Carcinogenesis vol.17 no.8 pp.1553-1559, 19%
ACCELERATED PAPER
Formation and accumulation of DNA ethenobases in adult
Sprague-Dawley rats exposed to vinyl chloride
Y.Guichard1, F.E1 Ghissassi1, J.Nair2, H.Bartsch2 and
A-Barbin1-3
'international Agency for Research on Cancer, 150, Cours Albert Thomas,
F-69372 Lyon Cedex 08, France and 2German Cancer Research Center,
Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
^To whom correspondence should be addressed
DNA ethenobases are promutagenic lesions formed by
carcinogens such as vinyl chloride (VC). Their formation
was investigated in 6-week old, male Sprague-Dawley rats
exposed to 500 p.p.m. VC by inhalation (4 h/day, 5 days/
week) for 1, 2, 4 or 8 weeks and in 7- and 14-week old,
matched control animals. l/v*-Ethenoadenine (eA) and
3, A^-ethenocytosine (eC) deoxyribonucleotides were analysed by immunoaffinity purification and 32P-postlabelling.
This postlabelling method was compared with a radioimmunoassay method, which yielded similar results. Background levels of ethenobases were found in DNA from
the liver, lungs, kidneys and circulating lymphocytes of
unexposed, control rats. In the liver, the following background molar ratios of ethenobase to parent base in DNA
were detected (mean valuesxlO"8): eA/A, 0.04-0.05; eC/C,
0.06-0.07. In the lungs, kidneys and circulating lymphocytes, background levels of eA and eC ranged from 1.7 to
4.2 XlO"8 and from 4.8 to 11.2X10-", respectively. Following
a 5-day exposure to VC, a significant increase of eA and
eC was measured in hepatic DNA from rats sacrificed
immediately after treatment Further, a dose-dependent
increase of both etheno adducts was observed in liver DNA
of VC-treated rats. Compared to the 5-day exposure, ~4fold higher levels of eA and eC were observed in the liver
of animals after 8 weeks of exposure. In contrast, there
was an accumulation of eC but not of eA in lungs and
kidneys. In circulating lymphocytes, no significant increase
of ethenobase levels above control values was observed
after 2 months of exposure to VC. Both etheno adducts
were found to be persistent in liver DNA, after 2 months
following the termination of VC exposure. These results
further support the notion that DNA ethenobases are
critical lesions in VC-induced carcinogenesis. The possible
contribution of lipid peroxidation products that also yield
ethenobases, on the formation and persistence of these
DNA adducts, remains to be clarified.
Introduction
Ethenobases are exocyclic adducts formed with nucleobases
by several carcinogens such as vinyl chloride (VC*) and
urethane (reviewed in 1,2). Recent data indicate that they
•Abbreviations: VC, vinyl chloride; eA, l.JV'-ethenoadenine; eC, 3,N*ethenocytosine; edAdo, l//-etheno-2'-deoxyadenosine; edCyd, B.yV'-etheno2'-deoxycytidine; RIA, radioimmuno-assay; 3'-£dAMP, l/i'6-etheno-2'-deoxyadenosine 3'-monophosphate; 3'-edCMP, 3,//t-etheno-2'-deoxycytidine 3'monophosphate; BSS, baJanced salt solution; PBS, phosphate buffered saline;
PEI, polyethylene imine.
© Oxford University Press
could also be formed endogenously through the stimulation of
lipid peroxidation (3-7). DNA ethenobases exhibit promutagenic properties (8-11; reviewed in 2,12), suggesting that
they could be critical miscoding DNA lesions in chemical
carcinogenesis. 7-(2-Oxoethyl)guanine, the major DNA adduct
formed by VC, did not display these promutagenic properties (13).
Following the identification of 7-(2-oxoethyl)guanine in
liver DNA from rodents exposed to VC (14,15), Green and
Hathway (16) were the first to present some evidence of the
formation of 1 .A^-ethenoadenine (eA) and S^-ethenocytosine
(eC) in liver DNA of rats exposed to VC for 2 years. In 1985,
Laib et al. (17) reported the detection of A^-ethenoguanine
in DNA hydrolysates from the liver of pre-weanling rats
treated with [14C]VC. Subsequently, A^-ethenoguanine was
determined by isotope dilution mass spectrometry following
electrophore-labelling in DNA hydrolysates from the liver,
lung and kidney of pre-weanling and adult Sprague-Dawley
rats exposed to 600 p.p.m. VC for 5 days, 4 h/day (18-20).
Following the production of monoclonal antibodies directed
against l//5-etheno-2'-deoxyadenosine (edAdo) and 3^/V4etheno-2'-deoxycytidine (edCyd), a sensitive technique, based
on HPLC purification and a competitive radioimmuno-assay
(RIA), permitted to demonstrate the formation of edAdo and
edCyd in liver and lung DNA from 11-day old SpragueDawley rats treated with 2000 p.p.m. VC for 10 days (21).
The analysis by HPLC/RIA was further used to measure edAdo
and edCyd in DNA samples from various tissues (liver, lung,
brain) of 7-day old and from liver of 13-week old BD VI rats
exposed to 500 p.p.m. VC, 7 h/day during 2 weeks (22).
Finally, a comparative study on the formation and persistence
of the four VC-DNA adducts was carried out with 10-day old
Sprague-Dawley rats exposed to 600 p.p.m. VC (4 h/day for
5 days). 7-(2-Oxoethyl)-guanine and the ethenobases were
quantitated in the liver, lung and kidney: all three etheno
adducts appeared to be persistent in liver DNA 2 weeks after
the end of exposure (20). At that time, a similar study on adult
animals was not feasible because of the lower levels of DNA
alkylation produced by VC in adult, as compared to young
rats (19,22,23).
