Chemico-Biological Interactions 94 (I 995) I35- I45
Oxidative modification of DNA bases in rat liver
and lung during chemical carcinogenesis and aging
Ying-Jan Wang, Yuan-Soon Ho, Ming-Jiang Lo, Jen-kun Lin*
Institute oj Biochemistry, College of Medicine. National Taiwan University, Taipei, Taiwan, Republic of
China
Received 27 January 1994; revision received I2 April 1994; accepted 14 April 1994
Abstract
The extent of DNA modification in cancerous rat liver and lung tissues was investigated
and compared to their respective normal tissues. Liver tumors were induced by 2-fluorenylacetamide (ZFAA) or N-nitroso-N-2-fluorenylacetamide
(N-NO-2-FAA), and lung tumors
were induced by sodium nitrite plus trimethylamine. In the DNA samples isolated from these
tissues, two pyrimidine-derived and four purine-derived modified DNA bases were identified
and quantified by gas chromatography/mass
spectrometry with selected-ion monitoring.
These compounds were characterized as Shydroxyuracil (5-OHUra), thymine glycol (TG),
4,6_diamino+formamidopyrimidine
(FapyAde), 2,6-diamino-4-hydroxy-S-formamidopyrimidine (FapyGua), 8-hydroxyadenine
(8-OHAde), and 8-hydroxyguanine
(8-OHGua).
Elevated amounts of modified DNA bases were found in most cancerous tissues when compared to the controls. Chemicals used for tumor induction were responsible for inducing DNA
lesions that could be promutagenic in vivo and could lead to various types of mutations. When
endogenous oxidative damage to DNA during aging was examined, a roughly 2-fold increase
of thymine glycol, 8-OHAde and 8-OHGua was found in aged (12 months) rat liver tissues
compared to young tissues (1 month). The same results were also found in lung tissues, except
that the amount of thymine glycol exhibited more than a IO-fold increase in aged tissues when
compared to young tissues. The association of the modified bases with the processes of aging
and carcinogenesis deserves further investigation.
Keywords:
Modified bases; Liver; Lung; Carcinogenesis;
Aging
* Corresponding author., Institute of Biochemistry, College of Medicine, No. I. Section
Jen-Ai Road, Tel.: (01 I)-886-2-3562213; Fax: (01 I)-886-2-3918944.
0009~2797/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved
SSDI 0009-2797(94)03327-5
I.
136
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1. Introduction
Free radicals generated in vivo by cellular processes have been implicated to play
an important role in a number of human diseases [ 11.The major oxygen-derived species generated by normal biochemical pathways are superoxide anion radical (02 :),
hydrogen peroxide (H202), and hydroxy radical (. OH) (reviewed in [2,3]). Under
physiological conditions, neither Oz : nor H202 appear to produce modifications in
DNA unless metal ions are present in the system. Thus, much of the toxicity of O2 ;
and HzOz is thought to result from their metal ion-catalyzed conversion into the
highly reactive -OH [2,3]. In normal cells, a primary defense against oxidative
damages is provided by antioxidants such as glutathione, as well as by DNA repair
enzymes that participate in the excision repair process [4,5]. However, these defense
mechanisms can be subverted by xenobiotics, which induce the production of excessive or untimely free radicals and result in damage to macromolecules, including
DNA [5-71. These lesions may have determinable biological consequences such as
cell death, mutagenesis, or carcinogenesis [8]. There is abundant evidence suggesting
that DNA damage by endogenous free radicals does occur and accumulate in vivo
[9- 111. It has been estimated that the genome of a human cell receives around lo4
oxidative hits/day on average, and this process is likely to be critical for both cancer
and aging [12,13].
