding species in microsomal incubations (DjuriC et al. 1986 a), which indicates that they may be important to tumourigenic activity in vivo. An adduct consistent with nitroreduction has been found in vivo along with, in some instances, other adducts (Stanton et al. 1985, Hashimoto and Shudo 1985, Mitchell
HO.
@
HO.'
0
@ 00,
H
6';" '-OH
0
Figure 2. 1 -Nitropyrene metabolites detected in in vitro incubations. From Beland et al. (1985).
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1988, El-Bayoumy et al. 1988a, Roy et al. 1989). However, it is not known if the treatment protocols used in these adduct studies will induce tumours. In order to gain insight into the pathways by which 1-nitropyrene is activated in vivo, we have performed a series of interrelated in vitro and in vivo experiments. Specifically, we determined the mutagenicities of ring-oxidized metabolites of 1nitropyrene in Chinese hamster ovary (CHO) cells and compared the results to those obtained from incubating C H O cells with 1-nitropyrene in the presence of rat liver S9 preparations. Since these data indicated that 1-nitropyrene 4,s-oxide and 1nitropyrene 9,lO-oxide played an important role in the mutagenic response, we examined the DNA adducts formed by these oxides following reactions with calf thymus DNA, both in the presence and absence of a nitroreductase, xanthine oxidase, and compared these adducts with those responsible for the mutagenicity of the oxides in CHO cells. Finally, we treated Sprague-Dawley rats, CD-1 mice, and A/J mice with I-nitropyrene according to three regimens reported to induce tumours (El-Bayoumy et al. 1984, Hirose et al. 1984, Wislocki et al. 1986) and compared the DNA adducts present in the target tissues with those obtained in the C H O cells.
Materials and methods Materials 1-Nitropyrene (lot number CSL-86-100-20; 99.85-9998% 1-nitropyrene, 0.15-0.02% 1,3-, 1,6- and l,S-dinitropyrene, by mass spectral analyses) was purchased from Chemsyn Science Laboratories, Lenexa, KS, USA. 1-Nitropyrene 4,s-oxide and 1-nitropyrene 9,lO-oxide (Fifer et al. 1986), and 3-, 6and 8-hydroxy-I-nitropyrene (El-Bayoumy and Hecht 1983) were synthesized as described in the indicated references. [y-"P]ATP was synthesized from carrier-free [3zP]phosphate (ICN Biomedicals, Inc., Irvine, CA, USA) (Gupta et al. 1982). DNA modified with dG-C8-AP was prepared by reacting N-hydroxy-1 -amin~[~H]pyrene with calf thymus DNA to give a level of modification of 8.3 adducts per 1 x lo3 nucleotides (Heflich et al. 1985 b). DNA modified with N-(deoxyguanosin-8-yI)-l-amino-6-nitropyrene was synthesized by reacting N-hydroxy-l-amin0-6-nitro[~H]pyrenewith pS189 DNA to give a modification level of 4 8 adducts per 1 x lo3 nucleotides (Djurii: et al. 1988). Rat liver S9 was prepared as described in Heflich et al. (1990). Mutagenicity assays Mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were measured by quantifying resistance to 6-thioguanine. Assays conducted in the presence and absence of S9 activation were performed as described in Heflich et al. (1990). Reaction of 1-nitropyrene 4,5-oxide and I-nitropyrene 9,IO-oxide with D N A Calf thymus DNA was incubated with the oxides in the presence and absence of xanthine oxidase and hypoxanthine as outlined in DjuriC et al. (1986a). Treatment of animals Animals were treated with 1-nitropyrene and DNA was isolated from target tissues as described in Smith et 01. (1990b). 32P-postlabelling of adducts 32P-postlabellingof DNA adducts was performed using modifications of the n-butanol enhancement procedure described by Gupta (1985). DNA digests were treated with nuclease P1 essentially as described by Gupta and Earley (1988) as modified by Smith et al. (1990 b). Adducts were treated with hydrazine as indicated in Smith et al. (1990 b). DNA adducts were separated by the contact-transfer method of Lu et al. (1986). Development in the first direction ( D l ) was with 0.65 M sodium phosphate, pH 6.0, or 0 9 M sodium phosphate, pH 6.8. Solvents used for adduct separation in D3 and D4 (on individual 10 x lOcm Merck PEI-cellulose t.1.c. plates) were 3.6 M lithium formate, 8.5 M urea, pH 3.5, and 1.2 M lithium chloride, 0.5 M Tric-HCI, 8.0M urea, pH 8.0,respectively. An additional development in 0.9 M sodium phosphate, pH 6.8, was performed in the direction of D4 onto a 3-cm Whatman 1 wick (Lu et al. 1986). Adducts were located by autoradiography, as described by Gupta et 01. (1982).
