Activation of propane 2-nitronate to a genotoxicant in V79-derived cell lines engineered for the expression of rat hepatic sulfotransferases

Article abstract of DOI:10.1016/S1383-5718(98)00194-6

2-Nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound has been attributed to a sulfotransferase-mediated formation of DNA-reactive species from the anionic form of 2-NP, propane 2-nitronate (P2N). Several observations have suggested that sulfotransferases (SULTs) 1A1 and/or 1C1 may be important in the activation of P2N to a genotoxicant in rat liver, but a definite proof is lacking. In order to identify the sulfotransferase(s) of rat liver that are capable of activating P2N, we have investigated the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of rat hepatic sulfotransferases. Genotoxicity was assessed by measuring the induction of DNA repair synthesis. 1-Hydroxymethylpyrene (HMP), which is metabolically activated by most sulfotransferases, served as a positive control. Neither P2N nor HMP induced DNA repair in the parental V79-MZ cells, which do not show any sulfotransferase activity. P2N was also inactive in V79-rHSTa and V79-rHST20 cells, which express specific hydroxysteroid sulfotransferases. By contrast, a clear and concentration-dependent induction of repair synthesis by P2N was observed in V79-rPST-IV and V79-rST1C1 cells, which express rat SULT1A1 and SULT1C1, respectively. HMP was genotoxic in all sulfotransferase-expressing cell lines. Acetone oxime (AO), the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results corroborate the essential role of sulfotransferases in the metabolic activation of P2N to genotoxic products and identify two rat sulfotransferases which are capable of catalyzing the activation step.

Full text of DOI:10.1016/S1383-5718(98)00194-6

Ž
.
Mutation Research 439 1999 191–197
Activation of propane 2-nitronate to a genotoxicant in
V79-derived cell lines engineered for the expression of rat hepatic
sulfotransferases
a
b
c
Ulrich Andrae a,), Patricia Kreis , Michael W.H. Coughtrie , Ulrike Pabel ,
c
d
c,d
Walter Meinl , Ingrid Bartsch , Hansruedi Glatt
GSF-National Research Center for EnÕironment and Health, Institute of Toxicology, D-85764 Neuherberg, Germany
Department of Molecular and Cellular Pathology, UniÕersity of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, UK
a
b
c
(
)
German Institute of Human Nutrition DIfE , Potsdam-Rehbrucke, Germany
¨
d
Department of Toxicology, UniÕersity of Mainz, Mainz, Germany
Received 22 October 1998; accepted 22 December 1998
Abstract
Ž
.
2-Nitropropane 2-NP is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound has been attributed to
Ž
.
a sulfotransferase-mediated formation of DNA-reactive species from the anionic form of 2-NP, propane 2-nitronate P2N .
Ž
.
Several observations have suggested that sulfotransferases SULTs 1A1 andror 1C1 may be important in the activation of
Ž .
P2N to a genotoxicant in rat liver, but a definite proof is lacking. In order to identify the sulfotransferase s of rat liver that
are capable of activating P2N, we have investigated the genotoxicity of P2N in various V79-derived cell lines engineered for
expression of individual forms of rat hepatic sulfotransferases. Genotoxicity was assessed by measuring the induction of
Ž
.
DNA repair synthesis. 1-Hydroxymethylpyrene HMP , which is metabolically activated by most sulfotransferases, served as
a positive control. Neither P2N nor HMP induced DNA repair in the parental V79-MZ cells, which do not show any
sulfotransferase activity. P2N was also inactive in V79-rHSTa and V79-rHST20 cells, which express specific hydroxysteroid
sulfotransferases. By contrast, a clear and concentration-dependent induction of repair synthesis by P2N was observed in
V79-rPST-IV and V79-rST1C1 cells, which express rat SULT1A1 and SULT1C1, respectively. HMP was genotoxic in all
Ž
.
sulfotransferase-expressing cell lines. Acetone oxime AO , the tautomeric form of the first reduction product of 2-NP,
2-nitrosopropane, was inactive in all cell lines. The results corroborate the essential role of sulfotransferases in the metabolic
activation of P2N to genotoxic products and identify two rat sulfotransferases which are capable of catalyzing the activation
step. q 1999 Elsevier Science B.V. All rights reserved.
Keywords: 2-Nitropropane; Propane 2-nitronate; V79 cell; Sulfotransferase; Genotoxicity
Abbreviations: AO, acetone oxime; BrdUrd, 5-bromo-2X-deoxyuridine; dCyd, deoxycytidine; DMSO, dimethyl sulfoxide; FdUrd,
5-fluoro-2X-deoxyuridine; HMP, 1-hydroxymethylpyrene; 2-NP, 2-nitropropane; P2N, propane 2-nitronate
)
Corresponding author. Tel.: q49-89-31872221; Fax: q49-89-31873449; E-mail: andrae@gsf.de
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
Ž
.
PII: S1383-5718 98 00194-6