Chemical characterization of free radical-induced modifications in DNA bases
can be achieved by the use of gas chromatography/mass spectrometry with selectedion monitoring (GC/MS-SIM) [14]. This method provides both precise identification
of molecules with structural evidence and quantification of a large number of modified bases [15-181. GC/MS-SIM has been used to investigate the purine-derived
modified bases from neoplastic livers of feral fish [ 191and from breast cancer tissues
of humans [20]. Both of these studies showed increased amounts of modified bases
when compared to the controls. The more recent report also showed a higher level
of purine- and pyrimidine-derived modified bases from various human cancerous tissues when compared with their cancer-free surrounding tissues [21]. However, very
little is known about tumor tissues in rodent species in this aspect. In the present
work, we identified and quantified two pyrimidine-derived (thymine glycol (TG),
5-hydroxyuracil
(5-OHUra)) and four purine-derived (8-hydroxyadenine
(8OHAde), 4,6-diamino-5-formamidopyrimidine
(FapyAde), 8-hydroxyguanine (8OHGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine
(FapyGua)) modified
bases in two types of liver cancer induced in rats by 2-fluorenylacetamide (2-FAA)
or N-nitroso-N-2-fluorenylacetamide
(N-NO-ZFAA), and in one type of lung
cancer induced by sodium nitrite plus trimethylamine. Some of the other pyrimidinederived base products were not measured because of the lack of authentic compound, (eg: 5-hydroxycytosine) [18] or because of the degradation during acid
hydrolysis (eg: 5-(hydroxymethyl)uracil)
1221. The endogenous amount of these
modified bases in different ages of rat liver and lung tissues were also examined to
see whether an age-dependent accumulation of oxidative DNA damage was present
in these two organs.
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137
2. Materials and methods
2.1. Materials
5Hydroxyuraci1, 4,6-diamino-5-formamidopyrimidine,
2,4,5-triamino-6-hydroxypyrimidine sulfate, thymine, 6-azathymine, 8-azaadenine, 8-bromoadenine, sodium
nitrite and trimethylamine were purchased from Sigma Chemical Co., St. Louis,
MO, 8-hydroxyguanine was obtained from Aldrich Chemical Co., Milwaukee, WI.
Acetonitrile and bis(trimethylsilyl)-trifluoroacetamide
(BSTFA) containing 1%
trimethylchlorosilane were obtained from Pierce Chemical Co., Rockford, IL. The
8-hydroxyadenine was synthesized by treatment of 8-bromoadenine with concentrated formic acid (95%) and purified by crystallization from water [18]. The 2,6diamino-4-hydroxy-5-formamidopyrimidine
was synthesized by treatment of 2,4,5triamino-6-hydroxypyrimidine
sulfate with boiling concentrated formic acid,
precipitated by ethanol and recrystallized from water [23]. Thymine glycol was synthesized from thymine by mild permanganate oxidation. The mixed reaction products were chromatographed on sephadex LH-20 in water, and thymine glycol was
eluted between the by-product, 5-hydroxy-5-methyl barbituric acid, and thymine as
determined by absorbance at 230 nm. Peak fractions were collected, concentrated
and recrystallized from water-ethanol [24]. N-NO-ZFAA was synthesized by the
nitrosation of 2-FAA according to the procedure described earlier [25] and purified
by recrystallization in 0.01% acetic acid of ethanol at -10 to -20°C. The purity of
the synthetic standards was measured by high-performance liquid chromatography
[18], and were more than 90% pure.
2.2. Animals and treatment
A total of 27 male, 5 week old Spraque-Dawley rats, were obtained from the Animal Center of National Taiwan University Hospital (Taipei). They were randomly
divided into three groups, FAA, NO-FAA, and control. After one week of acclimatization, the rats were treated with 2-FAA or N-NO-ZFAA (60 mg/kg weekly) by
i.p. injection. Hepatocellular carcinomas (HCC) were induced in both test groups
(819 in 2-FAA and 7/9 in N-NO-2-FAA) after ten months (Ho and Lin, unpublished
data). The cancerous lung tissues were obtained from male Wistar rats treated with
trimethylamine plus sodium nitrite (0.013% and 0.3% in their diets) for one year
(Lin, et al. unpublished data). Parts of the cancerous tissues were excised and quickly
frozen in liquid nitrogen and maintained at -70°C prior to extraction of the DNA.