Metabolic activation of 1 -nitropyrene
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Results Mutagenicity in CHO cells of 1 -nitropyrene and oxidized 1 -nitropyrene metabolites Mutagenicity assays were conducted with C H O cells exposed to 1-nitropyrene, 3-, 6- and 8-hydroxy-l-nitropyrene,1-nitropyrene 4,S-oxide) and 1-nitropyrene 9,lO-oxide (see figure 2 for structures) both in the presence and the absence of S9 activation. Data are summarized in table 1. In the absence of S9, 6-hydroxy-lnitropyrene and 1-nitropyrene 9,lO-oxide were the most mutagenic compounds. 3and 8-Hydroxy-1 -nitropyrene and 1-nitropyrene 4,s-oxide were weaker mutagens, while 1-nitropyrene was essentially non-mutagenic. T h e order of mutagenic potency with S9 was 1-nitropyrene 4,s-oxide 6-hydroxy-1 -nitropyrene 1-nitropyrene 9,lO-oxide > 3-hydroxy-1 -nitropyrene 1-nitropyrene > 8-hydroxy-1 -nitropyrene. S9 significantly decreased the mutagenicity of 6-hydroxy-1 -nitropyrene.
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N
N
Reaction of 1 -nitropyrene 4,5-oxide and 1 -nitropyrene 9,lO-oxide with D N A 32P-Postlabelling analysis of the 1-nitropyrene 4,s-oxide-modified calf thymus DNA indicated either one, or possibly several closely related, adducts (figure 3A). DNA reacted with 1-nitropyrene 4,s-oxide in the presence of xanthine oxidase and hypoxanthine also had these adducts as well as an additional adduct having migration characteristics identical to the bis-phosphate of dG-C8-AP (figure 3B). Calf thymus DNA was also reacted with 1-nitropyrene 9,lO-oxide and analysed by 32Ppostlabelling (figure 4A). Two adducts were observed that migrated in the vicinity of the 'direct-reaction' adducts obtained from 1-nitropyrene 4,s-oxide. A similar adduct pattern was obtained when calf thymus DNA was incubated with 1nitropyrene 9,lO-oxide in the presence of xanthine oxidase and hypoxanthine (figure 4B).
Table 1.
Summary of the mutagenicity of 1-nitropyrene and oxidized 1-nitropyrene metabolites in CHO cells. Minus S9 Compound 1-Nitropyrene 3-Hydroxy-1-nitropyrene 6-Hydroxy-1-nitropyrene 8-Hydroxy-1-nitropyrene 1-Nitropyrene 4,S-oxide 1-Nitropyrene 9,lO-oxide
Mutation frequency" 1.4f0.7 5.0 f 1.5"
52.2 k60'*d.' 4 0 f 1.2 6.7 3.1 286k 5.7'~~
Plus s 9 Nb
Mutation frequency
12 8 19 7 10 9
3.4f 1.0 4 5 1.0 13.3+2.6'*' 0.4 f2.4 18.9 k7.9 15.0+4.7'
*
Nb 8 5 8
7 8 9
a Data are from Heflich et al. (1990). Mutation frequencies (mutants per 1 x lo6 clonable cells per lOnmol compound per ml) were calculated from the increasing portions of dose-response curves. The spontaneous mutations ( 4 k 2, - S9, n = 21; 5 f 2, S9, n = 20) have been subtracted from each assay. Data are presented as the meanf SEM. Statistical comparisons were made by Student's t-test. Differences were considered significant when p < 0.05. Number of determinations. 'Significantly different from 1-nitropyrene. Significantly greater than in the presence of S9. Significantly greater than 3- and 8-hydroxy-1-nitropyrene. Significantly greater than 1-nitropyrene 4,s-oxide.
