Environmental Health Perspectives Volume
102, Supplement 6, October 1994
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in PubMed] [Related
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The Carcinogenicity of Methoxyl Derivatives of 4-Aminoazobenzene: Correlation
between DNA Adducts and Genotoxicity
Misaki Kojima,1 Masakuni Degawa,2 Yoshiyuki
Hashimoto,2 and Mariko Tada1
1Aichi Cancer Center Research Institute, Nagoya, Japan; 2Tohoku
University, Sendai, Japan
Abstract
To elucidate the cause of the difference in genotoxic activity between
carcinogenic 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and noncarcinogenic
2-methoxy-4-aminoazobenzene (2-MeO-AAB), we analyzed DNA adducts in the
livers of rats exposed to either of these chemicals and studied the resulting
biologic potential with the aid of in vitro modified M13 phage DNA.
32P-Postlabeling analysis revealed that the carcinogen 3-MeO-AAB
produced 20-fold higher amounts of adducts than did 2-MeO-AAB. Five adducts
were formed in the 3-MeO-AAB case whereas only one adduct was apparent in
2-MeO-AAB-treated rat. Studies of in vitro DNA replication using
N-hydroxy (N-OH)-aminoazo dye-modified M13 phage DNA as a template
demonstrated inhibition by 3-MeO-AAB adducts to be substantially greater
than in the 2-MeO-AAB-adducts. The specificity of mutagenesis induced in
M13mp9 phage DNA by these chemicals also was analyzed after transfection
into SOS-induced Escherichia coli JM103, mutation frequencies being
higher with N-OH-3-MeO-AAB- than N-OH-2-MeO-AAB-modified DNA. The mutation
spectra differed in each case. Our data suggest that the difference in hepatocarcinogenic
activity between the two chemicals depends not only on qualitative and quantitative
variation in adduct formation but also on conformation changes in modified
DNA. -- Environ Health Perspect 102(Suppl 6):191-194 (1994)
Key words: 3-methoxy-4-aminoazobenzene, 2-methoxy-4-aminoazobenzene,
DNA adduct, 32P-postlabeling, DNA synthesis, mutation M13 DNA
This paper was presented at the Fifth International Conference
on Carcinogenic and Mutagenic N-Substituted Aryl Compounds held
18-21 October 1992 in Würzburg, Germany.
This work was supported in part by grants-in-aid for cancer
research from the Ministry of Education, Science and Culture of Japan.
Address correspondence to M. Tada, Laboratory of Biochemistry,
Aichi Cancer Center Research Institute, Kanokoden, Chikusa-ku, Nagoya 464,
Japan. Telephone: 052 762 6111. Fax: 052 763 5233.
Introduction
Over several years, we have focused our attention on elucidation of the
cause of the difference in genotoxic activity between 3-methoxy-4-aminoazobenzene
(3-MeO-AAB) and 2-methoxy-4-aminoazobenzene (2-MeO-AAB) (1-4). The
carcinogenic potencies of these aminoazo dyes are known to be influenced
by the position of methoxy substituents on the aminoazobenzene molecule.
3-MeO-AAB is a hepatocarcinogen in the rat and a mutagen in Escherichia
coli and Salmonella typhimurium, whereas 2-MeO-AAB, differing
only in the position of the methoxy substituted on the same benzene ring,
is a noncarcinogen and nonmutagen (5-7).
This article concerns: a) 32P-postlabeling analysis
of DNA adducts in the liver after intraperitoneal (ip) administration of
3-MeO-AAB or 2-MeO-AAB, b) effects of DNA adducts on in vitro
DNA synthesis, and c) mutations induced by aminoazo dye adducts in
M13 viral DNA.
32P-Postlabeling Analysis of Hepatic DNA
Adducts in F344 Rats Treated with 3-MeO-AAB or 2-MeO-AAB
In vivo formation of hepatic DNA adducts after exposure to the
aminoazo dyes was studied using 32P-postlabeling analysis. Male
F344 rats (5-6 weeks old) were given a single ip injection of 3-MeO-AAB
or 2-MeO-AAB (50 mg/kg), and DNA was isolated from livers at 16, 24, 48,
72 hr and 1 week thereafter. Analysis of DNA adducts was carried out by
the butanol extraction procedure of the 32P-postlabeling assay
(1,8).
