Vol. 91, No. 4, 1979 December
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
28, 1979
Pages
1302-1308
PARAQUAT-INDUCED DNA DAMAGE IN MAMMALIAN CELLS W.E. Ross, M.D. E.R. Block, M.D. Rwei-Ying Chang University
Received SUMMARY
November
of Florida Gainesville,
College of Medicine FL 32610
lo,1979
:
We have examined the possibility that paraquat (PQ) may exert its toxicity by inducing DNA damage. Mouse lymphoblasts in culture exhibited inhibition of colony forming ability and DNA single strand breaks following a 2 hour exposure to PQ. These phenomenon are dose dependent and increase when a rat liver S9 fraction is included in the incubation mixture. The presence of superoxide dismutase and catalase did not prevent the effects of PQ. Our data indicate that DNA should be considered as a possibile macromolecular target for the lethal effects of paraquat. Paraquat rodents
(PQ) is
and man (1,2).
following
active
to a variety peroxide get
a bipyridinium
uptake
of toxic
(4).
for
these
as evidence
toxic for
certainty of alternative
lipids
are
peroxidation
thus
by which
reported
(7),
little
information
of thymidine
--in vivo
and may simply
(8),
into
diffuse
rat
Copyright All rights
@ I979
by Academic Press, Inc. in ony form reserved.
of reproduction
damage.
1302
tar-
has created
un-
and consideration
DNA represent A number
in man. (9),
DNA has been
0006-291X/79/241302-07$01.00/0
rise
has been presented
These
This
include
effect In other
for
(7).
There
is
Inhibition
up to 32 hours
is non-specific, work,
agents
alkylating
DNA.
reported
an im-
of these
and bleomycin
on PQ on intracellular
of PQ (10). cellular
PQ gives
macromolecular
however,
toxicity.
radiation
lung
that
and hydrogen
data
of intracellular
toxicity
the effects
in
(6).
produce
ionizing
published
peroxidation,
seems timely
pulmonary
administration
reflect
of lipid
anion
the major
injury
suggests
(3),
of malonaldehyde
Recently
many agents
regarding
incorporation
(5).
to date
superoxide
formation
pulmonary
parenchyma
to represent
and function
to cause
nitrosoureas
felt
causes
evidence
including
and the
role
which
of the pulmonary
species
in structure
agents
following
cells
mechanisms
mechanism
have been
into
the precise
Alterations portant
of the experimental
products
lipid
about
Most
chemical
Membrane
herbicide
George
however, and George
Vol. 91, No. 4, 1979
(11)
noted
of Vicia
that
BIOCHEMICAL
PQ causes This
faba.
uncoiling
result
of the
could
of this
cellular
DNA.
blasts
in culture.
dled, ular
well
We have This
characterized
work applied
chromatin
be explained
damage to the DNA such as strand The purpose
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fibers
in meiotic
by some form
of direct
chromosomes PQ-induced
breakage.
is
to directly
demonstrate
the DNA alkaline
mammalian
cell
and allows
direct
line
elution was chosen
correlation
that
PQ can damage intra-
technique because between
to mouse lymphoit
is
easily
han-
DNA damage and cell-
toxicity.