Recently, an ultra-sensitive method, based on immunoaffinity purification and 32P-postlabelling was developed to
determine l,A*-etheno-2'-deoxyadenosine 3'-monophosphate
(3'-edAMP) and 3,A*-etheno-2'-deoxycytidine 3'-monophosphate (3'-edCMP) in DNA (7,24). This technique permitted
the detection of background levels of eA and eC in liver
DNA samples from untreated rodents and from humans with
unknown exposure (7). In the present work, the accumulation
and persistence of eA and eC was investigated by immunoaffinity/32P-postlabelling in several tissues from adult SpragueDawley rats following chronic exposure to VC.
Materials and methods
Chemicals
VC (3% in N2, purity 99.9%) was obtained from Airgaz (SiJrezin du Rh6ne,
France). Nucleotides, nucleosides and nucleobases were purchased from Sigma
1553
Y.Guichard et al.
Chemical Co. (St Louis, MO) and from Pharmacia (Uppsala, Sweden). 3'edAMP and 3'-edCMP were prepared by reaction of the parent nucleotide
with 2-bromoacetaldehyde and purified by HPLC (24,25). EdCyd, [3H]edAdo
(specific activity, 13.3 Ci/mmol) and [3H]edCyd (18 Ci/mmol) were prepared
as described by Eberle et al. (21). [y-32P]ATP (specific activity >5000
Ci/mmol) was from Amersham France S.A. (Les Ulis, France).
Monoclonal antibodies EM-A-1 and EM-C-1, raised against edAdo and
edCyd, respectively, were obtained from Dr M.F.Rajewsky (University of
Essen, Germany). Protein A-Sepharose CL4B was purchased from Pharmacia,
cellulose MN300 from Macherey and Nagel (DUren, Germany), polyethylene
imine (PEI) Corcat P600 from Virginia Chemicals (Portsmouth, VA) and
hydroxyapatite (Bio-Gel HTP) from Bio-Rad Laboratories (Richmond, CA).
Micrococcal endonuclease (grade VI) and spleen phosphodiesterase (from
calf spleen) were from Sigma, and T4 polynucleotide kinase from Pharmacia.
The following enzymes were purchased from Boehringer-Mannheim
(Mannheim, Germany): proteinase K, RNase (from bovine pancreas), DNase
I (from bovine pancreas; grade II), snake venom phosphodiesterase (from
Crotalus dunssus), alkaline phosphatase (from calf intestine; grade I).
Animals
Male Sprague-Dawley (OFA) rats, obtained from Iffa-Credo (L'Arbresle,
France), were housed in a temperature and humidity controlled room with a
12 h light/dark cycle. They were given a commercial rat chow (Biscuits Extra
Labo from Socie'te' Pietrement, Provins, France) and water ad libitum.
Treatment was started after 1 week of adaptation to this chow.
VC exposure
For exposure to VC, animals were placed in an inhalation chamber with an
airflow of ~15 chamber volume changes per hour, as described previously
(22). VC concentration at four different positions inside the chamber was
monitored hourly by gas chromatography on Porapack QS (80-100 mesh,
from Waters Associates, Milford, MA), with flame ionisation detection. The
inhalation chamber was kept at the same housing conditions as described
above and the animals had free access to food and water.
To examine the accumulation of VC-DNA adducts with increasing length
of exposure, groups of five 6-week old rats were exposed to 500 p.p.m. VC,
4 h/day, 5 days/week, for 1, 2, 4 or 8 weeks. Animals were sacrificed
immediately after the end of exposure. In another experiment designed to
examine adduct persistence, 20 rats were exposed for 5 days to 500 p.p.m.
VC (4 h/day) and groups of five rats were killed at different time intervals
after the treatment, i.e. 0, 2, 4 or 8 weeks.
The control groups comprised male Sprague-Dawley rats that were housed
under the same conditions and were sacrificed at the age of 7 or 14 weeks.
Due to unavoidable constraints of space at the time this experiment was
initiated, control rats could not be kept in our laboratory at the same time as
treated animals. Therefore, we used control rats housed later in the same room
under similar conditions. These animals were matched to the VC-exposed
groups with regard to their source (Iffa Credo), age (7- and 14-week old), sex
and diet.
Preparation of tissues and DNA isolation
Animals were killed by exsanguination (cardiac puncture) under ether anaesthesia. Blood was collected in hepannized tubes. Organs (liver, lungs, kidneys)
were excised immediately, frozen in liquid nitrogen and kept at -80°C until
further processing.
Mononuclear leucocytes were separated as follows. A 5% (w/v) solution
of Dextran 200 000-400 000 (Fluka Chemie Ab, Buchs, Switzerland) was
prepared in a balanced salt solution (BSS) consisting in a 14-5 mM Tris-HCl
buffer (pH 7.6) containing 5 HM CaCI2, 98 uM MgCl2, 540 HM KC1, 126 mM
NaCl and 555 uM glucose.
Blood (5 to 10 ml from each animal) was mixed with one volume of
Dextran solution (by gentle inversion of the tube). After sedimentation of
aggregated erythrocytes (30 min at room temperature), the leukocyte-rich
supernatant was removed and layered onto a separation fluid (Mono-Poly
Resolving Medium from Flow Laboratories SA, Les Ulis, France). Following
centrifugation at 800 g for 15 min, mononuclear cells were recovered with a
Pasteur pipette from the sample/medium interface, washed twice in three
volumes of BSS, centrifuged at 400 g for 7 min, resuspended in 5 ml BSS
and stored frozen at -80°C.
DNA was purified on hydroxyapatite according to the procedure of
Mailer and Rajewsky (26), with some modifications (7). Frozen tissues were
fragmented in liquid nitrogen in a mortar, transferred as 500 mg aliquots to
50 ml polypropylene tubes containing 5 ml 1% (w/v) sodium dodecylsulfate
and homogenised with a Polytron apparatus (Kinematica, Lucerne,
Switzerland). The homogenate was incubated in presence of 2.5 mg proteinase
K for 1 h at 37°C and 10 h at room temperature. 0.5 mg of RNase (from beef
pancreas; pre-heated for 10 min at 80°C) was added and the mixture was
incubated for 1 h at 37°C. Proteins were extracted with a mixture of 20 ml
1554
of a urea buffer [180 mM sodium phosphate buffer (pH 6.8) containing 5 M
urea and 2 M NaCl] and 25 ml of chloroform/isoamyl alcohol (25:1, v/v).
