Mutagenesis vol. 18 no. 4 pp. 331-335,
July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Mutagenicity and cross-linking activity of chloroalkylnitrosamines, possible new antitumor lead compounds
Kyoritsu College of Pharmacy, Shibakoen 1–5-30, Minato-ku, Tokyo 105–8512, Japan
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Abstract |
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The mutagenicity of chlorinated
-acetoxy nitrosamines was assayed using nine bacterial strains with various DNA repair abilities and the mechanism of their reaction with DNA was evaluated. Three -acetoxy nitrosamines without a chloro group were used to investigate the effect of the chloro group on mutagenicity. Three nitrosamines having chloroethyl, chloropropyl and chlorobutyl groups were directly mutagenic in all tester strains used in this study, which showed that they damaged DNA in intact bacteria. Compared with Salmonella typhimurium TA1535, mutagenic activity was enhanced in ogt gene-deficient strains (YG7108 and YG7104), suggesting that an O6-alkylguanine adduct causes the mutation. The chlorinated nitrosamines showed stronger mutagenicity than non-chlorinated nitrosamines, indicating that alkylating activity was strengthened by the presence of a chloro group. The nitrosamines, especially the chloropropyl homolog, showed clear mutagenicity in the strains with an intact excision repair system, S.typhimurium TA92, TA1975 and G46. Further, chloropropyl and chlorobutyl homologs showed interstrand cross-linking activity towards plasmid DNA. These results suggest that some chlorinated nitrosamines can act on DNA to form DNA cross-links, as observed in antitumor chloroethylnitrosoureas. Environmental nitrosamines are usually dealt with as potential carcinogens, but introduction of a chloro group has added the possibility of in vivo cross-linking activity, which is a classical and essential mechanism for antitumor agents. Therefore, the novel chlorinated nitrosamines examined in this study are proposed as new bifunctional antitumor lead compounds. |
Introduction |
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Carcinogenic N-nitroso compounds are important because they are present in our environment; they can be formed in tobacco and also from dietary precursors in the stomach under acidic conditions (Preussmann and Eisenbrand, 1984
). They were also reported to be formed from amines by activated macrophages under neutral conditions in vivo (Marletta, 1984). Such N-nitroso compounds are divided into two groups; N-nitrosamines and N-nitrosamides. Generally, N-nitrosamines, such as N-nitrosodialkylamines, need to be metabolized in vivo by cytochromes P450 to achieve their biological activity, in contrast to N-nitrosamides, such as N-nitrosoureas, that do not need metabolic conversion. Activated metabolites of N-nitrosodialkylamines, -hydroxynitrosamines, decompose spontaneously to alkanediazohydroxides, then to alkyldiazonium ions, which can react with biological nucleophiles such as nucleic acids and proteins (Preussmann and Stewart, 1984). The resulting DNA alkylation is an important factor in the carcinogenic or mutagenic activities of N-nitroso compounds.
We have previously reported chemical and biological properties of alkanediazotates, precursors of alkanediazohydroxides, and revealed that their geometrical isomerism and alkyl chain length affected their alkylating and biological activities. The results supported the idea that -hydroxy nitrosamines degrade to alkanediazohydroxides which alkylate DNA (Ukawa-Ishikawa et al., 1998, 1999).
Although many N-nitroso compounds are carcinogens, N-nitroso-N-chloroethylureas, such as 1,3-bis(2-chloroethyl)-1-nitrosourea, show antitumor activity (Gnewuch and Sosnovsky, 1997). They have a chloroethyl moiety as a bifunctional group, which decomposes under physiological conditions to chloroethyldiazohydroxides, similar to the decomposition of carcinogenic N-nitroso compounds. Chloroethyldiazohydroxide can react with DNA to form an alkylated base (Lown et al., 1986), and a second alkylation reaction due to the chloro leaving group can lead to DNA cross-linking, which is a classical mechanism for antitumor agents.