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1. Introduction
in the presence of the nitronates of 2-NP and other
w
x Ž .
secondary nitroalkanes in vitro 4,7 ,
‘aryl sulfotransferase IV’ substrate N-hydroxy-2-
c that the
Ž
.
2-Nitropropane 2-NP is an important industrial
w
x
chemical, which is hepatocarcinogenic in rats 1,2 .
The carcinogenicity of the compound has been sug-
gested to be the consequence of DNA damage in-
duced by reactive metabolites formed from 2-NP in
the liver 3 . Sodum et al.
activation of 2-NP occurs via a sulfotransferase-
mediated pathway, which was postulated to convert
the anionic form of 2-NP, propane 2-nitronate P2N ,
into an aminating nitrenium ion through the interme-
diate formation of acetoxime-O-sulfonate and hy-
droxylamine-O-sulfonate Fig. 1 . 8-Aminoguanine
and 8-oxoguanine were identified as products of the
reaction of the aminating species with hepatic DNA
acetylaminofluorene inhibited the latter reaction, and
d that treatment of guanosine and tyrosine with
acetoxime-O-sulfonate and hydroxylamine-O-
sulfonate in vitro resulted in the production of 8-
aminoguanosine, N1-aminoguanosine, 8-oxoguano-
sine, 3-aminotyrosine and 3-hydroxytyrosine, respec-
Ž .
w x
w x
4 showed that the
w x
tively 6 . Further support for an essential role of
Ž
.
‘aryl sulfotransferase IV’ in the metabolic activation
of 2-NP was deduced from the findings that male
rats, which express higher activities of ‘aryl sulfo-
transferase IV’ than female rats 8 , show higher
levels of DNA modifications following exposure to
Ž
.
w x
w x
2-NP 9 and are more susceptible to the carcino-
genicity of the compound 10 than females.
w x
and RNA in vivo 5 , and 3-aminotyrosine was de-
w
x
w x
tected in liver proteins 6 . The crucial role of sulfo-
During the last decade, several breakthroughs have
been achieved in understanding the molecular basis
of sulfotransferase heterogeneity. The xenobiotic-
metabolizing sulfotransferases are cytosolic enzymes
encoded by a superfamily of genes 11–13 . A re-
vised, official nomenclature for cytosolic sulfotrans-
ferases, using SULT as the gene symbol, is currently
being devised R. Weinshilboum et al., in prepara-
tion . The genesrgene products of the SULT super-
family are classified into families e.g., SULT1,
Ž .
transferase s in the metabolic activation of 2-NP
was inferred from the findings a that the sulfotrans-
ferase inhibitors pentachlorophenol and 2,6-dichloro-
4-nitrophenol inhibited the 2-NP-induced formation
of DNA modifications in rat liver 4 ,
preparation of partially purified ‘aryl sulfotransferase
IV’ from rat liver catalyzed the production of 8-
aminoguanosine and 8-oxoguanosine from guanosine
Ž .
w
x
w x Ž .
b
that a
Ž
.
Ž
.
Ž
Ž
.
SULT2 , subfamilies e.g., SULT1A, SULT1C and
.
individual enzymes e.g., SULT1A1, SULT1C1 .
Most sulfotransferase enzymes are homodimers of
these gene products, but rat SULT1A1 and 1C1 may
w
x
also form heterodimers 14 . They are therefore diffi-
cult to separate from preparations of tissues, such as
the liver, in which both genes are expressed. The
name ‘arylsulfotransferase IV’ was initially used for
w
x
a purified enzyme 15,16 and was later associated
Ž
with a cDNA-expressed protein now termed ‘rat
.
SULT1A1’ which appeared to correspond to the
w
x
purified, hepatic ‘arylsulfotransferase IV’ 17,18 .
However, later it was detected that a characteristic
substrate of ‘arylsulfotransferase IV’ is only ineffi-
ciently conjugated by expressed rat SULT1A1 and
that another enzyme from male rat liver, SULT1C1,
w
x
shows very high activity with this substrate 19 .
Therefore, ‘hydroxamic acid sulfotransferase’ is
sometimes used as the trivial name for rat SULT1C1.
It is difficult to judge the molecular composition of
purified ‘arylsulfotransferase IV’ prepared in earlier
Fig. 1. Mechanism of the activation of 2-NP as proposed by
w
x
Sodum et al. 4,5 .