The remaining tissues were fixed in formaldehyde for histological examination. Five
tumor-bearing and five normal control rats in each group were chosen for the experiment, three separate tissue samples collected from each rat were used, and DNA was
extracted by the method described by Gupta [26]. Male Wistar rats were used for
the aging study and they were fed standard laboratory diets and water ad libitum.
The ages of the animals studied ranged from 1 to 12 months, and five rats of different
ages were examined.
138
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2.3. Hydrolysis, trimethylsilylation and gas chromatography/mass spectrometry
Modified DNA bases were quantitatively measured by GCYMS-SIM using 6azathymine and 8-azaadenine as internal standards [18]. In brief, known amounts
of the modified bases and internal standards were analyzed to measure the ion current ratios. This yielded a calibration plot and generated the relative molar response
factor for quantitative measurement [ 181. Aliquots of internal standards were added
in an aliquot solution containing 200 pg DNA in each sample as internal standards
for quantitative measurements. Samples were then evaporated under vacuum in a
Speed-Vat. The dried DNA was hydrolyzed to bases by treatment with 200 ~1 of formic acid (88%) at 140°C for 30 min in polytetrafluoroethylene-capped
hypovials
(Pierce). After cooling briefly, the samples were dried under a stream of nitrogen and
further dried overnight under vacuum. Then, they were trimethylsilylated in 80 ~1
of a mixture of BSTFA containing 1% trimethylchlorosilane and acetonitrile (1:l)
by heating for 30 min at 130°C [ 15,181. Analysis of the derivatized samples were performed by GC/MS-SIM. A Hewlett-Packard Model 5890 microprocessor-controlled
gas chromatograph interfaced to a Hewlett-Packard Model 597 1A mass selective detector was used. Separation was carried out using a fused silica capillary column
coated with 5% phenylmethyl silicone (DB-5, 15 M, 0.25 mm id.). Helium was used
as the carrier gas at an inlet pressure of 30 KPa. The injection port was kept at
25O”C, the GC/MS interface was maintained at 280°C. The column temperature was
increased from 120 to 250°C at a rate of 8°C per min after 2 min at 120°C. An aliquot
(1~1) of each derivatized sample was injected without any further treatment into the
NH;!
0
Thymine glycol
5Hydroxyuracil
(S-OHUra)
(S-OHAde)
(TG)
4,8Diamino-5lormamidopyrimidine
SHydroxyadenine
EHydroxyguanine
2.6-Diamino4hydroxy
Sformamidopyrimidine
(I-OHGua)
(Fapy Ade)
(FwGua)
Fig. 1. Chemical
structures
of the modified
bases.
Y.-J. Wang Ed al. / Chm.-Bid.
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IBY
injection port in the splitless mode. In the selected-ion-monitoring (SIM) mode,
characteristic ions of the trimethylsilyl (Me3Si) derivatives of base products of DNA
were monitored individually [ 151.
3. Results
3.1. Amounts of modified bases in rat liver tissues
The structures of the modified bases used in the present study are illustrated in
Fig. 1. In rats treated with 2-FAA or N-NO-2-FAA for ten months, liver tumors
were found as expected. The modified bases and their quantities are given in Table
1. Comparing the two types of tumor tissues with the normal controls, significantly
higher amounts of base lesions were found in the N-NO-ZFAA than in the 2-FAAinduced liver cancers. A 2-4.2-fold increase of 8-OHAde, 8-OHGua and thymine
glycol was found in almost all of the five rats treated with N-NO-2-FAA but only
a 1.3-2-fold increase was found in 3 to 4 rats out of the five rats treated with 2-FAA.
5-OHUra, FapyAde and FapyGua also presented similar results but in fewer
amounts and with lower frequencies.
3.2. Amounts of modified bases in rat lung tissues
In rats fed sodium nitrite plus trimethylamine for one year, lung cancers were induced. Table 2 shows the modified bases and their quantities for both lung cancer
tissues and the normal controls examined. In contrast to the results described above,
the amounts of purine-derived modified bases, 8-OHAde, FapyAde, 8-OHGua and
FapyGua,in tumour and normal tissues were found to be significantly different in
only 2 out of 5 rats. Additionally, the amounts of these bases only increased up to
1.8-fold, except for FapyGua (2-4-fold). Unlike the purine-derived moieties, thymine glycol was found to have dramatically increased in all live rats examined (ranging from 2.6-4.8-fold), and 5-OHUra also increased 1.4-2.3-fold in 3 out of 5 rats
examined.