After centrifugation, the aqueous phase was withdrawn carefully and mixed
with 1.5 g hydroxyapatite (pre-washed in urea buffer at 80°C for 20 min).
The suspension was incubated at room temperature for 30 min under gentle
shaking, centrifuged at 800 g for 2 min, and the supernatant was discarded.
The hydroxyapatite was washed six times with 30 ml of urea buffer, and once
with 30 ml of a 1 mM sodium phosphate buffer (pH 6.8). The DNA was
eluted three times with 3 ml of 0.5 mM sodium phosphate buffer. The
supernatants were pooled and dialysed against distilled water for 12 h. The
purity and concentration of DNA was estimated by the ratio of U V absorbencies
at 260 and 280 nm. 100 ug aliquots were dried by centrifugation under
vacuum with a Speed-Vac concentrator (Savant Instruments Inc., Farmingdale,
NY) and stored at -80°C.
Enzymatic hydrolysis of DNA
Hydrolysis to deoxyribonucleoside 3'-monophosphates was earned out in a
50 mM Tris-HCl buffer (pH 6.8) containing 10 mM CaCl2, micrococcal
nuclease (0.1 U/|ig DNA) and spleen phosphodiesterase (0.05 U/|ig DNA,
dialysed for 6 h against distilled water before use) (7). The reaction mixture
(total volume, 800 (ll) was incubated at 37°C for 4 h. After addition of 2 to
3 volumes of cold ethanol, proteins were precipitated at -20"C for 30 min
and removed by centrifugation. The supernatant was evaporated in a SpeedVac concentrator.
For the analysis by HPLC/RIA, DNA was hydrolysed using DNase I
(50 (ig/mg DNA, 30 min, 37°C), snake venom phosphodiesterase and alkaline
phosphatase (15 ng and 10 U/mg DNA, respectively, 2 h, 37°C), in a 0.05 M
Tris-HCl buffer (pH 6.75) containing 0.01 M MgCI2 (22).
Analysis of3'-edAMP and 3'-edCMP
Following hydrolysis of DNA to 2'-deoxyribonucleoside 3'-monophosphates,
3'-edAMP and 3'-£dCMP were purified by immunoaffinity chromatography
and quantitated by 32P-postlabelling, as described previously (7,24,25).
In brief, immunoaffinity columns retaining specifically 3'-edAMP and 3'edCMP were prepared by coupling monoclonal antibodies EM-A-1 and EMC-1 (21), respectively, to a gel of protein A/Sepharose CL4B (24,25). These
columns were stored at 4°C in phosphate buffered saline (PBS) containing
0.02% (w/v) sodium azide (PBS-azide). Before use, they were washed
sequentially in water (2X5 ml), methanol/water (1:1, v/v; 3X5 ml), water
(3X5 ml) and PBS (2X5 ml). A 175 nl aliquot from each DNA hydrolysate
was loaded onto an EM-A-1 column, another 175 |il aliquot onto an EM-C-1
column, at 4°C. After elution of normal nucleotides in PBS (8X5 ml), the
columns were washed in water (8X5 ml) and brought to room temperature
Etheno adducts were eluted in methanol/water (1:1, v/v; 2X2.5 ml). Eluates
of each sample from the two columns were pooled and dried by centrifugation
under reduced pressure. Dried samples were stored at -80°C. After use,
immunoaffinity columns were washed sequentially in methanol/water (1:1,
v/v; 5 ml), water (5 ml) and PBS-azide (10 ml).
32
P-Postlabelling analysis was carried out as follows (7,24,27). The dried
samples (equivalent to 100 Jig DNA) obtained from the immunoaffinity
columns were labelled in a 10 nl mixture containing 50 mM Tris-HCl
(pH 6.8), 10 mM MgCl2, 10 mM dithiothreitol, 100 amol 3'-dUMP (internal
standard), 10 nCi [f 32P]ATP (final concentration, -200 nM) and 10 U T4
polynucleotide kinase. At the pH used (6.8), T4 polynucleotide kinase
acts simultaneously as a 5'-kinase and 3'-phosphatase, thus converting
deoxyribonucleoside 3'-monophosphates to deoxynbonucleoside 5'-[32P]monophosphates (27,28). A solution of standards containing 100 amol each
of 3'-edAMP, 3'-edCMP and 3'-dUMP was labelled in parallel to the samples.
The reaction mixtures were incubated at 37°C for 90 min, centrifuged and
separated by two-dimensional TLC on PEI-cellulose plates (7,24,27). The
plates were developed in the first direction in 1 M acetic acid (pH 3.5, adjusted
with ammonia) and in the second direction in saturated ammonium sulphate
(pH 3.5, adjusted with H2SO4). After autoradiography, spots corresponding
to 5'-edAMP, 5'-edCMP and 5'-dUMP were cut out and the radioactivity was
determined by liquid scintillation counting.
Normal deoxyribonucleoside 3'-monophosphates in DNA hydrolysates
were quantitated by reverse-phase HPLC onto a Nucleosil Qg column
(4.6 mmX25 cm; particle size, 10 |im; Soci£t£ Francaise Chromato Colonne,
Gagny, France) (7,25). Elution was in 100 mM ammonium formate (pH 6.8)
at a flow rate of 1 rruVmin and quantitation was based on UV absorbance at
254 nm. RNA contamination was also determined on the same chromatograms.
Etheno adduct levels were calculated as molar ratios of adduct to parent
nucleotide in DNA (7). The amounts in amol of etheno nucleotides resolved
on the TLC plates are given by the formula 100X(F2/F,), where Fj and F2
are the ratios of radioactivity (c.p.m.) of the etheno adduct to the internal
standard (5'-dUMP), determined in the standard mixture and in the sample,
respectively. Molar ratios eA/A and eC/C are then obtained by dividing by
the amount of parent nucleotide measured in the HPLC analysis.
