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Induction of single-strand breaks in dna of mice by trichloroethylene and tetrachloroethylene
Letters, 31 (1986) 31-35
Toxicology
31
Elsevier
TOXLett.
1559
INDUCTION OF SINGLE-STRAND BREAKS IN DNA OF MICE BY TRICHLOROETHYLENE AND TETBACHLOROETHYLENE
(Trichloroethylene; S.A.S.
tetrachloroethylene;
DNA; single-strand breaks)
WALLES
Unit of Occupational Toxicology, Health, S-171 84 Solna (Sweden) (Received
December
(Revision
received
(Accepted
February
lOth,
National Board of Occupational Safety and
1985)
February 27th,
Research Department,
26th,
1986)
1986)
SUMMARY Trichloroethylene
(TRI) (4-10 mmol/kg
body wt) and tetrachloroethylene
wt) were given to male mice by i.p. injection. liver, kidney and lung was studied level of SSB in kidney completely
repaired
by the DNA unwinding
(PER) (4-8 mmol/kg
of single-strand
technique.
breaks
24 h after
injection.
The capability
to that of three other substances,
and styrene,
which have been studied
of SSB was in the following
earlier
There was a linear increase
i.e., methyl
methanesulfonate
order:
MMS
> styrene-7,8-oxide
was
SSB in liver DNA
(MMS),
The potency
of the
The damage
of TRI and PER to induce
by the same technique.
body
(SSB) in DNA of
and liver DNA but not in lung DNA 1 h after administration.
is compared induction
The induction
styrene-7,8-oxide
of the substances
> styrene
> PER
for
> TRI.
INTRODUCTION
TRI and PER are mainly used as industrial solvents. There is limited evidence that these substances are carcinogenic to mice [ 1,2]. TRI is mutagenic after microsomal activation in several test systems [ 11. The mutagenicity of PER is not sufficiently evaluated [2]. Both substances are metabolized to reactive intermediates [3]. TRI oxide has been postulated to be the metabolite responsible for covalent binding. However, Miller and Guengerich [4] have recently proposed another intermediate, i.e., a complex of
Abbreviations: DS, double-stranded DNA; MMS, methyl methanesulfonate; SS, single-stranded DNA; SSB single-stranded breaks; TRI, trichloroethylene.
PER, tetrachloroethylene;
0378-4274/86/S
Division)
03.50
0
1986 Elsevier
Science
Publishers
B.V. (Biomedical
32
TRI and cytochrome P-450. In the present investigation the induction of SSB in DNA of liver, kidney and lung of mice was studied after i.p. injection of TRI and PER, respectively. The induction of SSB was linearly dose-dependent for both substances in liver and kidney. There was no effect on lung. The damage of DNA was repaired in 24 h for both substances. MATERIALS
AND METHODS
TRI (99.5 (r/o) and PER (99.8 070)were from Merck-Schuchardt, Darmstadt, (F.R.G.), hydroxylapatite Bio-Gel HTP, DNA-grade Bio-Gel HTP from Bio-Rad Laboratories (Richmond, CA, U.S.A.), and 4’ ,6-diamidino-2-phenylindole.2HCl from Serva Feinbiochemica (Heidelberg, F.R.G.). Treatment of animals
Male mice (NMRI-type, 25-30 g) were injected i.p. with TRI (4-10 mmol/kg body wt) and PER (4-8 mmol/kg body wt), respectively, dissolved in 0.05 ml Tween-80, which was given to each mouse. Controls received Tween-80, only. The animals were killed by cervical dislocation 1 or 24 h after injection. Preparation
of cell nuclei
Cell nuclei were prepared from liver, kidney and lung according to the method described by Walles and Erixon [5]. Determination
of SSB
The level of SSB was determined by the DNA-unwinding technique [6] of Walles and Erixon [5]. The fraction of double-stranded DNA (Fns) was calculated as the ratio of the amount of DS and the sum of DS and SS: FDS
=
DS DS + SS
The negative logarithm of Fns is a linear function of the dose [5]. The difference between the value of -log Fnsx for the sample, and that of the control (-log Fuse) has been expressed as: A log Fns = -log
FDS~-
(-log
FDQ)
RESULTS
The level of SSB increased linearly in kidney and liver DNA with increasing dose of TRI (Fig. 1) and PER (Fig. 2). There was no effect on lung-DNA compared to
33 (Fig. 1)
4
0
(Fig. 2)
7
4 DOSE
6
8
10
8
-
DOSE
mmol/kg
mmol/kg
Fig. 1. Level of SSB expressed as - log Fns for DNA of liver ( q) kidney (0) and lung (A) 1 h after i.p. injection of different doses of trichloroethylene to mice. Mean values + SE of 3-9 animals are plotted. Fig. 2. Level of SSB expressed as -log Fns for DNA of liver (o), kidney (0) and lung (A) 1 h after i.p. injection of different doses of tetrachloroethylene to mice. Mean values f SE of 5-7 animals are plotted.