+
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Figure 3. 3zP-Postlabellingautoradiograms of adducts obtained from: A, calf thymus DNA reacted with 1-nitropyrene 4,s-oxide; B, calf thymus DNA reacted with 1-nitropyrene 4,s-oxide in the presence of xanthine oxidase and hypoxanthine; C, DNA from CHO cells incubated with 1nitropyrene 4,s-oxide. Data are from Smith et al. (1990a)
Figure 4. 32P-Postlabellingautoradiograms of adducts obtained from: A, calf thymus DNA reacted with 1-nitropyrene 9,lO-oxide; B, calf thyus DNA reacted with 1-nitropyrene 9,lO-oxide in the presence of xanthine oxidase and hypoxanthine; C, DNA from CHO cells incubated with 1nitropyrene 9,lO-oxide. Data are from Smith et al. (1990a).
D N A adduct analysis in CHO cells exposed to I-nitropyrene 4,5-oxide and 1nitropyrene 9,lO-oxide In C H O cells incubated with 1-nitropyrene 4,5-oxideY the major adducts detected corresponded with those observed by the direct reaction of the oxide with DNA (figure 3C). C H O cells exposed to 1-nitropyrene 9,lO-oxide also had adducts that migrated in the vicinity of the ‘direct-reaction’ adducts; in addition, there appeared to be a small amount of the bis-phosphate of dG-CS-AP (figure 4C). At a 10 PM dose, 1-nitropyrene 9,lO-oxide produced approximately four times as many mutations as 1-nitropyrene 4,s-oxide (table 2). Likewise, 1-nitropyrene 9,lO-oxide bound to the C H O cell DNA approximately four-fold more than 1-nitropyrene 4,soxide. D N A adduct analysis in animals treated with 1 -nitropyrene 32P-Postlabelling was used for the analysis of DNA adduct formation in tissues that have been demonstrated to be targets for tumourigenesis by I-nitropyrene. I n female Sprague-Dawley rats, two adducts were apparent in DNA extracted from the
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subcutaneous injection site (figure 5A). One of these was tentatively identified as dGC8-AP by comparison of its chromatographic behaviour with the adduct standard, dG-C8-AP, that was analysed in parallel (figure 6A). T h e second adduct migrated slightly below dG-Cg-AP. This less polar adduct appeared to be a dinitropyrene adduct, N-(deoxyguanosin-8-y1)-1-amino-x-nitropyrene (x = 3, 6 or 8; dG-C8ANP), based on the similarity of its chromatographic behaviour with that of the standard, N-(deoxyguanosin-8-y1)-1-amino-6-nitropyrene(figure 6D). Neither adduct was apparent in injection site DNA isolated from animals that received the vehicle only (figure 5B). In mammary gland DNA from the 1-nitropyrene-treated Sprague-Dawley rats, two adducts were found (figure SC) that were not observed in control animals (figure SD). T h e minor adduct migrated to the same position as the standard, dG-C8-AP, while the major adduct was more polar. Both liver DNA from newborn CD-1 mice (figure 5E) and lung DNA from A/J mice (figure 5G) contained a single major adduct that migrated to the same position as the standard, dG-C8ANP (figure 6D). This adduct was not detected in liver (figure 5F) or lung (figure 5H) DNA from control mice. T h e identification of the D N A adducts was facilitated by the fact that the two standards, dG-Cg-AP and dG-C8-ANP, responded differently following exposure to hydrazine in the presence of palladium on carbon or incubation with nuclease P1 (figure 6). When the dG-C8-AP standard was treated with hydrazine and palladium on carbon, the adduct was recovered in 76% yield (figure 6B), whereas < 1% of the dG-C8-ANP standard was recovered after a similar treatment (figure 6E). In contrast, incubation of the dG-C8-AP standard with nuclease P1 caused a 97% decrease in the intensity of this adduct (figure 6C), while the intensity of dG-C8ANP was decreased by < 20% (figure 6F). T h e in vivo adducts identified as dG-C8AP and dG-C8-ANP behaved in a manner identical to the standards upon treatment with nuclease P1 and hydrazine (not shown). T h e concentration of the DNA adducts in the target tissues was quantified through comparisons to the D N A standards modified with dG-C8-AP and dG-C8ANP. As shown in table 3, dG-C8-ANP was formed to approximately an eight-fold greater extent than dG-C8-AP in injection site D N A of Sprague-Dawley rats, while the unknown adduct in the mammary gland DNA was estimated to be present at 10 times the amount of dG-C8-AP.