DNA ethenobases, formation and accumulation after vinyl chloride exposure
Table L Comparison of two methods for the analysis of eA and eC in DNA
Source of liver DNA
VC-treated ratb
Untreated rat1
Immunoaffinity/32P-postlabelling°
HPLC/RIA1
(eA/AJXlO"8
(eC/QxlO- 8
(eA/AJXlfr 8
(eC/QXlfr 8
9.1,6.0
10.0, 6.7
24.7, 18.3
14.8, 13.0
4.7, 8.2
6.6, 5.4
34.4, 28.9
11.7, 11.0
"Duplicate analysis. Data are expressed as molar ratios of ethenobase to parent base in DNA.
b
Pre-weanling female Sprague-Dawley rat exposed to 600 p.p.m. vinyl chloride (4 h/day) between days 10 and 15 and killed at day 29.
Tre-weanling, 29-day old female Sprague-Dawley rat (housed under same conditions as the VC-treated animal).
RIA analysis of tdAdo and zdCyd
To further validate the immunoaffinity/32P-postlabelling technique, etheno
adducts in DNA were also determined as 2'-deoxyribonucleosides following
HPLC separation and competitive RIA (21,22). DNA hydrolysates were
separated on a Nucleosil C 18 reverse phase column (10 mmX25 cm, 5 u.m)
using a gradient elution previously described (13,22). The eluate was monitored
by UV absorbance at 254 nm and fractions containing individual 2'-deoxyribonucleosides were pooled. Normal nucleosides were quantitated by absorbance
at 260 nm. Fractions corresponding to ethenonucleosides were freeze-dried
and reconstituted in a small volume of RIA buffer. Competitive RIA for
quantitating edAdo and EdCyd were carried out in presence of monoclonal
antibodies EM-A-1 and EM-C-1, respectively, and of [3H]edAdo and
[3H]edCyd as tracers.
Statistics
Statistical analyses of the data were done using the one-way analysis of
variance (ANOVA), the multiple comparison methods of Dunnett and Tukey
and the two-sample Mest The regression lines for the accumulation of etheno
adducts in different organs were compared using the analysis of covanance.
Results
Comparison of two methods for the analysis of eA and eC in
DNA in vivo
The analysis by immunoaffinity/32P-postlabelling developed
previously (7,24,25) was further validated by comparison with
a method based on HPLC pre-separation of the etheno-2'deoxyribonucleosides and quantitation by a competitive RIA
(21,22). Two liver DNA samples from 29-day old rats were
analysed in duplicate. As shown in Table I, both methods
yielded very similar values. Because the dosage by HPLC/
RIA required more than 2 mg of DNA, it was not possible to
carry out a triplicate analysis of these samples.
Reproducibility of the analysis by immunoaffinity/32 F'-postlabelling
For both eA and eC, a coefficient of variation ranging from
9 to 26% was obtained after repeated analyses of liver
DNA samples.
Endogenous formation of etheno adducts in DNA in vivo
The analysis of DNA samples from untreated adult rats (7- to
14-weeks old) revealed the presence of background levels of
ethenobases in all the tissues investigated (Tables II and III).
In the lungs, kidneys and circulating lymphocytes, eA and eC
levels (mean values) ranged from 1.7 to 4.2 and from 4.8
to 11.2 adducts/108 parent bases, respectively. Under these
experimental conditions, the liver exhibited 40 to 100 times
less eA and 70 to 180 times less eC than the other tissues
examined. There were no significant increases in adduct levels
between 7 and 14 weeks of age in any of the tissues examined
in untreated animals. Therefore age-related changes in endogenous levels of etheno adducts over this time period are
unlikely to explain the accumulation of adducts observed
following VC treatment (see below).
Ttoble II. Background levels of eA and eC in DNA from rat tissues"
Liver
Age
(weeks)
7
eA/A
EC/C
14
eA/A
eC/C
0.048
0.069
0.043
0.062
Lungs
±
±
±
±
0.020b
0.012b
0.025b
0.027b
Kidneys
2.05 ± 0.78b
7.25 ± 2 07 b
1.68 ± 0.84c
11.10 ± 1.79°
3.26
8.67
2.91
11.20
±
±
±
±
0.35 b
0.6O>>
0.39°
1.37*
"Male Sprague-Dawley rats (OFA rats, Iffa-Credo, L'Arbresle, France) fed a
standard diet (Biscuits Extra Labo from Soci£t£ Piitrement, Provins,
France). Adduct levels are expressed as molar ratios (X 10~*) of ethenobase
to parent base in DNA.
b
Mean ± SD of four samples.
°Mean ± SD of three samples.
Table m . Analysis of 3' -edAMP and 3'-edCMP in DNA from mononuclear
leucocytes of control and VC-treated rats"
Group
Unexposed
Treated for 4 weeks'1
Treated for 8 weeks'1
nb
6
3
3
Molar ratio X10"8 c
eA/A
eC/C
4.25 ±: 1.16
4.51 ±: 0.25
6.24 ±: 2.05c
4.81 ± 1.43
4.02 ± 0.48
13.27 ± 5.99*
"Male Sprague-Dawley rats, 8- to 14-weeks old, fed a standard pelleted
diet. Unexposed and treated animals were housed under similar conditions
(see Materials and methods).
b
n, number of individual samples analyzed (one analysis per sample).
^ e a n ± SD.
d
500 p.p.m. VC, 4 h/day and 5 days/week. Animals were killed immediately
after the end of exposure.
e
Not significantly different from control values (P > 0.05, two-sample
/-test).
Accumulation of DNA etheno adducts following exposure to
vinyl chloride
The formation and accumulation of ethenobases was analysed
in adult rats exposed to 500 p.p.m. VC for various lengths of
time (Figures 1 to 3). The data points in Figures 1 to 3
represent the mean ± SD (error bars) of 3 to 8 analyses per
group. Three to five individual samples were analysed within
each group, some samples were analysed several times (up to
3). All analyses within a group were combined because
variations between individual samples within a group were
smaller than variations between analyses of the same sample.