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U. Andrae et al.rMutation Research 439 1999 191–197
193
Ž
.
studies. It is likely that most of them were mixtures
of SULT1A1 and 1C1.
Aldrich Steinheim . All other chemicals were of the
highest purity available.
SULT1A1 and 1C1 show male-dominant expres-
w
x
sion in the liver of adult rats 20 . However, the
sexual dimorphism is much higher for SULT1C1
than for SULT1A1. Western blot analysis indicates
that the levels of SULT1A1 and 1C1 proteins are
about 2.0 and at least 20 times higher in the liver of
2.2. Cells
The construction and applications of the cell lines
V79-rHSTa clone V79-STa-2
Ž
.
w
x
30–33 , V79-
Ž
rHST20 preliminary clone V79-rHST20-1, showing
Ž
. w
x
adult male Wistar rats than in female rat liver W.
Meinl and H.R. Glatt, unpublished result .
a relatively low expression level 32 and V79-
.
w
x
rST1C1 32 have been reported previously. Cell line
Ž .
Ž
In order to identify rat hepatic sulfotransferase s
V79-rPST-IV expressing rat SULT1A1 isolated from
.
Ž
capable of activating 2-NPrP2N to a genotoxicant,
we have assessed the genotoxicity of P2N in various
V79-derived cell lines engineered for expression of
individual sulfotransferases from rat liver. Genotoxi-
city was determined by measuring the capacity of
P2N to induce DNA repair synthesis. So far, 2-
NPrP2N has elicited DNA repair only in rat hepato-
cytes 3,7,21–24 , certain differentiated rat hepatoma
cell lines 25,26 and cultured ovine seminal vesicle
cells 27 . All these cells are known to contain
sulfotransferase activity. In parental V79 cells, which
do not show any xenobiotic-metabolizing sulfotrans-
a rat liver cDNA library was constructed using the
same expression vector pMPSV and very similar
.
w
x
x
w
w
x
w
x
ferase activities 28,29 , 2-NPrP2N does not induce
w
x
repair 3,25 . Thus, the induction of DNA repair
synthesis by P2N in a V79 line engineered to express
a sulfotransferase would clearly indicate the ability
of this enzyme to activate 2-NPrP2N.
Since the proposed mechanism of the metabolic
w
x
activation of 2-NP 4,5 involves initial deoxygena-
tion and sulfonation steps which result in the forma-
tion of acetoxime-O-sulfonate as an intermediate
Ž
.
Fig. 1 , we also investigated whether acetone oxime
Ž
.
AO might be a substrate for the sulfonation and
induce DNA damage.
2. Materials and methods
Fig. 2. DNA repair synthesis in parental V79 cells exposed to
Ž
.
P2N, AO and HMP. Cells clone V79-MZ were preincubated
2.1. Chemicals
Ž
.
Ž
.
with FdUrd 2 mM and BrdUrd 10 mM for 1 h. Subsequently
they were treated with the test compounds in the presence of
w3
x
Ž
Ž
.
2-NP CAS no. 79-46-9; purity )99% was do-
Ž
.
.
FdUrd, BrdUrd, hydroxyurea 2 mM and H dCyd 10 mCi
mly1 for 5 h. Controls Con were exposed to medium only.
Ž
.
Ž
nated by Angus-Chemie Ibbenburen-Uffeln, Ger-
many . P2N was prepared by mixing equal amounts
of 2-NP and NaOH in water and allowing the mix-
ture to stand at room temperature for 30 min. AO
¨
Ž
.
Repair synthesis in these cells s100% corresponded to 65"8
.
y2
cpm mgy1 DNA. Irradiation with UV light 20 J m
Ž
.
immedi-
ately after preincubation increased repair synthesis to 2640"185%
of the control value. Numbers below the abscissa indicate the
concentrations of the test compounds in mM. Results are the
means"SEM from 3 independent experiments.
Ž
.
Ž
.
98% was purchased from Riedel-de Haen Seelze ,
. Ž
Ž
.
and 1-hydroxymethylpyrene HMP 98% from