Discussion In this study we have used both in vitro mutagenesis and in vivo D N A binding assays to obtain a better understanding of the pathways by which I-nitropyrene is metabolized to a mammalian-cell mutagen and carcinogen. Initially, C H O cells were incubated with a series of 1-nitropyrene metabolites (figure 2). Of these, 6-hydroxy1-nitropyrene, 1-nitropyrene 9,10-oxide, and 1-nitropyrene 4,S-oxide were the strongest mutagens in C H O cells. These mutagenic responses were produced both in the presence and absence of S9 activation (table l), which indicates that these compounds are the metabolites primarily responsible for the S9-mediated mutagenicity of 1-nitropyrene in CHO cells. T h e direct-acting mutagenicity of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,lO-oxide could be due to DNA binding either through the ring epoxide or via an N-hydroxy arylamine intermediate produced by nitroreduction. Both routes of DNA binding have been demonstrated with these compounds in cell-free incub-
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Figure 5. Autoradiograms of 32P-postlabelled adducts in wiwo in the DNA of target tissues for 1 nitropyrene tumourigenesis. Injection site DNA from Sprague-Dawley rats treated with 1-nitropyrene (A) or solvent alone (B). Mammary gland DNA from Sprague-Dawley rats treated with 1-nitropyrene (C) or solvent alone (D). Liver DNA from CD-1 mice treated with 1-nitropyrene (E) or solvent alone (F). Lung DNA from A/J mice treated with I-nitropyrene (G) or solvent alone (H). Data are from Smith et al.
(1990b).
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Figure 6. Autoradiograms of 32P-postlabelled DNA adduct standards dG-C8-AP (A-C) and dG-C8ANP (D-F), before (A, D) and after treatment with hydrazine (B, E) or nuclease P1 (C, F). Data are from Smith et al. (1990b).
Table 2.
Mutations and DNA adducts produced in CHO cells by 1-nitropyrene 4,s-oxide and 1nitropyrene 9,lO-oxide. Compound
Mutants per 1 x lo6 clonable C H O cells’
DNA adduct concentration (adducts/106 nucleotides)b
6.7 k 3.1 28.6 & 5.7
3.8 147
1-Nitropyrene 4,s-oxide ( 1 0 ~ ~ ) 1-Nitropyrene 9,lO-oxide ( 1 0 ~ ~ )
Data are from table 1. b T h e concentration of DNA adducts was determined by 3zP-postlabelling through comparison to a standard modified with dG-C8-AP at a level of 0.5 adducts per 1 x lo6 nucleotides. Data are from Smith et al. (1990a). a
Table 3.
Concentration of DNA adducts formed in target tissues of animals treated with 1-nitropyrene. DNA adduct concentration (adducts/lO’ nuc1eotides)a
Species
Tissue
Sprague-Dawley rats
Injection site Mammary gland Liver Lung
CD-1 mice A/J mice
dG-C8-AP 0.8 k 0.4 0-2k0 0 6
dG-C8-ANP
Unknown
6.2 0.9 1.8 0.4b 7.0k3.1 7.9 f 2.9
a Data are from Smith et al.(1990 b). Adduct levels were quantified by comparison to DNA standards modified with dG-C8-AP and dG-C8-ANP. Results are reported as the meankstandard deviation of individual determinations from 4 to 8 animals. Quantified through comparison to DNA standards modified with dG-C8-ANP.