METHODS: Mouse leukemia L1210 cells, grown in suspension in EPMl 1630 medium with 20% fetal calf serum were employed in all experiments. Details of tissue culture technique and the labeling of cells with radioactive thymidine have been published (12). Colony forming ability was determined using the soft agar forming technique of Chu and Fisher (13). Paraquat (Aldrich Chemicals) was dissolved in sterile water just prior to use. Superoxide dismutase (Palosein, Diagnostic Data, Inc.) and catalase (Sigma Chemicals) were dissolved in culture medium just prior to use. Cells were resuspended in fresh warm medium at 7~10~ cells/ml just before treatment with PQ. Drug treatment was for 2 hours at 37O after which the cells were washed twice with fresh cold medium and resuspended. When cells were studied for repair of the drug-induced DNA damage, they were washed free of PQ and allowed to incubate in the fresh medium at 37O for various periods of time prior to analysis. In some experiments, metabolic activation of PQ was attempted by including an aliquot of reconstituted lyophylized 9OOOg supernatant (S9) from the livers of phenobarbital-induced rats along with the co-factors NADP (2.4mg/ml) and isocitric a&d (4.5mg/ml). Lyophylized material was prepared according to the method of Leibman (14). DNA damage in the form of single strand breaks was determined by the method of alkaline elution. The theoretical basis for and technical details of DNA alkaline elution have been discussed elsewhere (12). Briefly, cells containing 14C-.labeled DNA are exposed to drug, washed, deposited on a membrane filter and lysed with a solution containing 2M NaCl, O.OlM Na2EDTA, 0.2% SDS, and proteinase K (.5mg/ml). The DNA is then eluted from the filter using tetrapropylatmnonium hydroxide at pH 12.1. Fractions are collected for 15 hours at 90 minute intervals and counted in Aquasol liquid scintillation fluid (New England Nuclear, Boston, MA). The fraction of the total counts remaining on the filter at each collection interval is calculated and plotted against the simultaneous elution of an internal standard (vide infra). The rate of DNA elution from the filter increases quantitatively with increasing frequency of strand breaks. The 3H-labeled reference cells (abscissa, Figure 2), henceforth referred to as internal standard DNA, received 150 rads irradiation prior to elution. These cells provided DNA within each experiment with a known frequency of strand breaks and consistent elution kinetics which are not influenced by the elution rate of the experimental 14C-labeled DNA. Normalization of the 14C-labeled NA to this internal standard reduces quantitative variability. The shapes of the ?4 C-labeled DNA elufion curves are the same whether plotted against time or elution of the internal standard because the elution of the 3H-labeled DNA is first order with respect to time. RESULTS: There malian
cells
is
little in culture.
information Thus,
regarding it
paraquat
was important
1303
toxicity to establish
in non-pulmonary a dose response
mamre-
Vol. 91, No. 4, 1979
BIOCHEMICAL
0.001 1
I 1.0
AND BIOPHYSICAL
I 2.0
I 3.0
I 4.0
RESEARCH COMMUNICATIONS
‘p 5.0
mM PARAQUAT Figure
1:
lationship ations. ability
Effect of paraquat Following a 2 hour absence of S9, cells untreated cells as (f S9) is 80-90X. 3 experiments.
--in vitro For
this
which purpose,
of mouse leukemia
of untreated
cells
is
forming
ability
is
little
increase
in
is
an increase
is
important
to note
provide
we have cells
80-90%.
of colony
in drug
would
on colony forming ability by L1210 cells. exposure to paraquat in the presence or are seeded in soft agar. Survival of determined by colony forming efficiency Each point represents the mean of at least
a basis
studied
in soft
effect that
agar
(Fig.
In the absence
When S9 is and the biphasic
S9 without
subsequent
the effect
1304
1).
Colony
increased
present quality
PQ has no effect
biochemical
correl-
of PQ on the colony
of S9, there
as the dose of PQ is toxicity.
for
is
forming
during
efficiency
a progressive
to 3mM after drug
on survival.
which
incubation
of the curve
is
forming
loss there there
lost.
It
BIOCHEMICAL
Vol. 91, No. 4, 1979
The possibility explored
using
on the
the
presence
a dose-related
strand
breaks
The presence
PQ although
it
in elution biphasic
shape
or regions tion
the
which
of the elution
can also
ed by the method
is
curves
of Ewig
order
are
(15)
cells
which colony
forming 2B) by
strand
breaks
result
to time
(12).