Representative results are shown in Figure 1. DNA adducts formed by 3-MeO-AAB
consisted of one major (no. 1) and four minor (nos. 2-5) spots, while 2-MeO-AAB
gave only one spot (no. 1).
Figure 1. Autoradiographs
of PEI-cellulose maps of DNA adducts in rat livers formed at 24 hr and 48
hr after treatment with 3-MeO-AAB (A) and 2-MeO-AAB (B), respectively.
About 70 µCi and 140 µCi of 32P-labeled digests for
3-MeO-AAB and 2-MeO-AAB, respectively, were chromatographed. Development
was carried out in the first two dimensions as described (1,8); then
in 3.5 M lithium formate, 8.5 M urea, pH 3.5 (D3), followed by 0.8 M LiCl,
0.5 M Tris-HCl, 8.5 M urea, pH 8.0 (D4). Autoradiograms were developed for
24 hr at -80ºC.
Maximal DNA binding of 3-MeO-AAB and 2-MeO-AAB was observed after approximately
24 and 48 hr, respectively, and thereafter decreased gradually (Table 1).
After 7 days, 40% of the maximum adduct levels were still present in both
cases. The maxima were 3.8 for 3-MeO-AAB and 0.16 for 2-MeO-AAB per 107
nucleotides. The carcinogen 3-MeO-AAB generated more than 20-fold higher
amounts of adducts in the liver than did 2-MeO-AAB at all time points examined.
Effects of 3-MeO-AAB- and 2-MeO-AAB-Adducts on in Vitro DNA
Synthesis
To determine the effects of DNA adducts caused by 3-MeO-AAB and by 2-MeO-AAB
on the replication of DNA, we analyzed the reaction products of E. coli
DNA polymerase I (pol I) action on aminoazo dye-modified M13mp10 DNA templates
by DNA sequencing gel electrophoresis.
Single-stranded M13mp10 DNA was modified with N-hydroxy (N-OH)
derivatives of 3-MeO-AAB or 2-MeO-AAB in the presence of seryl-AMP, whereby
adducts have been proposed to be formed through electrophilic intermediate
N4-O-seryloxyamino derivatives (9,10). The
extents of aminoazo dye adducts were calculated from spectrophotometric
analyses (2,11).
Figure 2 shows representative sequencing gel-electrophoresis bands of
primer elongation products. From the evidence of stronger arrested bands
and shorter DNA products observed with the N-OH-3-MeO-AAB-modified template,
a conclusion can be drawn in this case of more effective inhibition of DNA
chain elongation. In clear contrast, the arrested bands with N-OH-2-MeO-AAB-modified
template were much fainter and the DNA products generally were longer, suggesting
that pol I was able to read through 2-MeO-AAB adducts.
Figure 2. Analysis
of products synthesized by pol I using M13 DNA template containing DNA lesions.
One pmole of each template DNA containing 8 to 10 adducts per DNA molecule
was annealed with 1 pmole of the primer, and elongation by pol I was carried
out as described previously (2). Lane 1, N-hydroxy-3-methoxy-4-aminoazobenzene;
lane 2, N-hydroxy-2-methoxy-4-aminoazobenzene; lane 3, 4-hydroxyaminoquinoline-1-oxide;
lane 4, unmodified DNA; lanes 5 to 8, as for lanes 1 to 4, respectively,
except that a chase reaction for 20 min was carried out. The lanes T, A,
C, and G represent standard Sanger dideoxyribonucleotide sequence ladders.
Elongation was arrested extensively at one base prior to every 3-MeO-AAB
guanine adduct and at -GGGG- sequences but not always at 2-MeO-AAB-guanine
adducts, but it was blocked at adenines in -GAG- sequences. The differences
between 3-MeO-AAB and 2-MeO-AAB guanine adducts might be because of their
chemical structures or surrounding altered structures of the DNA.