Thus we were interested to determine whether bifunctional nitrosamines may act as DNA cross-linking agents in vivo and have antitumor activity. Recently, we synthesized N-nitroso-N-(acetoxymethyl)-
-chloroalkylamines having chloroethyl, chloropropyl and chlorobutyl groups (NAMCE, NAMCP and NAMCB, respectively) (Figure 1) (Ishikawa et al., 2000). These compounds showed positive results in the mutation assay using Salmonella typhimurium TA1535 and TA92, suggesting that they hydrolyze and decompose to chloroalkyldiazohydroxides in aqueous solution, then alkylate DNA and form DNA cross-links (Figure 1; Ishikawa et al., 2000). In this present study, we assayed their mutagenicity using S.typhimurium strains of various genotypes and the effects of DNA repair systems on mutagenicity were examined to confirm the mechanism of action of these chlorinated nitrosamines. Furthermore, their cross-linking activity was assayed using plasmid DNA to reveal whether these compounds act as DNA cross-linking agents.
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Materials and methods |
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Chemicals
N-nitroso-N-(acetoxymethyl)-2-chloroethylamine (NAMCE, 76215–00–4), N-nitroso-N-(acetoxymethyl)-3-chloropropylamine (NAMCP, 312304–87–3) and N-nitroso-N-(acetoxymethyl)-4-chlorobutylamine (NAMCB, 312304–89–5) were synthesized as described previously (Ishikawa et al., 2000
). N-nitroso-N-(acetoxymethyl)ethylamine (NAME, 65986–80–3), N-nitroso-N-(acetoxymethyl)propylamine (NAMP, 66017–91–2) and N-nitroso-N-(acetoxymethyl)butylamine (NAMB, 56986–36–8) were also synthesized as described previously (Mochizuki et al., 1978). Dimethyl sulfoxide (DMSO) was freshly distilled from calcium hydride before use (bp7 58°C). Sodium ammonium hydrogenphosphate was purchased from Merck & Co. (Rahway, NJ). Bacto agar and bacto nutrient broth were purchased from Difco Laboratories (Detroit, MI). Sodium azide (26628–22–8) was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). N-nitroso-N-methylurea (NMU, 684–93–5) and N-nitroso-N-ethylurea (NEU, 759–73–9) were kindly provided by Toshin Chemical Industries (Tokyo, Japan). 4-Nitroquinoline N-oxide (4NQO, 56–57–5) was purified by recrystallization before use. Cisplatin (Briplatin®) was purchased from Bristol-Myers Squibb (Tokyo, Japan) and glycogen was purchased from Roche Diagnostics (Mannheim, Germany). Most reagents used were purchased from Wako Pure Chemical Industries (Osaka, Japan) as the purest grade available. In mutation assay, NMU, NEU and 4NQO were diluted in DMSO and NaN3 and mitomycin C were diluted in water.
Bacterial strains
The genotypes of bacterial strains used in this study are listed in Table I. A culture of S.typhimurium TA1535 was kindly provided by Dr B.N.Ames (University of California, Berkeley, CA). Salmonella typhimurium YG7108, YG7100, YG7104, TA1975, G46, YG7155, TA100 and TA92 were kindly provided by Dr T.Nohmi (National Institute of Health Sciences, Tokyo, Japan).
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Mutation assay
The mutation assay was carried out according to the plate incorporation method reported by Maron and Ames (1983
), with slight modification (Ishikawa et al., 2000). Test compounds were diluted in DMSO and their concentration was expressed as µmol/plate. As positive controls, the following compounds were used: 0.5 µg/plate NaN3 for TA1535; 5 µg/plate NEU for YG7108; 250 µg/plate NEU for YG7100; 10 µg/plate NEU for YG7104; 100 µg/plate NMU for TA1975; 10 µg/plate NMU for G46; 1 µg/plate NMU for YG7155; 0.1 µg/plate 4NQO for TA100; 2.5 µg/plate mitomycin C for TA92. The colonies on the plates were counted manually and the number of revertant colonies of the solvent control was subtracted from the total count to derive the number of induced revertants. Under these conditions, the numbers of solvent control (s.c.) and positive control (p.c.) revertants were as follows: TA1535, s.c. 20 ± 8, p.c. 480 ± 160; YG7108, s.c. 47 ± 15, p.c. 460 ± 60; YG7100, s.c. 17 ± 7, p.c. 61 ± 6; YG7104, s.c. 24 ± 5, p.c. 250 ± 22; TA1975, s.c. 2 ± 1, p.c. 200 ± 80; G46, s.c. 4 ± 2, p.c. 860 ± 170; YG7155, s.c. 43 ± 11, p.c. 600 ± 130; TA92, s.c. 29 ± 9, p.c. 380 ± 50; TA100, s.c. 190 ± 28, p.c. 940 ± 140. All data reported are representative of at least three experiments using duplicate plates for each dose level.