the control. Statistical analyses were performed with Student’s t-test. Already at a dose of 6 mmol/kg body wt (l/4 of LD~o) of TRI the value of -log FDS differed significantly from control (P
Trichloroethylene (7.5 mmol/kg)
Liver Kidney Lung
0.059 f 0.006 0.061 f 0.006 0.058 + 0.004
OF TRICHLOROETHYLENE
Tetrachloroethylene (7.2 mmoI/kg)
AND TETRA-
Control
- Log Fos 0.066 f 0.004 0.067 * 0.005 0.068 + 0.002
0.064 f 0.004 0.060 + 0.006 0.063 f 0.003
34
TABLE
II
COMPARISON
OF THE CAPABILITY
SSB (A log F&mmol/kg
OF DIFFERENT
BODY WT) AND THE
AGENTS
TO INCREASE
CORRESPONDING body wt
AMOUNT
Agent
Alog F&mmol/kg
MMS
0.14
Styrene-7,8-oxide
0.013
20
Styrene
0.0084
13
PER
0.0037
5.6
TRI
0.0029
4.4
y-Radiation
0.20
THE LEVEL OF OF SSB/IO”
Da
SSB/IO”Da 210
Alog F&krad
corresponds to been calculated Only a small tion. Therefore,
300
about 3 SSB/ lo9 Da per krad [8]. The numbers of SSB/ 10” Da have from these data for different agents and are tabulated in Table II. part of TRI and PER is metabolized during 1 h after i.p. administrathe effects of these agents are probably underestimated in Table II.
DISCUSSION
The results of this investigation show that TRI and PER induce SSB in DNA of kidney and liver in mouse. There are different possibilities for the origin of SSB induced by TRI and PER: (1) repair of alkylated bases, (2) influence of oxygen radicals formed during the biotransformation of the substances and (3) destruction of DNA by autolyzes of cells at toxic doses. None of these possibilities can be excluded in the present experiments. The induction of active oxygen species by TRI could be characteristic of promotor activity, which has been shown for benzoylperoxide and phorbol myristate acetate [ 12,131. Covalent binding of TRI or PER to DNA in vivo has not yet been verified [ 10,111. However, the degree of alkylation could be below detection limit, which was 1 alkylation/ 10’ nucleotides in experiments with PER [ 111. The detection limit for the unwinding technique is 1 SSB/S x lo6 nucleotides (K. Erixon, personal communication). In this case that method is more sensitive than the quantification of alkylated adducts after treatment with PER. Prolonged exposure to TRI and PER should be performed to see whether the SSB are persistent or not after repeated administration. ACKNOWLEDGEMENTS
I wish to thank Pia Hanserkers for technical assistance. My thanks are also due to Eskil Hjort for drawing the figures. This work was supported by the Swedish Work Environmental Fund (No. 84-1269).
35
REFERENCES 1 IARC Monographs tional
Agency
on the evaluation
for Research
2 IARC Monographs tional
Agency
activity, 4 R.E.
on the evaluation
for Research
3 D. Henschler, Miller
and
Lyon, Lyon,
and mutagenicity
Health
Perspect.,
F.P.
Guengerich,
and reconstituted
risk of chemicals
to man, Vol. 20, Interna-
1979, pp. 545-567.
of the carcinogenic
on Cancer,
Metabolism
Environ.
microsomes,
of the carcinogenic
on Cancer,
risk of chemicals
to man, Vol. 20, lnterna-
1979, pp. 491-514.
of halogenated
olefins - a comparison
of structure
and
21 (1977) 61-64. Metabolism
of
trichloroethylene
enzyme systems containing
cytochrome
in isolated P-450, Cancer
hepatocytes, Res., 43 (1983)
1145-1152. 5 S.A.S.