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ations utilizing mammalian nitroreductases and isolated D N A (DjuriC et al. 1986 a). T h e presence of the mammalian nitroreductase, xanthine oxidase, and hypoxanthine had no obvious effect on the adduct pattern produced by 1-nitropyrene 9,lO-oxide (figure 4), but an additional adduct with properties consistent with dG-C8-AP was observed in reactions conducted with 1-nitropyrene 4,s-oxide (figure 3). It appears that dG-C8-AP was produced from the reduction of 1-nitropyrene 4,s-oxide to 1nitropyrene followed by nitroreduction to N-hydroxy-1 -aminopyrene. Sugiura et al. (1980) have shown that xanthine oxidase and hypoxanthine will reduce arene oxides, while we (Howard et al. 1983) have demonstrated that the same system will catalyse the reduction of 1-nitropyrene to N-hydroxy-1 -aminopyrene, which will react with DNA to give dG-C8-AP. I-Nitropyrene 4,s-oxide produced a DNA adduct profile in C H O cells (figure 3C) similar to that found by reacting this compound directly with DNA (figure 3A). T h e adduct profile produced in CHO cells by 1-nitropyrene 9,lO-oxide (figure 4C) also included a small amount ( 5%) of an adduct that migrated in the vicinity of dGC8-AP. This adduct was not seen when the 9,lO-oxide was incubated with calf thymus DNA either in the presence or absence of xanthine oxidase and hypoxanthine. 1-Nitropyrene 9,lO-oxide also bound to C H O cell DNA to a much greater extent and was more mutagenic than 1-nitropyrene 4,S-oxide, while binding of the two oxides to calf thymus DNA was nearly equal (DjuriC et al. 1986a). T h e differential activities of these compounds in C H O cells could reflect the fact that 1nitropyrene 4,s-oxide is more readily detoxified than 1-nitropyrene 9,lO-oxide through epoxide hydrase-catalysed hydration (DjuriC et al. 1987). T h e results obtained with the CHO cells indicated that ring-oxidized metabolites could be responsible for the mutations induced in mammalian cells by 1nitropyrene; therefore, we ascertained if adducts derived from these metabolites were present in target tissues for 1-nitropyrene-induced tumours. In the injection site DNA from Sprague-Dawley rats, two major adducts were observed by 32Ppostlabelling (figure 5A). Based upon their chromatographic behaviour and their sensitivity to nuclease P1 and hydrazine relative to adduct standards, these were identified as adducts derived from the nitroreduction of 1-nitropyrene and a dinitropyrene. Two adducts also were detected in mammary gland DNA from Sprague-Dawley rats (figure SC). One of these had chromatographic characteristics and hydrazine and nuclease P1 sensitivities similar to dG-C8-AP. T h e identity of the other adduct is unknown, but it may result from ring oxidation. In the other two animal models, newborn CD-1 mice and A/J mice, the only DNA adduct detected in liver and lung, respectively, had characteristics consistent with dG-C8-ANP (figures 5E and 5G). Although the major adducts detected in CD-1 and A/J mice and at the injection site in Sprague-Dawley rats resulted from the nitroreduction of dinitropyrenes (table 3), mass spectral analyses of the I-nitropyrene used in these experiments indicated very low levels of dinitropyrene contamination. This indicates that dinitropyrenes are activated very efficiently to electrophilic metabolites, to an extent far better than 1-nitropyrene. There appear to be a number of reasons for this. First, as noted previously, the major metabolic pathways for 1-nitropyrene in vivo are through ring oxidation. Although ring-oxidized metabolites have been reported to bind to DNA (DjuriC et al. 1986 a, Smith et al. 1990a), the data we obtained in the present study indicate that, with the possible exception of the mammary gland in Sprague-Dawley rats, this does not occur to a large extent in target tissues for 1-
-
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nitropyrene tumourigenesis. In contrast, the only metabolic pathways that have been reported for dinitropyrenes are through nitroreduction (DjuriC et al. 1985, 1986c, 1988, Heflich et al. 1986), which presumably results in the formation of reactive N-hydroxy arylamine intermediates. Second, 1-nitropyrene undergoes nitroreduction primarily via a one-electron route, while the initial reduction of 1,6and 1-8-dinitropyrene is mainly by a two-electron pathway (Eddy et al. 1986). In addition, the one-electron reduction product of 1-nitropyrene, a radical anion, is quite sensitive to oxygen and readily undergoes redox cycling to regenerate 1nitropyrene (DjuriC et al. 1986 c). This will result in decreased concentrations of the electrophile, N-hydroxy-1-aminopyrene. Third, the N-hydroxy arylamine metabolites of dinitropyrenes are converted by 0-acetyltransferases to N-acetoxy arylamines (figure l ) , with a further increase in reactivity. For example, in in vitro studies with rat liver cytosol, 0-acetylation of N-hydroxy-1 -amino-3-, 6-, or 8nitropyrene increased the extent of D N A binding by 2W0-fold (DjuriC et al. 1985). This does not occur with the reduced 1-nitropyrene metabolite, N-hydroxy-laminopyrene, for reasons that are presently unknown. In summary, mutagenesis assays using C H O cells indicate that ring-oxidized metabolites of 1-nitropyrene can contribute to the mutagenic response of this compound in mammalian cells. However, the presence of dG-C8-AP in the DNA from the injection site and mammary glands of Sprague-Dawley rats indicates that the metabolic pathway responsible for activation of 1-nitropyrene to a tumourigen in these tissues is nitroreduction. An unidentified adduct present in mammary gland DNA indicates that pathways other than nitroreduction are also involved in the metabolic activation of 1-nitropyrene in this tissue. Nevertheless, when the mammary gland data are compared with the CHO cell results, it appears that this unidentified pathway does not involve 1-nitropyrene 4,s-oxide or 1-nitropyrene 9,10-oxide. Finally, the fact that only dinitropyrene-derived adducts were found in DNA from the livers of CD-1 mice and the lungs of A/J mice raises the question as to whether or not the previously reported tumour formation in these tissues can be attributed to 1-nitropyrene.