3.3. Amounts of modified bases in different ages of rat liver and lung tissues
The basal level of endogenous modified bases in different ages (1,4,8,12 months)
of Wistar rats was evaluated as a function of aging. In these cases, in both of the
two organs examined, the amount of modified bases increased with the age of the
animal (Fig. 2). The steady-state levels of thymine glycol, 8-OHAde and 8-OHGua
in DNA isolated from the livers of 12-month-old rats were 0.29 f 0.05,0.36 f 0.04
and 0.47 f 0.08 nmol per mg of DNA, respectively. These values represent an
increase of 120, 90 and 140%, respectively, over the values observed in l-month-old
rats (0.12 i 0.02,O. 19 f 0.03 and 0.20 f 0.05). In DNA isolated from the lung, the
increase in the levels of 8-OHAde and 8-OHGua over the same period was 160 and
lOO%, respectively, increasing from 0.29 f 0.10 and 0.42 f 0.11 in l-month-old
rats to 0.75 i 0.24 and 0.88 f 0.16 nmol per mg of DNA in 12-month-old rats. The
level of thymine glycol increased from 0.01 f 0.01 to 0.19 f 0.02, representing a
more than IO-fold increase. Nevertheless, a significant change of 5-OHUra,
FapyAde and FapyGua between the different ages (data not shown) was not found.
0.09
0.08
0.13
0.18
0.22
0.14
NOFAA-I d
NOFAA-2
NOFAA-3
NOFAA-4
NOFAA-5
Average
0.58
0.69
0.36
0.62
0.68
0.59
0.25
0.38
0.44
0.36
0.04
0.06*
0.08*
0.09
zt 0.13*
zt 0.06”
zt 0.08
ZJZ
0.11’
f 0.18*
* 0.13
zt
zt
f
f
0.26 zt 0.03
0.28 f 0.06
0.46 f 0.12’
TG
0.21
0.10
0.08
0.06
0.16
0.12
0.12
0.09
0.06
0.08
0.05*
0.04
0.03
0.03
ztz0.08*
* 0.02;
f 0.02
zt 0.03
+ 0X14*
l 0.06
f
*
f
+
0.06 zt 0.03
0.05 l 0.03
0.06 f 0.02
FapyAde
0.92 f 0.21*
0.68 f 0.14*
0.73 h 0.22,
1.16 f 0.06*
1.29 ziz0.13;
0.96 zt 0.27
0.56 f 0.12;
0.64 f 0.13*
0.53 l 0.14;
0.51 f 0.11
0.37 f 0.08
0.35 f 0.07
0.47 f 0.07*
I-OHAde
0.02
0.04
0.15
0.06
0.12
0.08
0.03
0.05
0.12
0.06
0.01
0.02
0.03;
0.04
0.01
f 0.02
f 0.04’
f 0.01*
f 0.03;
ZJZ
0.05
l
f
f
f
f
0.04 * 0.01
0.04 f 0.01
0.08 f 0.02’
FapyGua
0.15*
0.14*
0.26*
0.20*
0.28*
0.42
ZIZ0.06
zt 0.12*
ztz0.18*
f 0.15
0.74 *
0.62 zt
1.12 f
1.22 l
1.68 zt
1.08 f
0.48
0.65
0.79
0.62
0.40 zt 0.08
0.74 f 0.04*
0.45 f 0.1 I
8-OHGua
al nmol of a modified base/mg of DNA = 320 modified base residues/IO6 DNA base residues. Each value represents the mean f SD. from three separate
tissue samples.
bControl rat liver DNA (10 months old), five rats and three separate tissue samples per rat were used for the measurement.
‘2-FAA-induced liver cancer.
dN-NO-2-FAA-induced liver cancer.
*Significantly different from controls (P < 0.05 by Student’s r-test).