Walles and K. Erixon,
methyl
methane
sulfonate
Carcinogenesis,
Walles
styrene
in alkaline
normal
oxide,
Radiation solution,
and I. Or&t,
and styrene
8 K. Erixon Res.,
and K. Erixon,
separation
7 S.A.S.
breaks
in DNA of various
determined
organs
by the alkaline
of mice induced unwinding
by
technique,
5 (1984) 3 19-323.
6 G. Ahnstrom strand
Single-strand
and dimethylsulfoxide induced
Single-strand
Cancer
and G. Ahnstrom,
strand
Int. J. Radiat.
Lett.,
breaks
breakage
Biol.,
in DNA
in DNA from
cells,
of various
organs
of mice induced
by
21 (1982) 9-15.
Single-strand
breaks
in DNA during
repair
and Xeroderma pigmentosum cells and hydroxylapatite
human
mammalian
23 (1973) 285-289.
of UV-induced
damage
chromatography,
in
Mutation
59 (1979) 257-271.
9 D.E. Brash and R.W. Hart,
DNA damage
and repair in vivo, J. Environ.
Pathol.
Toxicol.,
2 (1978)
79-l 14. 10 K. Bergman, mouse 11 R.H.
Reitz,
macokinetic Suppl., 12 H.C.
of trichloroethylene
J.F.
Quast,
Toxicol.,
A.M.
parameters
with DNA in vitro and with RNA and DNA of various
54 (1983) 181-193.
Schumann,
P.G.
need to be considered
Watanabe in high
and
dose/low
P.J.
Gehring,
Nonlinear
extrapolation,
phar-
Arch.
Toxicol.,
promoter,
phorbol
3 (1980) 79-94. Birnboim,
myristate 13 H.L.
Interactions
tissues in vivo, Arch.
Gensler
latestage
DNA strand
acetate,
Science,
and G.T.
promotion
breakage
in human
leukocytes
exposed
to a tumor
215 (1982) 1247-1249.
Bowden,
of tumor
Evidence
cell phenotype,
suggesting
a dissociation
Carcinogenesis,
of DNA strand
4 (1983) 1507-1511.
scissions
and
Toxicology
31
Elsevier
TOXLett.
1559
INDUCTION OF SINGLE-STRAND BREAKS IN DNA OF MICE BY TRICHLOROETHYLENE AND TETBACHLOROETHYLENE
(Trichloroethylene; S.A.S.
tetrachloroethylene;
DNA; single-strand breaks)
WALLES
Unit of Occupational Toxicology, Health, S-171 84 Solna (Sweden) (Received
December
(Revision
received
(Accepted
February
lOth,
National Board of Occupational Safety and
1985)
February 27th,
Research Department,
26th,
1986)
1986)
SUMMARY Trichloroethylene
(TRI) (4-10 mmol/kg
body wt) and tetrachloroethylene
wt) were given to male mice by i.p. injection. liver, kidney and lung was studied level of SSB in kidney completely
repaired
by the DNA unwinding
(PER) (4-8 mmol/kg
of single-strand
technique.
breaks
24 h after
injection.
The capability
to that of three other substances,
and styrene,
which have been studied
of SSB was in the following
earlier
There was a linear increase
i.e., methyl
methanesulfonate
order:
MMS
> styrene-7,8-oxide
was
SSB in liver DNA
(MMS),
The potency
of the
The damage
of TRI and PER to induce
by the same technique.
body
(SSB) in DNA of
and liver DNA but not in lung DNA 1 h after administration.
is compared induction
The induction
styrene-7,8-oxide
of the substances
> styrene
> PER
for
> TRI.
INTRODUCTION
TRI and PER are mainly used as industrial solvents. There is limited evidence that these substances are carcinogenic to mice [ 1,2]. TRI is mutagenic after microsomal activation in several test systems [ 11. The mutagenicity of PER is not sufficiently evaluated [2]. Both substances are metabolized to reactive intermediates [3]. TRI oxide has been postulated to be the metabolite responsible for covalent binding. However, Miller and Guengerich [4] have recently proposed another intermediate, i.e., a complex of
Abbreviations: DS, double-stranded DNA; MMS, methyl methanesulfonate; SS, single-stranded DNA; SSB single-stranded breaks; TRI, trichloroethylene.