The
a subpopulation
to drug
effect.
of cells
Biphasic
of DNA crosslinking.
and no crosslinking
pro-
(Fig.
distributed
resistant
with
indicating
formation
respect
remains
treated
decreased
PQ suggests
by the presence
and Kohn
cells
the filter
break
with
following
DNA which
be caused
which
Randomly first
DNA damage was
The PQ concentrations
increased
by itself.
filter
from
to those
of SY significantly
of intracellular
curves
2A).
comparable
are
DNA from
in elution
(Fig.
with
DNA from untreated
By contrast,
breaks
had no effect
from
technique.
increase
strand
RESEARCH COMMUNICATIONS
of PQ may be associated
the elution.
of single
duce single
toxicity
elution
throughout
exhibits
ability.
the
the alkaline
filter
paraquat
that
AND BIOPHYSICAL
This
was detected
elu-
was test(data
not
shown). Potent induced
mechanisms
DNA strand
lowing
a 6 hour
exist
in mammalian
breaks.
However,
post-treatment
cells
for
the repair
when cellular
incubation,
of x-ray
DNA was examined
there
was no evidence
and drug-
by elution
fol-
of damage repair
(Fig.2B). Montgomery H202 in
(4)
the presence
strand
breaks
vented
by superoxide
ation
it
of rat
that
lung
was of interest
mixture.
strand
has shown
dismutase
Inclusion
breakage
(data
the
microsomes.
Since
to determine
if
(300Ug/ml)
of these not
PQ stimulates
formation either
enzymes
of these
the effects
and catalase during
of superoxide
may cause DNA
of PQ could
(750Ounits/ml)
PQ exposure
and
be
pre-
in the
failed
incubi
to prevent
shown).
DISCUSSION: Using
an --in vitro
cells
to PQ results
These
breaks
following while
drug higher
system, in significant
increase removal. than
those
in a dose
we have demonstrated
exposure
damage to DNA in the form dependent
fashion
The PQ concentrations typically
that
described
1305
and are not required
in vivo,
of mammalian
of strand rapidly
to produce correlate
well
breaks. repaired
strand with
breaks, the
con-
; : c
)
i I ,
i 0 1
f
A
06 3H DNA
06
+s9
bearing 14C-labeled DNA are 2 ho&s, washed, and analyzed 150 rads x-irradiation and
ON FILTER
FRACTION
L121O cells (B) of S9 for DNA are given
0.8 RETAINED
0.2
04
Figure 2: DNA strand breakage by paraquat. to paraquat in the absence (A) or presence alkaline elution. Cells bearing 3H-labeled in each filter as an internal standard.
08
3.6
I i 0
04
0.6
0
,
I.C
I
-s9
exposed by DNA included
04
25mM 5mM + 6 hr recar
J
1
BIOCHEMICAL
Vol. 91, No. 4, 1979
centrations within
necessary
the limits
our data
to inhibit
a fresh
The mechanism to participate system
(e.g.,
02,
Although
effects
unlikely
tance.
were that
intracellular
that
PQ treatment
strand
to the lung
This via
have
peroxidation that
for
mine)
protected
lung
demonstrating warrants
further
of free
doses
unknown
proposed
peroxidation lipid
of PQ to rats. rats
with
them a high microsomes
from
the association investigation
controversial cast
peroxidation
of PQ-induced
It
is
following with
is
DNA also
toxic
at present.
While
microsomes
--in vivo
following
Shu
on the lipid confirming in vitro,
they
administration
lethality
was not
(N,N1-diphenyl-p-phenylene
dia-
although
both
of these
by paraquat
DNA damage with
as an alternative
it
cells
some doubt (5).
to lung
an anti-oxidant
lipid
by
by causing
(17).
paraquat-induced
diet
curves
growth
by Bus et al
products
carbohydrate
it
of impor-
however,
of L1210
radicals
which
Further,
or H202 is
regard,
cell
is
when added
peroxidation
Thus,
(7).
observations
originally
by pre-treating or feeding
generation
published
lipid
demonstrate
of supralethal
inhibits
peroxides.
of involvement
treatment
antibiotic
is
anion
and survival
after
radicals,
and DNA damage,
likelihood
of the elution
shown
of a NADPH regener-
dismutase.