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U. Andrae et al.rMutation Research 439 1999 191–197
After preincubation the medium was removed and
the cells were provided with fresh medium addition-
3
ally containing 10 mCi mly1 5- H deoxycytidine
w
x
Žw3
x
.
y1
H dCyd, 25.6 Ci mmol ; NEN , 2 mM hydro-
Ž
. w x
xyurea Sigma 36 and the test compound. P2N and
AO were dissolved in medium, HMP in dimethyl
Ž
.
sulfoxide DMSO . The final DMSO concentration
in the medium was 0.5%. For UV irradiation, cells
were rinsed with phosphate buffered saline, drained,
y2
and irradiated with UV light 254 nm, 1 J m sy1
Ž
.
.
After 5 h of incubation the DNA was isolated and
Ž
.
unreplicated ‘light’ DNA strands were separated
Ž
.
from replicated, density-labeled ‘heavy’ strands by
equilibrium centrifugation in alkaline CsClrCs2 SO4
gradients. Repair synthesis was determined by mea-
suring the incorporation of radioactivity into unrepli-
cated DNA strands and expressed as cpm mgy1
DNA.
Fig. 3. DNA repair synthesis in V79-rHSTa cells exposed to P2N,
AO and HMP. See legend to Fig. 2. Repair synthesis in untreated
cells was 82"7 cpm mgy1 DNA. Irradiation with UV light 20 J
Ž
my2 immediately after preincubation increased repair synthesis
.
to 2300"970% of the control value. )), significantly different
from the control; P-0.01.
Ž
methods U. Pabel, W. Meinl, M. Hagen, M.W.H.
.
Coughtrie and H.R. Glatt, manuscript in preparation .
Cells were grown as monolayer cultures in Dul-
Ž
.
becco’s modification of Eagle’s medium from PAA
Ž
supplemented with 10% fetal calf serum Gibcor
y1
.
BRL , 100 U ml
penicillin and 100 mg mly1
Ž
.
streptomycin Biochrom at 378C in a humidified
atmosphere of 93% airr7% CO2.
2.3. DNA repair synthesis
DNA repair synthesis in V79 cells was deter-
X
Ž
.
mined using the 5-bromo-2 -deoxyuridine BrdUrd
w
x
density-shift method 34 with the modifications de-
scribed 3,35 . Cells were preincubated in 5 ml Basal
medium Earle Biochrom supplemented with 20
Fig. 4. DNA repair synthesis in V79-rHST20 cells exposed to
P2N, AO and HMP. See legend to Fig. 2. Repair synthesis in
w
x
Ž
.
untreated cells was 36"7 cpm mgy1 DNA. Irradiation with UV
Ž
.
mM 4- 2-hydroxyethyl -1-piperazine ethane sulfonic
y2
Ž
.
light 20 J m
immediately after preincubation increased repair
Ž
.
.
acid HEPES ,
2
mM 5-fluoro-2X-deoxyuridine
synthesis to 7700"880% of the control value. ), significantly
different from the control; P-0.05.
Ž
Ž
.
FdUrd; Sigma and 10 mM BrdUrd Sigma for 1 h.