PER, tetrachloroethylene;
0378-4274/86/S
Division)
03.50
0
1986 Elsevier
Science
Publishers
B.V. (Biomedical
32
TRI and cytochrome P-450. In the present investigation the induction of SSB in DNA of liver, kidney and lung of mice was studied after i.p. injection of TRI and PER, respectively. The induction of SSB was linearly dose-dependent for both substances in liver and kidney. There was no effect on lung. The damage of DNA was repaired in 24 h for both substances. MATERIALS
AND METHODS
TRI (99.5 (r/o) and PER (99.8 070)were from Merck-Schuchardt, Darmstadt, (F.R.G.), hydroxylapatite Bio-Gel HTP, DNA-grade Bio-Gel HTP from Bio-Rad Laboratories (Richmond, CA, U.S.A.), and 4’ ,6-diamidino-2-phenylindole.2HCl from Serva Feinbiochemica (Heidelberg, F.R.G.). Treatment of animals
Male mice (NMRI-type, 25-30 g) were injected i.p. with TRI (4-10 mmol/kg body wt) and PER (4-8 mmol/kg body wt), respectively, dissolved in 0.05 ml Tween-80, which was given to each mouse. Controls received Tween-80, only. The animals were killed by cervical dislocation 1 or 24 h after injection. Preparation
of cell nuclei
Cell nuclei were prepared from liver, kidney and lung according to the method described by Walles and Erixon [5]. Determination
of SSB
The level of SSB was determined by the DNA-unwinding technique [6] of Walles and Erixon [5]. The fraction of double-stranded DNA (Fns) was calculated as the ratio of the amount of DS and the sum of DS and SS: FDS
=
DS DS + SS
The negative logarithm of Fns is a linear function of the dose [5]. The difference between the value of -log Fnsx for the sample, and that of the control (-log Fuse) has been expressed as: A log Fns = -log
FDS~-
(-log
FDQ)
RESULTS
The level of SSB increased linearly in kidney and liver DNA with increasing dose of TRI (Fig. 1) and PER (Fig. 2). There was no effect on lung-DNA compared to
33 (Fig. 1)
4
0
(Fig. 2)
7
4 DOSE
6
8
10
8
-
DOSE
mmol/kg
mmol/kg
Fig. 1. Level of SSB expressed as - log Fns for DNA of liver ( q) kidney (0) and lung (A) 1 h after i.p. injection of different doses of trichloroethylene to mice. Mean values + SE of 3-9 animals are plotted. Fig. 2. Level of SSB expressed as -log Fns for DNA of liver (o), kidney (0) and lung (A) 1 h after i.p. injection of different doses of tetrachloroethylene to mice. Mean values f SE of 5-7 animals are plotted.
the control. Statistical analyses were performed with Student’s t-test. Already at a dose of 6 mmol/kg body wt (l/4 of LD~o) of TRI the value of -log FDS differed significantly from control (P
Trichloroethylene (7.5 mmol/kg)
Liver Kidney Lung
0.059 f 0.006 0.061 f 0.006 0.058 + 0.004
OF TRICHLOROETHYLENE
Tetrachloroethylene (7.2 mmoI/kg)
AND TETRA-
Control
- Log Fos 0.066 f 0.004 0.067 * 0.005 0.068 + 0.002
0.064 f 0.004 0.060 + 0.006 0.063 f 0.003
34
TABLE
II
COMPARISON
OF THE CAPABILITY
SSB (A log F&mmol/kg
OF DIFFERENT
BODY WT) AND THE
AGENTS
TO INCREASE
CORRESPONDING body wt
AMOUNT
Agent
Alog F&mmol/kg
MMS
0.14
Styrene-7,8-oxide
0.013
20
Styrene
0.0084
13
PER
0.0037
5.6
TRI
0.0029
4.4
y-Radiation
0.20
THE LEVEL OF OF SSB/IO”
Da
SSB/IO”Da 210
Alog F&krad
corresponds to been calculated Only a small tion. Therefore,
300
about 3 SSB/ lo9 Da per krad [8]. The numbers of SSB/ 10” Da have from these data for different agents and are tabulated in Table II. part of TRI and PER is metabolized during 1 h after i.p. administrathe effects of these agents are probably underestimated in Table II.