Acknowledgements We thank Nancy F. Fullerton for technical assistance, M. Matilde Marques for providing the 1-nitropyrene-modified DNA, Marcia Mah for preparing the 1,6dinitropyrene-modified DNA, Glenn Talaska for giving advice on 32P-postlabelling assays, Walter A. Korfmacher for conducting the mass spectral analyses, and Cindy Hartwick for helping prepare this manuscript. Research in this article was conducted, in part, under contract to the Health Effects Institute (HEI), an organization jointly funded by the United States Environmental Protection Agency (EPA) (Assistance Agreement X-812059) and automotive manufacturers. T h e contents of this article do not necessarily reflect the views of the HEI, nor do they necessarily reflect the policies of the EPA, or automotive manufacturers.
References ANDREWS, P. J., QUILLIAM, M. A,, MCCARRY, B. E., BRYANT, D. W., and MCCALLA, D. R., 1986, Identification of the DNA adduct formed by metabolism of 1,8-dinitropyrene in Salmonella typhimurium. Carcinogenesis, 7 , 105-1 10. BELAND, F. A,, and KADLUBAR, F. F., 1990, Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons. In Handbook of Experimental Pharmacology, Vol. 94/I, edited by C. S. Cooper and P. L. Grover (Berlin: Springer-Verlag), pp. 267-325.
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BELAND, F. A., HEFLICH, R. H., HOWARD, P. C., and Fu, P. P., 1985, The in vitro metabolic activation of nitro polycyclic aromatic hydrocarbons. In Polycyclic Hydrocarbons and Carcinogenesis, ACS Symposium Series, No. 283, edited by R. G. Harvey (Washington, DC: American Chemical Society), pp. 371-396. DJURIC,Z., COLES, B., FIFER,E. K., KETTERER, B., and BELAND, F. A,, 1987, I n wiwo and in witro formation of glutathione conjugates from the K-region epoxides of 1-nitropyrene. Carcinogenesis, 8, 17811786. DJURIC, Z., FIFER,E. K., and BELAND, F. A., 1985,Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA. Carcinogenesis, 6, 941-944. DJURIC,Z., FIFER, E. K., HOWARD, P. C., and BELAND, F. A,, 1986 a, Oxidative microsomalmetabolism of 1-nitropyrene and DNA-binding of oxidized metabolites following nitroreduction. Carcinogenesis, 7, 1073-1079. DJURIC,Z., HEFLICH, R. H., FIFER, E. K., and BELAND, F. A,, 1986 b, Metabolic activationof mutagenic and tumorigenic dinitropyrenes. In Biochemical and Molecular Epidemiology of Cancer, edited by C. Harris (New York: Alan R. Liss), pp. 4 4 1 4 7 . D. W., HEFLICH, R. H., and BELAND, F. A,, 1986c, Aerobic and anaerobic reduction DJURIC,Z., POTTER, of nitrated pyrenes in vitro. Chemico-Biological Interactions, 59. 309-324. DJURIC,Z., FIFER,E. K., YAMAZOE, Y., and BELAND, F. A,, 1988, DNA binding by 1-nitropyreneand 1,6dinitropyrene in witro and in wiwo: effects of nitroreductase induction. Carcinogenesis, 9, 357-364. R., 1986, Dichotomy in the EDDY,E. P., McCoy, E. C., ROSENKRANZ, H. S., and MERMELSTEIN, mutagenicity and genotoxicity of nitropyrenes: apparent effect of the number of electrons involved in nitroreduction. Mutation Research, 161. 