An accumulation of both eC and eA was observed in the liver
during exposure (Figure 1). In contrast, in lungs and kidneys,
eC but not eA accumulated in DNA (Figures 2 and 3).
The regression lines representing the accumulation of DNA
ethenobases (eA and eC in liver, Figurel; eC in lungs, Figure 2;
1555
Y.Gulchard et at.
32
12
28 --
O, eA/A; V. eC/C (tr«at«d)
1
1
e ^/A;
^ eC/C (treat • d)
o.
• , eA/A; T , eC/C (controls)
1
r
-
• , e A/A; T , eC/C (cont rols)
24 -
10
20 --
11
1"
r=0.84
o
z
r=0.95
16 --
o 5
3
a
12 -
3
o
I
8 -
o
r=0.95
S
T
0.05
2
1
0.00
-- y 2
i
I
I
i
10
10
LENGTH OF EXPOSURE (WEEKS)
LENGTH OF EXPOSURE (WEEKS)
Fig. 1. Accumulation of eA and eC in liver DNA from adult rats exposed to
500 p.p.m. VC (4 h/day, 5 days/week).
B0
1
1
1
1
O, eA/A; V. eC/C (tr«ot«d)
70 --
-*
• , eA/A; T . eC/C (controls)
K
w
r=0.65
50 -
i
2
o
i
60 -
o
o
40 -
i
7
30 I
5 1
o °-
20 -
I
o
1r
- ¥
10
I
0
5
i
2
4
I
a
o
i
*
6
8
10
LENGTH OF EXPOSURE (WEEKS)
Fig. 2. Accumulation of eA and eC in lung DNA from adult rats exposed to
500 p.p.m. VC (4 h/day, 5 days/week).
eC in kidneys, Figure 3) were obtained using first order linear
regression. The P values were =£ 0.001. Curve fitting was not
significantly improved after transformation (e.g. log transformation) of the data. The following mean values were
computed for the slopes, expressed as (molar ratio of ethenobase to parent base)X10~8X(week)"1 : eA in liver, 0.682; eC
in liver, 0.809; eC in kidneys, 1.12; eC in lungs, 3.87.
Comparison of the regression curves by the analysis of
covariance showed that they were all significantly different
1556
Fig. 3. Accumulation of eA and eC in kidney DNA from adult rats exposed
to 500 p.p.m. VC (4 h/day, 5 days/week).
from each other (P < 0.0001; Table IV). However, the rates
of accumulation (slopes) of eA and eC in liver, and of eC in
kidneys, were not very significantly different (fluctuation due
to lack of parallelism, F2; 49 = 3.30, P = 0.045). The
accumulation rate of eC was higher in the lungs as compared
to the kidneys (P = 0.035) and liver (P = 0.012).
Levels of eA found in lung DNA after various periods of
exposure to VC were compared by ANOVA and Dunnett's
test, using for the latter the control values measured in 7-week
old animals. eA levels were significantly different from controls
after 2, 4 or 8 weeks (P < 0.001) but not after 1 week (P >
0.05) of treatment. Levels measured between 2 and 8 weeks
of exposure were not different (Tukey's test, P > 0.05),
suggesting a steady state in the formation of eA after 2 weeks.
Several samples of mononuclear leucocytes from rats
exposed to VC for 4 or 8 weeks were analysed (Table III). As
compared to controls, no significant increase of the levels of
eA and eC was observed after 8 weeks of treatment (P >
0.05, Student's Mest), with this limited number of analyses.
Persistence of DNA etheno adducts in liver
The persistence of eA and eC in liver DNA was investigated
in rats following a 5 day exposure to 500 p.p.m. VC. Adduct
levels measured immediately after the treatment were: eA/
A = (0.71 ± 0.06) X10"8, eC/C = (2.86 ± 0.30) X10"8
(n = 3). After 2 months, they were not significantly different
(P > 0.05, Student's Mest): eA/A = (0.68 ± O.17)X1O-*, eC/
C = (2.34 ± 0.24)X10-8 (n = 3). These molar ratios being
significantly higher than in 14-week old controls (P < 0.05),
where eA/A = (0.043 ± 0.025) X10"8 and eC/C = (0.062 ±
0.027) X10"8 (n = 4) (Table II), the present data suggest that
ethenobases formed during exposure to VC are persistent in
hepatic DNA.
Discussion
The aim of this study was to measure the formation of
ethenobases in DNA from adult rats exposed to VC. Previously,
DNA ethenobases, formation and accumulation after vinyl chloride exposure
Table IV. Analysis of covariance of the accumulation of DNA etheno adducts in adult rats exposed to VC
Comparison"
eA
eC
eA
eA
eC
eC
(liv), eC (liv)
(liv), eC (kid)
0iv), eC (kid)
(liv), eC (liv), eC (kid)
(lun), eC Giv)
(lun), eC (kid)
Fluctuation F (P value)
Between organs and/or adducts
Parallelism6
F, 34 = 48.99 (<0.0001)
F
Z 3 o = 94.17 (<0.0001)
F Z 3 0 = 170.79 (<0.0001)
F 4 47 = 123.79 (<0.0001)
F Z 4 , = 26.07 (<0.0001)
F, 37 =
9.95 (0.0003)
F, 35 =
F,; 3 1 =
F, 31 =
F^ 49 =
F, 42 =
F,' 3g =
2.03 (0.16)
2.36(0.13)
4.54 (0.041)
3.30 (0.045)
6.91 (0.012)
4.76 (0.035)
*Liv, liver, kid, kidneys; lun, lungs
''Fluctuation between the slopes of the regression lines.
this type of study was only feasible in pre-weanling animals,
which exhibit higher levels of VC-DNA adducts than adults
(20,22). The ultra-sensitive immuno-affinity/32P-postlabelling
method for measuring 3'-edAMP and 3'-edCMP residues in
DNA (7,24,25) was further validated by comparison with an
independent method based on HPLC separation and competitive RIA of the corresponding ethenodeoxyribonucleosides
(21,22). As shown in Table I, both methods yielded very
similar values. The immunoaffinity/32P-postlabelling method
also exhibited good reproducibility.