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)
U. Andrae et al.rMutation Research 439 1999 191–197
195
2.4. Statistical analysis
Statistical analysis of the data was performed
using Student’s t-test.
3. Results
Ž
The results obtained with parental V79 cells clone
.
V79-MZ , which do not contain any sulfotransferase
activity, are shown in Fig. 2. None of the chemicals
induced a detectable increase in DNA repair synthe-
sis, indicating that they did not cause DNA damage
in the cells. By contrast, UV-irradiation of the cells
strongly induced repair, as was the case with all
Ž
.
other V79-derived cell lines employed Figs. 3–6 .
Figs. 3 and 4 show the results obtained with
Ž
.
Ž
.
V79-rHSTa Fig. 3 and V79-rHST20 cells Fig. 4 ,
engineered for expression of two different rat hepatic
Ž
hydroxysteroid sulfotransferases rHSTa and
Fig. 6. DNA repair synthesis in V79-rST1C1 cells exposed to
P2N, AO and HMP. See legend to Fig. 2. Repair synthesis in
untreated cells was 35"13 cpm mgy1 DNA. Irradiation with UV
y2
Ž
.
light 20 J m
immediately after preincubation increased repair
synthesis to 6130"280% of the control value. ), )), signifi-
cantly different from the control; ), P-0.05; )), P-0.01.
.
rHST20 . Neither P2N nor AO caused increased
repair in these cells. Exposure to HMP resulted in a
Ž
.
very strong about 20-fold induction of repair in
Ž
.
V79-rHSTa cells Fig. 3 , but in a relatively low
increase, which was statistically significant only at
Ž
.
30 mM, in V79-rHST20 cells Fig. 4 .
Figs. 5 and 6 show the results obtained with
Ž
.
Ž
V79-rPST-IV Fig. 5 and V79-rST1C1 cells Fig.
.
6 , which express SULT1A1 and 1C1, respectively.
In both cell lines, P2N induced statistically signifi-
cant increases in repair synthesis at concentrations
01 mM. AO did not induce repair. The effects were
very similar in both cell lines. At the concentration
of 10 mM, HMP stimulated DNA repair synthesis
about 9-fold above the control level. Increasing the
concentration to 30 mM did not lead to an enhance-
ment of the effect; the induction of repair was even
slightly decreased to about 7-fold above the control
level.
Fig. 5. DNA repair synthesis in V79-rPST-IV cells exposed to
P2N, AO and HMP. See legend to Fig. 2. Repair synthesis in
untreated cells was 47"4 cpm mgy1 DNA. Irradiation with UV
y2
Ž
.
light 20 J m
immediately after preincubation increased repair
synthesis to 4390"280% of the control value. ), )), signifi-
cantly different from the control; ), P-0.05; )), P-0.01.

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U. Andrae et al.rMutation Research 439 1999 191–197
.
4. Discussion
1 occurs after, not before the sulfonation step. The
exact mechanisms of both of these reactions remain
to be elucidated.
The present study was performed in order to
examine four different rat hepatic sulfotransferases
for their ability to activate the rat hepatocarcinogen
2-NP to a genotoxicant. To this end, we have investi-
gated the genotoxicity of the anionic form of 2-NP,
P2N, in V79 Chinese hamster cell lines engineered
for expression of these sulfotransferases. P2N in-
duced a concentration-dependent induction of DNA
repair synthesis in two of the cell lines, V79-rPST-IV
and V79-rST1C1, which express rat SULT1A1 and
1C1, respectively. This result indicates that both of
these enzymes are capable of metabolizing 2-
NPrP2N to a reactive, DNA damaging metabolite.
The result is in accord with the conclusions of
Acknowledgements
The expert technical assistance of Ms. Silke Groitl
and Ms. Stefanie Brandner is gratefully acknowl
edged. This work was financially supported by
the Bundesministerium fur Bildung, Wissenschaft,
¨
Ž
Forschung und Technologie BMBF, grant 0311243;
.
to the Potsdam-Rehbrucke laboratory and the Com-
mission of the European Communities BMH1-
CT92-0097; to the Potsdam-Rehbrucke and Dundee
¨
Ž
¨
.
laboratories .
w x
w x
Ž
Sodum et al. 4 and Fiala et al. 7 who had pro-
posed that aryl sulfotransferase IV which nominally
is identical with SULT1A1, but in practice, when
purified from rat liver, is likely to comprise a mix-
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