0.03
0.02
0.02*
0.05’
l 0.06*
* 0.06
0.08 f 0.03
0.06 * 0.01
0.12 f 0.038
0.10 l 0.04
FAA-3
FAA-4
FAA-5
Average
*
zt
*
zt
0.07 * 0.02
0.06 * 0.01
0.16 f 0.04’
Control b
FAA-lC
FAA-2
5-OHUra
nmol DNA moditication/mg DNAa
Table 1
Amounts of modified DNA bases in the liver tissues of rats treated with the carcinogen FAA or NOFAA
base/mg
f
*
f
f
f
f
0.12*
0.22’
0.14*
0.25*
0.18*
0.16
of DNA = 320 modified
0.49
0.68
0.63
0.92
0.71
0.69
0.19 f 0.02
TG
DNAa
base residues/lo’
zt
f
f
rt
f
f
0.08
0.12*
0.22
0.16*
0.14
0.14
f
f
zt
*
f
l
l
0.01
0.03*
0.01
0.02*
0.01
0.05
0.02
Each value represents
0.03
0.08
0.05
0.16
0.04
0.07
0.04
0.84
0.79
0.68
1.23
1.28
0.96
f
f
*
f
f
f
from three separate
0.12
0.28
0.14
0.16*
0.21’
0.27
0.88 f 0.16
I-OHGua
the mean f SD.
per rat were used for the measurement.
DNA base residues.
0.68
0.97
0.73
0.98
0.81
0.83
0.75 f 0.24
FapyGua
plus trimethylamine
8-OHAde
nitrite
tissue samples
0.10 f 0.02
0.15 f 0.03*
0.09 f 0.01
0.11 * 0.04
0.12 f 0.02’
0.1 I f 0.02
0.08 f 0.02
Fapy Ade
with sodium
bControl rat lung DNA (12 months old ), five rats and three separate
%odium nitrite plus trimethylamine-induced
lung cancer.
*Significantly different from controls (P < 0.05 by Student’s r-test).
al nmol of a modified
tissue samples.
0.01
0.04*
0.03
0.03*
l 0.02*
f 0.04
zt
+
zt
f
0.06
0.16
0.08
0.12
0.10
0.10
NTA-IC
NTA-2
NTA-3
NTA-4
NTA-5
Average
5-OHUra
nmol DNA modilication/mg
DNA bases in the lung tissues of rats treated
0.07 f 0.02
of modified
Control b
Table 2
Amounts
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12
A
m
1 month
4
q
m
’
months
:.
> R months
;:;12
months
I
1
4
a
,lZ
month
months
m0ntk
months
T
I
00
TG
a-OHAde
a-OiIGua
TG
a-OHAde
a-OHGua
Fig. 2. (A) Content of thymine glycol, I-OHAde and 8-OH&a in the liver of Wistar rats with different
ages. (B) Content of thymine glycol, I-OHAde and I(-OHGua in the lung of Wistar rats with different
ages. Each value represents the mean f SD. from five animals.
When compared with the different organs of rats from the same age, lung tissues had
a larger amount of 8-OHAde and 8-OHGua than liver tissues (about a 2-fold
increase). In all cases examined, the amounts of 8-hydroxypurines were higher than
those of formamidopyrimidines which are formed via reductive pathways from the
respective 8-0~~1 derivatives [27].
4. Discussion
In the present study we attempted to determine whether an elevated amount of
DNA lesions existed in rat cancerous tissues, and tried to find any difference between the tumor tissues induced by different carcinogens. It has been shown that one
of the DNA base lesions could be induced in livers by a single administration of the
classical hepatocarcinogen, 2-FAA [28]. By the nitrosation of 2-FAA, a new chemical, N-NO-ZFAA, was synthesized [25]. Without metabolic activation, N-NO-2FAA exhibited more direct and stronger damaging effects on DNA than its parent
compound [29]. When rats were treated with N-NO-2-FAA or 2-FAA by subcutaneous (s.c) injection, subcutaneous lesions consisting of an inflammatory reaction
were observed in N-NO-2-FAA but not 2-FAA-treated animals (Ho and Lin, unpublished observation). DNA base modifications in tumor tissues induced by these
two chemicals were examined, and the results indicated that more lesions were present in N-NO-ZFAA than in 2-FAA-treated rats. It is not known whether the DNA
base lesions identified in the present work play a role in carcinogenesis or are formed
in great amounts in cancerous tissues as a result of the disease. However, there is
no doubt that free radicals and DNA damage play an important role in carcinogenesis (reviewed in [30]). A number of free radical-induced DNA base lesions
have been examined for their biological consequences, and some of these have been
found to possess mutagenic properties [31,32]
Y.-J. Wang el al. /Chem.-BinI.