Using this new method, the formation of ethenobases was
investigated in DNA from adult Sprague-Dawley rats exposed
to 500 p.p.m. VC (4 h/day, 5 days/week) for up to 8 weeks.
Accumulation curves were established for eA and eC in liver
DNA (Figure 1), lung DNA (Figure 2) and kidney DNA
(Figure 3). All these curves were significantly different, as
demonstrated by analysis of covariance of the regression lines
(Table IV). Both ethenobases accumulated in liver DNA and
eC, but not eA, accumulated in lung and kidney DNA during
exposure to VC. These data could suggest a poor repair of eA
and eC in liver and of eC in lungs and kidneys, under these
experimental conditions, an explanation supported by the
apparent persistence of eA and eC in liver DNA after the end
of exposure. These data are in apparent contradiction with
in vitro experiments showing an efficient release of ethenobases
from DNA by a mammalian repair enzyme, the human
N-alkylpurine-DNA glycosylase (29) or in presence of cell
homogenates of mononuclear blood cells from humans (30).
More recently, it was found that eA and eC are excised by
separate human DNA glycosylases, purified from HeLa cellfree extracts (31). This raises the question of whether these
separate repair activities could explain the differential accumulation of eA and eC observed in DNA from lungs (Figure 2)
and kidneys (Figure 3) of adult rats exposed to VC.
However, the interpretation of the observed accumulation
and persistence of etheno adducts in DNA is not straightforward, due to the existence of a natural background (Tables
II and III); this background level varies significantly in rat
liver and can be higher than reported in Table II and Fig. 1
(7). Several lines of evidence point to lipid peroxidation
products, presumably mwu-4-hydroxy-2-nonenal, as a possible
endogenous source of this background (3-6) which was also
observed in human liver samples and mouse tissues (2,7). In
rodents, background levels of DNA ethenobases seem to vary
with diet (7,32). Possibly they can be affected by exposure to
environmental agents as oxidative metabolism of xenobiotics
is often accompanied by an oxidative stress and stimulation
of lipid peroxidation (33-36). Reactive oxygen species are
produced as a result of uncoupling of cytochrome P450catalysed monooxygenase reactions (33), the cytochrome P450
2E1 isoenzyme appearing as particularly effective in releasing
such species and in inducing lipid peroxidation (34). Treatment
of rats with A^-nitrosoamines or aflatoxin B| leads to an
enhanced pro-oxidant state and stimulation of lipid peroxidation, these effects appear to persist long after the treatment
(35,36). In Sprague-Dawley rats (unpublished results), and
humans (37), VC is mainly oxidised by cytochrome P450 2E1,
yielding the ultimate carcinogenic metabolite, chloroethylene
oxide, which rearranges spontaneously into 2-chloroacetaldehyde. 2-Chloroacetaldehyde has been shown to stimulate
lipid peroxidation in isolated rat hepatocytes (38). These data
suggest that exposure to VC could be associated with a
stimulation of lipid peroxidation in experimental animals.
Therefore, the accumulation and persistence of ethenobases in
DNA from rats exposed to VC could result both from a
direct pathway involving chloroethylene oxide (39—41) and an
indirect pathway involving lipid peroxidation products. Our
previous data suggesting the persistence of ethenobases in
liver DNA from pre-weanling rats after exposure to VC (20)
also need to be reinterpreted in view of this endogenous
formation. In fact, more recent analyses with the newly
developed techniques indicated the presence of ethenobases in
liver DNA from unexposed pre-weanling rats (Table I).
The existence of a natural background in DNA will hamper
the dosimetry of ethenobases at low levels of exposure to VC
(or to other etheno adduct-forming carcinogens). In addition
our analyses failed to reveal increased levels of DNA etheno
adducts in easily accessible tissues, such as mononuclear blood
cells, after prolonged inhalation exposure of rats to a high
concentration of VC. Thus, biomonitoring of human exposure
to VC on the basis of DNA etheno adducts in lymphocytes
may not be feasible.
It has been suggested that ethenobases are critical promutagenic lesions involved in chemical carcinogenesis (2). Our
data do not show a correlation between total etheno adduct
levels and organospecificiry of VC-induced carcinogenesis: the
liver, the main target organ in Sprague-Dawley rats (42), did
not exhibit higher levels of etheno adducts after VC exposure
than the other tissues examined (Figures 1-3). However, the
accumulation of eA in liver DNA (Figure 1) could be implicated
in VC-induced hepatocarcinogenesis. This accumulation was
not observed in organs less susceptible to tumorigenesis such
as lungs and kidneys (Figures 2 and 3). Furthermore, VCinduced liver tumours in rats exhibit point mutations at AT
base pairs in the Ha-ras (43) or p53 (unpublished results)
1557
Y.Gulchard et al.
genes, suggesting the involvement of eA as a promutagenic
lesion (9).
Further studies on the formation and repair of DNA ethenobases during VC-induced carcinogenesis are needed in order
to elucidate the contribution from endogenous sources and the
cell specificity of these processes.
Acknowledgements
This work was supported in part by contracts with INSERM (Lyon) and Elf
Atochem (Paris), the Weisbrcm-Benenson Foundation (Paris), the European
Union (EV5V-CT94-O4O9) and the NIEHS (Grant ES05948). The authors
wish to thank Dr J.Esteve for advice on statistical analyses, and Ms Z.Schneider
for typing this manuscript.
References
l.Leonard.NJ. (1992) Etheno-bridged nucleotides in structural diagnosis and
carcinogenesis. Chemtracts-Biochem. Mol. Biol, 3, 273-297.
2.Bartsch,H., Barbin.A., Marion,M.-J., NairJ. and Guichard.Y. (1994)
Formation, detection and role in carcinogenesis of ethenobases in DNA.
Drug Metab. Rev., 26, 349-371.
3.Sodum,R.S. and Chung,F.-L. (1991) Stereoselective formation of in vitro
nucleic acid adducts by 2,3-epoxy-4-hydroxynonanal. Cancer Res., 51,
137-143.