109-1 11. EL-BAYOUMY, K., and HECHT,S. S., 1983, Identification and mutagenicity of metabolites of 1nitropyrene formed by rat liver. Cancer Research, 43, 3132-3137. EL-BAYOUMY, K., HECHT,S. S., SACKL, T., and STONER, G. D., 1984, Tumorigenicity and metabolism of 1-nitropyrene in A/J mice. Carcinogenesis, 5 , 1449-1452. EL-BAYOUMY, K., SHIUE,G.-H., and HECHT,S. S., 1988a, Metabolism and DNA binding of 1 nitropyrene and 1-nitrosopyrene in newborn mice. Chemical Research in Toxicology, 1 , 243-247. EL-BAYOUMY, K., RIVENSON, A., JOHNSON, B., DIBELLO,J., LITTLE,P., and HECHT,S. S., 1988b, Comparative tumorigenicity of 1-nitropyrene, 1-nitrosopyrene, and 1-aminopyrene administered by gavage to Sprague-Dawley rats. Cancer Research, 48, 42564260. FIFER,E. K., HOWARD, P. C., HEFLICH, R.H., and BELAND, F. A., 1986, Synthesis and mutagenicity of 1nitropyrene 4,S-oxide and 1-nitropyrene 9,10-oxide, microsomal metabolites of 1-nitropyrene. Mutagenesis, 1, 433438. GUPTA,R. C., 1985, Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen : DNA adducts. Cancer Research, 45, 5656-5662. GUPTA,R. C., and EARLEY, K., 1988, 32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis, 9, 1687-1693. GUPTA,R. C., REDDY, M. V., and RANDERATH, K., 1982, 32P-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis, 3, 1081-1092. HASHIMOTO, Y., and SHUDO,K., 1985, Modification of nucleic acids with 1-nitropyrene in the rat: identification of the modified nucleic acid base. Japanese Journal of Cancer Research (Gann), 76, 253-256. HEFLICH, R. H., DJURIC,Z., FIFER, E. K., CERNIGLIA, C. E., and BELAND, F. A., 1986, Metabolism of dinitropyrenes to DNA-binding derivatives in witro and in wiwo. In Carcinogenic and Mutagenic Effects of Diesel Engine Exhaust, edited by N. Ishinishi, A. Koizumi, R. 0. McClellan and W. Stober (Amsterdam: Elsevier Science Publishers B.V. (Biomedical Division)), pp. 185-1 97. HEFLICH, R. H., FIFER,E. K., DJURIC,Z., and BELAND, F. A,, 1985a, DNA adduct formation and mutation induction by nitropyrenes in Salmonella and Chinese hamster ovary cells: relationships with nitroreduction and acetylation. Enwironmental Health Perspectiwes, 62, 135-143. R. H., HOWARD, P. C., and BELAND, F. A,, 1985b, 1-Nitrosopyrene: an intermediate in the HEFLICH, metabolic activation of 1-nitropyrene to a mutagen in Salmonella typhimurium TA1538. Mutation Research, 149, 25-32. HEFLICH, R. H., THORNTON-MANNING, J. R.,KINOUCHI, T., and BELAND, F. A,, 1990, Mutagenicity of oxidized microsomal metabolites of 1 -nitropyrene in Chinese hamster ovary cells. Mutagenesis, 5 , 151-157. HIROSE, M., LEE,M.-S., WANG,C. Y., and KING,C. M., 1984, Induction of rat mammary gland tumors by 1-nitropyrene, a recently recognized environmental mutagen. Cancer Research, 44.11 58-1 162. HOWARD, P. C., HEFLICH, R. H., EVANS, F. E., and BELAND, F. A,, 1983, Formation of DNA adducts in witro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1nitropyrene. Cancer Research, 43, 2052-2058. I A R C Monographs on the Ewaluation of Carcinogenic Risks to Humans: Diesel and Gasoline Engine Exhausts and Some Nitroarenes, Volume 46, 1989 (Lyon: IARC), pp. 1-458. KING,C. M., 1988, Metabolism and biological effects of nitropyrene and related compounds. Health Effects Institute Research Report Number 16 (Cambridge, Health Effects Institute), pp. 1-22.