DNA cross-linking assay
The assay procedure was according to the method reported by Hartley et al. (1991) with a slight modification as follows. pBluescript® II KS– DNA was linearized by digestion with HindIII and BamHI. Linearized DNA (160 ng) was treated with test compound (final concentration 0.01–10 mM) dissolved in DMSO at 37°C in 25 mM phosphate-buffered solution (pH 7.4). After reactions were terminated, the DNA was precipitated and dried by lyophilization.
The DNA samples were re-dissolved in strand separation buffer, heated at 95°C for 5 min and then immediately chilled in an ice bath. After loading of the samples with gel loading buffer, electrophoresis was performed on a 1% agarose gel at 50 V for 150 min in running buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) using a Mupid® system. The DNA bands were visualized with ethidium staining and the gel images were recorded on Polaroid film. The band intensity of double-stranded DNA was quantified by densitometric analysis using the Scion Image B4 program.
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Results |
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Mutagenicity
Mutagenicities of six nitrosamines were assayed in the nine S.typhimurium strains listed in Table I. All nitrosamines were directly mutagenic and the mutagenic potency was linearly related to the concentration of the compound. Figure 2 shows the dose–mutagenicity relationship of chlorinated nitrosamines in the YG7108 (rfa,
uvrB, ada, ogt) and TA1975 (rfa, uvrB+, ada+, ogt+) strains. For all compounds, no cytotoxicity was detected in the range of concentrations tested. To compare the mutagenicity of nitrosamines, specific mutagenicity was defined as revertants per µmol of chemical according to the slope of the linear portion of the dose–mutagenicity relationship and was calculated by the least squares method. Table II shows the relative mutagenicities for the six nitrosamines in all strains.
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, NAMCP; 
, NAMCB; 
, NAME; 
, NAMP; 
, NAMB.
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The mutagenicity was influenced by alkyl chain length of nitrosamines. For chlorinated nitrosamines (NAMCE, NAMCP and NAMCB), an increase in alkyl chain length increased the mutagenicity in the TA1535 and YG7100 strains, which possess the ogt gene, and in TA100, in contrast to other strains in which NAMCP showed the strongest activity. Conversely, among non-chlorinated nitrosamines (NAME, NAMP and NAMB), an increase in alkyl chain length generally decreased mutagenicity.
Loss of excision repair ability increased the nitrosamine-induced mutagenicity; relative mutagenicity in TA1975 (uvrB+) versus TA1535 (uvrB) was 46 times for NAMCE, 11 times for NAMCP, 43 times for NAMCB and 9–16 times for non-chlorinated nitrosamines. In addition, mutagenicity in TA100 was stronger than that in TA92. Under these conditions, the mutagenicity was further increased by loss of O6-alkylguanine-DNA alkyltransferase (AGT) repair ability, i.e. when the mutagenicity in TA1535 was compared with that in YG7108 (uvrB, ada, ogt), an increase in mutagenicity was observed; 4 times for NAMCE, 31 times for NAMCP, 5 times for NAMCB and 24–94 times for non-chlorinated nitrosamines. For almost all nitrosamines, mutagenicity in YG7104 (uvrB, ogt) was similar to that in YG7108 and mutagenicity in YG7100 (uvrB, ada) was similar to that in TA1535. Comparing the mutagenicity in G46 with that in YG7155 (rfa+, uvrB, ada, ogt), the increase in mutagenicity was relatively small; 6 times for NAMCE and 2 times for NAMCP and NAMCB. A deficiency of rfa had little effect on the nitrosamine-induced mutagenicity, i.e. mutagenicity in TA1975 (rfa, uvrB) was similar to that in G46 (rfa+, uvrB). The presence of plasmid pKM101 had no effect on the observed mutagenicity, and the relative mutagenicity in TA100 was similar to that in TA1535.
DNA cross-linking activity
The ability of chlorinated nitrosamines to form DNA interstrand cross-links was determined using plasmid DNA. As shown in Figure 3A, band intensity of double-stranded DNA due to cross-linking was increased dose-dependently after treatment with NAMCP. When the intensity of the bands of double-stranded DNA was densitometrically analyzed, a dose–response relationship and a time-dependence were observed (Figure 3B). The NAMCB showed weaker activity than the NAMCE and the chloroethyl homolog showed no activity under the reaction conditions used in this study (data not shown). The three non-chlorinated nitrosamines did not show any cross-linking activity.