4.Barbin,A., El Ghissassi.F., NairJ. and Bartsch,H. (1993) Lipid peroxidation
leads to formation of iyv*-ethenoadenine and 3^V4-ethenocytosine in DNA
bases. Proc. Am. Assoc. Cancer Res., 34,136.
5.Chen,H.-J.C. and Chung,F.-L. (1994) Formation of etheno adducts in
reactions of enals via autoxidation. Chem. Res. Toxicol., 7, 857-860.
6. El Ghissassi.F., Barbin.A., NairJ. and Bartsch.H. (1995) Formation of
1 .A^-ethenoadenine and S^'-ethenocytosine by lipid peroxidation products
and nucleic acid bases. Chem. Res. Toxicol, 8, 278-283.
7.Nair,J., Barbin.A., GuichanLY and Bartsch.H. (1995) l^-Ethenodeoxyadenosine and 3/i^-ethenodeoxycvtidine in liver DNA from humans
and untreated rodents detected by immunoaffinity/32P-postlabelling.
Carcinogenesis, 16, 613-617.
8.Cheng,K.C, Preston,B.D., Cahill.D.S., Dosanjh,M.K., Singer.B. and
Loeb.L.A. (1991) The vinyl chloride derivative, iV2,3-ethenoguanine,
produces G—>A transitions in Escherichia colt. Proc. NatlAcad. Sci. USA,
88, 9974-9978.
9. Basu,A.K., Wood.M.L., Neidernhofer.LJ., Ramos,L.A. and Essigmann,
J.M. (1993) Mutagenic and genotoxic effects of three vinyl chlondeinduced DNA lesions: 1 JV*ethenoadenine, S^-ethenocytosine and
4-amino-5-(imidazol-2-yl)imidazole. Biochemistry, 32, 12793-12801.
10.Palejwala,V.A., Rzepka,R.W., Simha,D. and Humayun.M.Z. (1993)
Quantitative multiplex sequence analysis of mutational hot spots.
Frequency and specificity of mutations induced by a site-specific
ethenocytosine in M13 viral DNA. Biochemistry, 32, 4105-4111.
ll.Moriyaid., Zhang.W., Johnson.F. and Grollman,A.P. (1994) Mutagenic
potency of exocyclic DNA adducts: marked differences between
Escherichia coli and simian kidney cells. Proc. NatlAcad. Sci. USA, 91,
11899-11903.
12,Grollman,A.P. and Shibutani.S. (1994) Mutagenic specificity of chemical
carcinogens as determined by studies of single DNA adducts. In
Hemminki,K., DippleA, Shuker,D.E.G., Kadlubar.F.F., Segerback,D. and
Bartsch,H. (eds), DNA adducts: Identification and Biological Significance.
IARC Scientific Publications no. 125. IARC, Lyon, pp. 385-397.
13.Barbin,A., Laib.RJ. and Bartsch.H. (1985) Lack of miscoding properties
of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA adduct. Cancer
Res., 45, 2440-2444.
14.Osterman-Golkar,S., Hultmark,D., Segerback,D., Calleman,CJ., GOthe.R.,
Ehrenbergl. and Wachtmeister,C.A. (1977) Alkylation of DNA and
proteins in mice exposed to vinyl chloride. Biochem. Biophys. Res.
Commun., 76, 259-266.
15.Laib,RJ., Gwinner.L.M. and Bolt.H.M. (1981) DNA alkylation by vinyl
chloride metabolites: etheno derivatives or 7-alkylation of guanine? Chem. Biol. Interactions, 37, 219-231.
16.Green,T. and Hathway.D.E. (1978) Interactions of vinyl chloride with rat
liver DNA in vivo. Chem. -Biol. Interactions, 22, 211-224.
17.Laib,RJ., Doerjer,G. and Bolt.H.M. (1985) Detection of JV2,3ethenoguanine in liver-DNA hydrolysates of young rats after exposure of
the animals to I')C-vinyl chloride. J. Cancer Res. Clin. Oncol., 109, A7.
18.Fedtke,N., Boucheron,J.A.,Tumer,MJ.,Jrand SwenbergJA. (1990a) Vinyl
chloride-induced DNA adducts. I: Quantitative determination of N2,3-
1558
ethenoguanine based on electrophore labeling. Carcinogenesis, 11,
1279-1285.
19. Fedtke,N., BoucheronJ.A., Walker,V.E. and SwenbergJ.A. (1990b) Vinyl
chloride-induced DNA adducts. 11: Formation and persistence of 7-(2'oxoethyl)guanine and N2,3-ethenoguanine in rat tissue DNA.
Carcinogenesis, 11, 1287-1292.
20. SwenbergJA., Fedtke.N., Ciroussel,F, Barbin.A. and Bartsch.H. (1992)
Etheno adducts formed in DNA of vinyl chloride-exposed rats are highly
persistent in liver. Carcinogenesis, 13, 727-729.
21.Eberle,G., Barbin.A., Laib.RJ., Ciroussel.E, ThomaleJ., Bartsch.H. and
Rajewsky,M.F. (1989) lA*-etheno-2'-deoxyadenosine and 3^-etheno2'-deoxycytidine detected by monoclonal antibodies in lung and liver
DNA of rats exposed to vinyl chloride. Carcinogenesis, 10, 209-212.
22.Ciroussel,F., Barbin.A., Eberle.G. and Bartsch.H. (1990) Investigations on
the relationship between DNA ethenobase adduct levels in several organs
of vinyl chloride-exposed rats and cancer susceptibility. Biochem.
Pharmacol, 39, 1109-1113.
23.Laib,R.J., Cartier.R., Bartsch,H. and Bolt.H.M. (1983) Influence of age on
induction of preneoplastic foci and on alkylation of rat liver DNA by
vinyl chloride (VQ. /. Cancer Res. Clin. Oncol., 105, A21.