DISCUSSION
The results of this investigation show that TRI and PER induce SSB in DNA of kidney and liver in mouse. There are different possibilities for the origin of SSB induced by TRI and PER: (1) repair of alkylated bases, (2) influence of oxygen radicals formed during the biotransformation of the substances and (3) destruction of DNA by autolyzes of cells at toxic doses. None of these possibilities can be excluded in the present experiments. The induction of active oxygen species by TRI could be characteristic of promotor activity, which has been shown for benzoylperoxide and phorbol myristate acetate [ 12,131. Covalent binding of TRI or PER to DNA in vivo has not yet been verified [ 10,111. However, the degree of alkylation could be below detection limit, which was 1 alkylation/ 10’ nucleotides in experiments with PER [ 111. The detection limit for the unwinding technique is 1 SSB/S x lo6 nucleotides (K. Erixon, personal communication). In this case that method is more sensitive than the quantification of alkylated adducts after treatment with PER. Prolonged exposure to TRI and PER should be performed to see whether the SSB are persistent or not after repeated administration. ACKNOWLEDGEMENTS
I wish to thank Pia Hanserkers for technical assistance. My thanks are also due to Eskil Hjort for drawing the figures. This work was supported by the Swedish Work Environmental Fund (No. 84-1269).
35
REFERENCES 1 IARC Monographs tional
Agency
on the evaluation
for Research
2 IARC Monographs tional
Agency
activity, 4 R.E.
on the evaluation
for Research
3 D. Henschler, Miller
and
Lyon, Lyon,
and mutagenicity
Health
Perspect.,
F.P.
Guengerich,
and reconstituted
risk of chemicals
to man, Vol. 20, Interna-
1979, pp. 545-567.
of the carcinogenic
on Cancer,
Metabolism
Environ.
microsomes,
of the carcinogenic
on Cancer,
risk of chemicals
to man, Vol. 20, lnterna-
1979, pp. 491-514.
of halogenated
olefins - a comparison
of structure
and
21 (1977) 61-64. Metabolism
of
trichloroethylene
enzyme systems containing
cytochrome
in isolated P-450, Cancer
hepatocytes, Res., 43 (1983)
1145-1152. 5 S.A.S.
Walles and K. Erixon,
methyl
methane
sulfonate
Carcinogenesis,
Walles
styrene
in alkaline
normal
oxide,
Radiation solution,
and I. Or&t,
and styrene
8 K. Erixon Res.,
and K. Erixon,
separation
7 S.A.S.
breaks
in DNA of various
determined
organs
by the alkaline
of mice induced unwinding
by
technique,
5 (1984) 3 19-323.
6 G. Ahnstrom strand
Single-strand
and dimethylsulfoxide induced
Single-strand
Cancer
and G. Ahnstrom,
strand
Int. J. Radiat.
Lett.,
breaks
breakage
Biol.,
in DNA
in DNA from
cells,
of various
organs
of mice induced
by
21 (1982) 9-15.
Single-strand
breaks
in DNA during
repair
and Xeroderma pigmentosum cells and hydroxylapatite
human
mammalian
23 (1973) 285-289.
of UV-induced
damage
chromatography,
in
Mutation
59 (1979) 257-271.
9 D.E. Brash and R.W. Hart,
DNA damage
and repair in vivo, J. Environ.
Pathol.
Toxicol.,
2 (1978)
79-l 14. 10 K. Bergman, mouse 11 R.H.
Reitz,
macokinetic Suppl., 12 H.C.
of trichloroethylene
J.F.
Quast,
Toxicol.,
A.M.
parameters
with DNA in vitro and with RNA and DNA of various
54 (1983) 181-193.
Schumann,
P.G.
need to be considered
Watanabe in high
and
dose/low
P.J.
Gehring,
Nonlinear
extrapolation,
phar-
Arch.
Toxicol.,
promoter,
phorbol
3 (1980) 79-94. Birnboim,
myristate 13 H.L.
Interactions
tissues in vivo, Arch.
Gensler
latestage
DNA strand
acetate,
Science,
and G.T.
promotion
breakage
in human
leukocytes
exposed
to a tumor
215 (1982) 1247-1249.
Bowden,
of tumor
Evidence
cell phenotype,
suggesting
a dissociation
Carcinogenesis,
of DNA strand
4 (1983) 1507-1511.
scissions
and