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143
It is a surprising finding that sodium nitrite plus trimethylamine fed to rats causes
a high incidence of lung cancer (Lin, et al., unpublished data). It has been reported
[331 that the alkylating agent, dimethylnitrosamine, usually causes liver, but not
lung, cancer in rats. In addition, this N-nitroso compound can be produced in vitro
by the reaction of sodium nitrite plus trimethylamine (data not shown). The role of
these two chemicals in lung carcinogenesis is still unknown and remains to be determined. The amounts of modified bases examined in cancerous lung tissues were only
slightly higher than in the normal control tissues except for thymine glycol. This
result is very different from that found with the liver tumors because the liver tumors
exhibited a more significant change in the amounts of modified bases when compared to the control tissues. The reason for this phenomenon may be due in part to
the different activities of the antioxidant enzymes among the individual tumors and
due to the different chemicals used in this study. Together, these two effects may account for the different amounts of modified DNA bases found in various cancerous
tissues. Abnormal levels and activities of antioxidant enzymes have been reported
in tumor cells [30]. Low levels of antioxidant enzymes, such as superoxide dismutase
or catalase, may result in an accumulation of O2 : and Hz02 with subsequent . OHinduced damage to DNA.
When the basal level of endogenous modified bases in different ages of rats were
compared, a roughly 2-fold increase of oxidative damage in DNA isolated from the
liver and lung (except for thymine glycol in the lung) was found. These results are
consistent with previous studies [ 111,which indicate that the age-dependent increase
in the level of 8-OHGua could be the result of an increase in the formation of this
oxidized base, a decrease in the repair activity, or a combination of both. Aerobic
organisms are subject to an unavoidable background risk of activated oxygen species
due to living in an oxygen atmosphere [34]. From the electron transport chain of
mitochondria and normal metabolic process that utilize oxygen, free radicals can be
produced continuously [35]. When these activated oxygen species react with nuclear
DNA, modified bases are formed and thus oxidative mutations could be produced
in somatic cells. The fact that the levels of 8-OHAde and 8-OHGua in the lungs of
the control rats are higher than those in the livers (about 2-fold) may be due to the
content of antioxidants in these two organs. It has been reported that the content
of glutathione in normal rat livers is much higher than in the lungs (reviewed in [36]).
The same level of thymine glycol in these two organs may be caused by the different
content of repair enzymes that recognize specifically-damaged pyrimidine bases [37].
Epidemiologic studies have indicated that of humans who reach the age of 50,
about 20-25% will eventually die of cancer [38]. The incidence of most cancers in
human populations was found to be intimately associated with the increase of age.
Damage to DNA could be a ubiquitous process occurring under normal physiological conditions and could be responsible for aging and cancer. The effort of this
study is to examine purine- and pyrimidine-derived base modifications in chemicallyinduced tumor tissues in rats on a structural and quantitative basis. Our results show
a higher level of modified DNA bases in rat tumor tissues than in the respective
normal controls and show that the chemicals used for tumor induction are related
to the amount of these DNA lesions. Little is known about the role of individual
base modifications in carcinogenesis. However, high levels of modified bases may
144
Y.-J. Wang et al. / Chem.-Biol.
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94 (199s)
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contribute to the genetic instability of tumor cells and thus increase metastatic
potential [39]. Measurement of purine- and pyrimidine-derived DNA lesions in tissues may be useful for understanding the putative role of base lesions in the etiology
of cancer and their potential use as biomarkers for cancer risk assessment [40,41].
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