24.Guichard,Y, NairJ., Barbin.A. and Bartsch.H. (1993) Immunoaffinity
clean-up combined with 32P-postlabelling analysis of 1 .A^-ethenoadenine
and S^-ethenocytosine in DNA. In Phillips.D.H., Castegnaro,M. and
Bartsch.H. (eds), Postlabelling Methods for Detection of DNA Adducts.
IARC Scientific Publications no. 124, IARC, Lyon, pp. 263-269.
25. Guichard.Y. (1994) Development of an ultra-sensitive method for the
analysis of ethenobases in DNA. Application to the study of their role in
vinyl chloride carcinogenesis. Ph.D. thesis, Lyon, France.
26.MUller,R. and Rajewsky.M.F. (1980) Immunological quantification by
high affinity antibodies of O*-ethyldeoxyguanosine in DNA exposed to
W-ethylnitrosourea. Cancer Res., 40, 887-8%.
27. Hollstein,M., NairJ., Bartsch.H., Bochner.B. and Ames.B.N. (1986)
Detection of DNA base damage by 32P-postlabelling: TLC separation of
5'-deoxynucleoside monophosphates. In Singer.B. and Bartsch,H. (eds),
The Role of Cyclic Nucleic Acid Adducts in Carcinogenesis and
Mutagenesis. IARC Scientific Publications no. 70, IARC, Lyon, pp.
437-448.
28.Cameron,V. and Uhlenbeck.O.C. (1977) 3'-Phosphatase activity in T4
polynucleotide kinase. Biochemistry, 16, 5120-5126.
29.Dosanjh,M.K., ChennaA, Kim.E., Fraenkel-Conrat,H., SamsonJ^. and
Singer.B. (1994) All four known cyclic adducts formed in DNA by the
vinyl chloride metabolite chloroacetaldehyde are released by a human
DNA glycosylase. Proc. Natl. Acad. Sci. USA, 91, 1024-1028.
30.Oesch,F, Weiss,C.-M. and Klein,S. (1994) Use of oligonucleotides
containing ethenoadenine to study the repair of this DNA lesion.
Determination of individual and collective repair activities. Arch. Toxicol,
68, 358-363.
31.Hang,B., Chenna,A., Rao,S. and Singer^- (1996) 1 ^-Ethenoadenine and
3^*-ethenocytosine are excised by separate human DNA glycosylases.
Carcinogenesis, 17, 155-157.
32.Fernando,R.C, NairJ., Barbin.A., MillerJ.A. and Bartsch.H. (1996)
Detection of 1 .A^-ethenodeoxyadenosine and S^-ethenodeoxycytidine
by immunoaffinity/32P-postlabelling in liver and lung DNA of mice treated
with ethylcarbamate (urethane) or its metabolites. Carcinogenesis, 17,
1711-1718.
33. KanizinaJ.I. and Archakov.A.I. (1994) The oxidative inactivation of
cytochrome P450 in monooxygenase reactions. Free Radical Biol. Med.,
16, 73-97.
34.Ekstr0m,G. and Ingerman-Sundberg,M. (1989) Rat liver microsomal
NADPH-supported oxidase activity and lipid peroxidation dependent on
ethanol-inducible cytochrome P-450 (P-450 IIE1). Biochem. Pharmacol,
38, 1313-1319.
35.Hietanen.E., Bartsch.H., Ahotupa,M., BereziaU-C., Bussacchini-Griot,V,
CabraU.R., Camus,A.-M., LaiUnenJvl. and Wild.H. (1991) Mechanisms
of fat-related modulation of /V-nitrosodiethylamine-induced tumors in
rats: organ distribution, blood lipids, enzymes and pro-oxidant state.
Carcinogenesis, 12, 591-600.
36.Shen,H.-M., Shi,C.-Y, Lee,H.-P. and Ong,C.-N. (1994) Aflatoxin B,induced lipid peroxidation in rat liver. Toxicol Applied Pharmacol, 127,
145-150.
37.Guengerich,F.P., Kim,D.-H. and Iwasaki.M. (1991) Role of human
cytochrome P-450 HE 1 in the oxidation of many low molecular weight
cancer suspects. Chem. Res. Toxicol, 4, 168—179.
38.Sood,C. and O'BrienJU. (1993) Molecular mechanisms of chloroacetaldehyde-induced cytotoxicity in isolated rat hepatocytes. Biochem,
Pharmacol, 46, 1621-1626.
DNA ethenobases, formation and accumulation after vinyl chloride exposure
39. Barbin.A., Br6sil,H., Croisy^A., Jacquignon,P., Malaveille,C, MontesanoJ*.
and Bartschji. (1975) Liver-microsorae-raediated formation of alkylating
agents from vinyl bromide and vinyl chloride. Biochem. Biophys. Res.
Commun., 67, 596-603.
40. Guengerich,F.P. (1992) Roles of vinyl chloride oxidation products 1chlorooxirane and 2-chloroacetaldehyde in the in vitro formation of etheno
aducts of nucleic acid bases. Chem. Res. Toxicol., 5, 2—5.
41.Bolt,H.M. (1994) Vinyl halides, haloaldehydes and monohaloalkanes. In
Hemroinki.K., Dipple.A., Shuker.D.E.G., Kadlubar^.E, Segerbflck,D. and
Bartsch,H. (eds), DM4 adducts: Identification and Biological Significance.
IARC Scientific Publications no. 125, IARC, Lyon, pp. 141-150.
42.Maltoni,C, Lefemine.G., Ciliberti.A., Cotti.G. and Caretti,D. (1984)
Experimental research on vinyl chloride carcinogenesis. In Maltoni.C. and
Mehlman,M.A. (eds), Archives of Research on Industrial Carcinogenesis.
vol. 2, Princeton Scientific Publishers Inc., Princeton.
43.Froment,O., Boivin,S., Barbing., Bancel,B., Tr6po,C. and Marion>IJ.
(1994) Mutagenesis of ras proto-oncogenes in rat liver tumors induced by
vinyl chloride. Cancer Res., 54, 5340-5345.
Received on March 8, 1996; revised on May 24, 1996; accepted on May
29, 1996
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