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Discussion |
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We have synthesized chlorinated
-acetoxy nitrosamines as novel cross-linking agents (Ishikawa et al., 2000). In the present study, we assayed their mutagenicity in bacterial strains with various DNA repair abilities and compared the mutagenicities with those of non-chlorinated -acetoxy nitrosamines. Since mutagenicity in the TA1975 strain was the same as that in the G46 strain, we assumed that the rfa genotype did not affect bacterial entry of the nitrosamines. Below we also discuss the effect of repair ability of each strain employed.
All nitrosamines assayed were directly mutagenic in the TA1535 strain, which showed that they damaged bacterial DNA and induced base pair change mutations. The GC
AT transition mutation detected in TA1535 was mainly induced by O6-alkylguanine at the hisG46 locus, which is repaired by AGT. When we used Salmonella strains deficient for the AGT genes ada and ogt (Yamada et al., 1997), the nitrosamines showed stronger mutagenicities in YG7108 and YG7104 than in TA1535 and YG7100. These results indicate that only ogt deficiency increased sensitivity of bacteria to nitrosamine-induced mutagenicity, i.e. Ogt protein, but not Ada protein, is responsible for repair of DNA alkylation damage induced by the nitrosamines having an alkyl group other than methyl. This agreed well with the reported results that Ogt protein mainly repaired DNA damage induced by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (Yamada et al., 1995) and revealed that NAMCE, NAMCP and NAMCB alkylated the O6 position of guanine, as do classical N-nitroso compounds, including non-chlorinated nitrosamines. The effect of AGT on nitrosamine-induced mutagenicity differed according to alkyl chain length. AGT affected the mutagenicity of NAMCP more than that of NAMCE or NAMCB, which suggests that NAMCP may induce more O6-alkylguanine adducts. Furthermore, the chlorinated nitrosamines showed stronger mutagenicity than non-chlorinated nitrosamines. From these results, it is evident that the chloro group enhances the reactivity of nitrosamines with biological nucleophiles. The mutagenicity of nitrosamines in TA1535 was similar to that in TA100 which possesses plasmid pKM101, suggesting that lack of the SOS repair system in TA1535 is not responsible for nitrosamine-induced mutation.
Excision repair mechanisms are also important in the repair of alkylated DNA and deficiency of excision repair genes increased the mutagenicity of the nitrosamines; the mutagenicity in TA1535 was greater than that in TA1975. Although loss of the uvrB gene enhanced the mutagenicity of NAMCE and NAMCB, the effect on the mutagenicity of NAMCP was relatively small. This tendency was opposite to the effect of AGT deficiency described above. Since the excision repair system acts on alkylated adducts which induce a distortion of the DNA strands (Murray, 1979), a suitable repair system for such adducts may need to account for alkyl chain length, which affects the type and amount of DNA adduct formed. Indeed, the action of AGT was affected by the presence of excision repair. Although Ogt protein plays a major protective role in alkylating damage in S.typhimurium, the effect of ogt gene deficiency was small in the presence of the excision repair gene uvrB. Among six nitrosamines, only NAME showed stronger mutagenicity than NAMCE in ogt-deficient strains, and this result may be explained by the high alkylating activity of NAME and also by the high specificity as a substrate of AGT.
Excision repair is also responsible for mutations induced by cross-linking damage and excision repair-proficient strains, such as TA1975, showed revertants due to DNA cross-linking; this agrees with previous findings, where chlorinated nitrosamines were mutagenic in S.typhimurium TA92 (Ishikawa et al., 2000). In the present study, we also tested the activity of non-chlorinated nitrosamines in TA92 for comparison with that of chlorinated nitrosamines. In spite of no cross-linking activity, the non-chlorinated nitrosamines showed weak but clear mutagenicity in strain TA92. Since strain TA92 possesses a hisG46 mutation, as does TA1535, this result was due to more alkylated DNA that could not be repaired by the excision repair system. In comparing the mutagenicities of non-chlorinated nitrosamines with those of chlorinated nitrosamines, the influence of excision repair deficiency was most evident with NAMCP exposure, and the mutagenicity of NAMCP in strain TA92 was the strongest among all nitrosamines tested. This suggests that NAMCP has the strongest cross-linking activity among the chlorinated nitrosamines. Despite a report that chloropropylnitrosourea did not have cross-linking activity (Lown et al., 1979), NAMCP showed clear mutagenicity towards three strains with an intact excision repair system; TA1975 and G46, in addition to TA92.