Downloaded by [RMIT University Library] at 22:22 05 May 2016
Metabolic activation of 1 -nitropyrene
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LI,A. P., and DUTCHER, J. S., 1983, Mutagenicity of mono-, di- and tri-nitropyrenes in Chinese hamster ovary cells. Mutation Research, 119, 387-392. Lu, L.-J. W., DISHER, R. M., REDDY, M. V . , and RANDERATH, K., 1986, 32P-Postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene. Cancer Research, 46, 3046-3054. MAEDA, T., IZUMI,K., OTSUKA, H., MANABE, Y., KINOUCHI, T., and OHNISHI, Y., 1986, Induction of squamous cell carcinoma in the rat lung by 1,6-dinitropyrene. Journal of the National Cancer Institute, 76, 693-701. MITCHELL, C. E., 1988, Formation of DNA adducts in mouse tissues after intratracheal instillation of 1nitropyrene. Carcinogenesis, 9, 857-860. NESNOW, S., TRIPLETT, L. L., and SLAGA, T. J., 1984, Tumor initiating activities of 1-nitropyrene and its nitrated products in SENCAR mice. Cancer Letters, 23, 1-8. H., NEGISHI, C., WAKABAYASHI, K., KUSAMA, K., SATO,S., and SUCIMURA, T., 1984, Induction OHGAKI, of sarcomas in rats by subcutaneous injection of dinitropyrenes. Carcinogenesis, 5, 583-585. T . , 1985, Absence of OHGAKI, H., HASEGAWA, H., KATO,T., NEGISHI,C., SATO,S., and SUGIMURA, carcinogenicity of 1-nitropyrene, correction of previous results, and demonstration of carcinogenicity of 1,6-dinitropyrene in rats. Cancer Letters, 25, 239-245. OTOFUJI, T., HORIKAWA, K., MAEDA, T., SANO,N., IZUMI,K., OTSUKA, H., and TOKIWA, H., 1987, Tumorigenicity test of 1,3- and 1,8-dinitropyrene in BALB/c mice. Journal ofthe National Cancer Institute, 79, 185-188. ROY,A. K., EL-BAYOUMY, K., and HECHT,S. S., 1989, 32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female SpragueDawIey rats. Carcinogenesis, 10, 195-1 98. R. H., OHNISHI, Y., OHUCHIDA, A., KINOUCHI, T.,THORNTON-MANNING, J. R., SMITH,B. A,, HEFLICH, F. A., 1990a, DNA adduct formation by 1-nitropyrene 4 3 - and 9,lO-oxide. In and BELAND, Nitroarenes: Occurrence, Metabolism, and Biological Impact edited by P. C. Howard, S. S. Hecht, and F. A. Beland (New York: Plenum Press), pp. 181-187. SMITH,B. A., KORFMACHER, W. A,, and BELAND, F. A,, 1990 b, DNA adduct formation in target tissues of Sprague-Dawley rats, CD-1 mice and A/J mice following tumorigenic doses of 1-nitropyrene. Carcinogenesis, 11, 1705-1710. STANTON, C. A., CHOW,F. L., PHILLIPS, D. H., GROVER, P. L., GARNER, R. C., and MARTIN, C. N., 1985, Evidence for N-(deoxyguanosin-8-yI)-l-aminopyrene as a major DNA adduct in female rats treated with 1-nitropyrene. Carcinogenesis, 6, 535-538. SUCIURA, M., YAMAZOE, Y., KAMATAKI, T., and KATO,R., 1980, Reduction of epoxy derivatives of benzo(a)pyrene by microsomal cytochrome P-450. Cancer Research, 40,291CL2914. TAKAYAMA, S., ISHIKAWA, T., NAKAJIMA, H., and SATO,S., 1985, Lung carcinoma induction in Syrian golden hamsters by intratracheal instillation of 1,6-dinitropyrene. Japanese Journal of Cancer Research ( C a m ) , 76, 457461. S., OTSUKA, H., MANABE, Y., KINOUCHI, T., and TOKIWA, H., OTOFUJI, T., HORIKAWA, K., KITAMORI, OHNISHI, Y., 1984,1,6-Dinitropyrene: mutagenicity in Salmonella and carcinogenicity in BALB/c mice. Journal of the National Cancer Institute, 73, 1359-1363. E. S., Lu, A. Y. H., DOOLEY, K. L., Fu, P. P., HAN-HSU, H., BELAND, F. A., and WISLOCKI, P. G., BAGAN, KADLUBAR, F. F., 1986, Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis, 7, 1317-1 322.