Mutations Induced by 3-MeO-AAB and 2-MeO-AAB Adducts in M13 Viral
DNA
The ability of 3-MeO-AAB and 2-MeO-AAB adducts to induce mutations was
analyzed using M13mp9 viral DNA modified by these chemicals as described
above (2). Modified DNA containing 20 to 40 adducts/molecule was
transfected into E. coli host cell JM103 and mutants were screened
for expression of the marker enzyme, ß-galactosidase.
Mutation frequencies were increased up to 7-fold (21 x 10-4)
for N-OH-3-MeO-AAB-modified DNA and 4-fold (8 x 10-4) for N-OH-2-MeO-AAB-modified
DNA in SOS-induced host cells, as compared to the uninduced cells. The mutagenesis
induced by aminoazo dye adducts therefore seems to be largely dependent
on SOS functions.
DNA sequence analysis was performed by Sanger's methods (12) within
the lacZ´ region base pairs 6200 to 6400, corresponding to
the N-terminal sequence of the ß-galactosidase (Figure 3). The mutational
hotspots were G at position 6300 in both modified DNAs. The most frequent
mutation being G
A transition, but GT and GC transversions also occurred with 3-MeO-AAB adducts.
Figure 3. Spectra
of mutations induced by 3-MeO-AAB adducts and 2-MeO-AAB adducts under SOS-induced
conditions. The DNA sequence of the viral strand of M13mp9 in the lacZ´
fragment is given. Mutation changes induced by N-OH-3-MeO-AAB-modified DNA
are presented above and those of N-OH-2-MeO-AAB-modified DNA are below the
sequence.
The most frequent events were base substitutions; 60% (38/63) for 3-MeO-AAB
adducts and 82% (39/47) for 2-MeO-AAB-adducts (Table 2). Frameshift mutations--one
base deletion and large deletion (about 120 base pairs)--were observed more
frequently in N-OH-3-MeO-AAB-modified DNA than in N-OH-2-MeO-AAB-modified
DNA. Both base substitution and -1 frameshift mutations occurred preferentially
at guanine residues in agreement with our previous finding that these aminoazo
dyes specifically form adducts with guanine bases (11).
Discussion
The aim of this study was to elucidate differences in genotoxic activity
between 3-MeO-AAB and 2-MeO-AAB. 32P-Post- labeling analysis
of DNA adducts from the liver of rats exposed to these chemicals revealed
that the carcinogen 3-MeO-AAB produced 20-fold higher amounts of adduct
than the noncarcinogen 2-MeO-AAB. Moreover, five spots were detected on
TLC sheets from 3-MeO-AAB-treated rats, whereas only one adduct was found
in 2-MeO-AAB-treated rats. Essentially similar results were obtained with
DNA of E. coli uvrA strains treated with N-OH-3-MeO-AAB or N-OH-2-MeO-AAB
(3).
Although the adducts formed by 3-MeO-AAB and 2-MeO-AAB have not yet been
fully characterized, C8-substituted deoxyguanosine (spot no. 1) may well
be a major product. Minor spots (no. 2-5) in 3-MeO-AAB-modified DNA may
be deoxyguanosin N2-yl-substituted and deoxyadenosin N6-yl-substituted
4-aminoazobenzene derivatives (13,14). In this context, it is of
particular importance that the kind of DNA damage--the nature of the adduct
itself or the conformational changes in DNA--which is responsible for the
biologic activity of 3-MeO-AAB be clarified.
We therefore compared the biologic potential of M13 phage DNA in vitro
modified by these chemicals. As a result, DNA replication by pol I was found
to be blocked at or one base prior to guanine bases in the templates, 3-MeO-AAB
adducts having a significantly greater inhibitory effect on DNA synthesis
than 2-MeO-AAB adducts. The arrested pattern also differed in each case.
While 3-MeO-AAB adducts induced mutations more frequently than did 2-MeO-AAB
adducts, they were both mainly derived from guanine base. However, 3-MeO-AAB
adducts induced a higher percentage of frameshift mutations as well as base
substitutions, suggesting a possibly greater alteration in DNA conformation.
In contrast, 2-MeO-AAB adducts induced mostly base substitutions.
Our data thus suggest that the difference in hepatocarcinogenic activity
between 3-MeO-AAB and 2-MeO-AAB depends not only on qualitative and quantitative
variations in adduct formation, but also on conformation changes in DNA
modified by these chemicals.
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