To form DNA cross-links, DNA monoalkylation is needed as the first step. It is reported that chloroethylnitrosourea forms O6-chloroethylguanine and that it forms DNA cross-links through an N1,O6-ethanoguanine intermediate (Gnewuch and Sosnovsky, 1997). Our results, which indicate that the chlorinated nitrosamines form O6-alkylguanine adducts, suggest that a similar mechanism may be responsible for cross-link formation. A proposed structure of the cyclic intermediate formed from NAMCP is N1,O6-propanoguanine, which may be more stable than N1,O6-ethanoguanine due to its six-membered ring. There are other possibilities: for example, O6-chloroalkylguanine may participate directly to form cross-links by nucleophilic attack on the chloroalkyl group on the O6 position or the chloroalkyldiazonium ion may alkylate the N7 position of guanine to form a cross-link. As the decomposition rate of NAMCB in aqueous solution was similar to that of NAMCP (Ishikawa et al., 2000), similar toxicological behaviors of both nitrosamines may be expected. However, formation of the presumed cyclic intermediate derived from NAMCB through O6-chlorobutylguanine is thought to be difficult because of the seven-membered ring, and the likelihood of cross-link formation due to NAMCB was less than for NAMCP. Further study is needed to elucidate more precisely the mechanism involved in adduct formation by chlorinated nitrosamines. This could be achieved by reacting these compounds with oligonucleotides and then analyzing the type of cross-linked adduct formed.
Of the three chlorinated nitrosamines, NAMCE showed the weakest mutagenicity in all strains used. One of the reasons why the activity of NAMCE is relatively low is that the chloroethyl group endowed NAMCE with extremely high reactivity due to the neighboring group effect and reaction with nucleophiles other than DNA was thus accelerated. In other words, there is a possibility that hydrolysis of NAMCE competed with DNA alkylation and that the amount of reaction with DNA was insufficient to impart heightened mutagenicity. Another reason is that NAMCE formed more cross-links on the DNA molecule, but no transition mutation in the hisG46 locus.
Since the results of the mutation assays described above suggested a hypothesis that not only NAMCE but also NAMCP and NAMCB could act on DNA to form DNA cross-links, as observed with antitumor chloroethylnitrosoureas, we examined their cross-linking activities using plasmid DNA to test the hypothesis. When we used triethanolamine as a buffer component in the reaction solution (Hartley et al., 1991), the solution was strongly acidified by acetic acid due to hydrolysis of acetate ester in the nitrosamines. Consequently, spontaneous depurination occurred under acidic conditions and interstrand cross-links formed (Burnotte and Verly, 1972). To prevent additional cross-links, we used phosphate, with stronger buffer capacity than triethanolamine, and confirmed no cross-link formation from non-chlorinated nitrosamines. NAMCP was clearly demonstrated to have a dose-related cross-linking activity. This result indicated that in TA92, NAMCP formed cross-links which led to mutagenicity. The weaker cross-linking activity of NAMCB may be explained by lack of an appropriate bond length between the two cross-linkable bases in the DNA strand, while NAMCE was too unstable to show cross-linking activity in this assay system. From these overall results, we conclude that chlorinated nitrosamines possess DNA cross-linking activity. We are now challenged to design new compounds having elevated affinities for DNA and higher abilities to form DNA cross-links effectively.
Environmental nitrosamines have to-date been considered as carcinogens, but introduction of a chloro group as a second leaving group has meant that these chemicals behave as bifunctional agents that cross-link DNA. Therefore, these novel chlorinated nitrosamines provide possible novel antitumor compounds and merit testing as such in the future.
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Acknowledgements |
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We thank Dr T.Nohmi of the National Institute of Health Sciences, Japan, for the supply of S.typhimurium YG7108, YG7100, YG7104, TA1975, G46, YG7155, TA100 and TA92. This work was supported in part by the Nishi Cancer Research Fund, Japan, and the Promotion of Foundation and Mutual Aid Corporation for Private Schools of Japan.
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Notes |
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1To whom correspondence should be addressed. Tel: +81 3 5400 2695; Fax: +81 3 5400 2695; Email: ishikawa-st{at}kyoritsu-ph.ac.jp
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References |
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Received on May 18, 2002; accepted on April 11, 2003.
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