In this
pulmonary toxicity
PQ-induced
mechanism
not
reduced
antitumor
has been
lethality
of superoxide
system,
on DNA by free
and superoxide
observed
the mechanism
PQ stimulates
could
to those
recently
both
peroxides.
shape
intracellular
although
The basis (6)
or lipid
The drug
that
cells.
such as H202 or lipid
us to comment on the
the biphasic
(16). breaks
et al
radicals
--in vitro
the presence
increased
by catalase
we believe
mammalian
of attack
species
generation
allow
are similar
bleomycin
chemical
prevented
do not
in
the possibility
of the S9 mixture
not
free
of interest
toxic
extracellular
Our data
reactions
suggests
*OH) or other
the presence
these is
This
on how PQ may injure
transfer
basis,
by the use of this
PQ damages DNA is unclear.
electron
(5).
RESEARCH COMMUNICATIONS
On this
formation.
imposed
perspective
by which
in
ating
colony
of interpretation
provide
AND BIOPHYSICAL
mechanism
maneuvers
in vitro. cellular for
lung
Our data, toxicity,
injury.
REFERENCES 1. 2.
Bullivant, Kimbrough,
C.J. R.D.
(1966) Br. and Gaines,
J. Med. 1: 1272-1273. T.B. (1970) Toxicol.
1307
Appl.
Pharmacol.
17:
679-690.
Vol. 91, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13. 14. 15. 16.
Rose, M.S., Smith, L.L., Wyatt, I. (1974) Nature 252: 314-315. Montgomery, M.R. (1976) Toxicol. Appl. Pharmacol. 36: 543. Bus, J.S., Aust, S-D., Gibson, J.E. (1974) Biochem. Biophys. Res. Comm. 58: 749-755. Shu, H., Talcott, R.E., Rice, S.A., Wei, E.T. (1979) Biochem. Pharmacol. 28: 327-331. Green, M.R.(1977) Western J. Med. 127: 292-298. Durant, J.R., Norgard, M.J., Murad, T.M., Bartolucci, A.A., Langford, K.H. (1979) Ann. Int. Med. 90: 191-194. Rubin, P. and Casarett, G.W. (1968) Clinical Radiation Pathology, pp. 423-470 W.B. Saunders, Philadelphia. Van Osten, G.K. and Gibson, J.E. (1975) Food Cosmet. Toxicol. 13: 47-54. George, K., George, M., Indian, .J.I (1977) Biochem. Biophys. 14: 7. L.C., Ewig, R.A.G., Friedman, C.A. (1976) Biochemistry Kohn, K.W., Erickson, 15: 4629-4737. Chu, M.Y., Fischer, G.A. (1968) Biochem. Pharmacol. 17: 753-767. Leibman, K.C. (1965) Mol. Pharmacol. 1: 239-246. Kohn, K.W. (1977) Cancer Res. 37: 2122-2144. Ewig, R.A.G., Ighal, Z.M., Kohn, K.W., Ewig, R.A.G., Fornace, A.J. (1978) Cancer Res. 36:
17.
Oberly,
3. 4. 5. 6. 7. 8. 9. 10.
11. 12.
3834-3838.
L.W.,
Buettner,
G.R.
(1979)
FEBS Lett.
1308
97:
47-49.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
28, 1979
Pages
1302-1308
PARAQUAT-INDUCED DNA DAMAGE IN MAMMALIAN CELLS W.E. Ross, M.D. E.R. Block, M.D. Rwei-Ying Chang University
Received SUMMARY
November
of Florida Gainesville,
College of Medicine FL 32610
lo,1979
:
We have examined the possibility that paraquat (PQ) may exert its toxicity by inducing DNA damage. Mouse lymphoblasts in culture exhibited inhibition of colony forming ability and DNA single strand breaks following a 2 hour exposure to PQ. These phenomenon are dose dependent and increase when a rat liver S9 fraction is included in the incubation mixture. The presence of superoxide dismutase and catalase did not prevent the effects of PQ. Our data indicate that DNA should be considered as a possibile macromolecular target for the lethal effects of paraquat. Paraquat rodents
(PQ) is
and man (1,2).
following
active
to a variety peroxide get
a bipyridinium
uptake
of toxic
(4).
for
these
as evidence
toxic for
certainty of alternative
lipids
are
peroxidation
thus
by which
reported
(7),
little
information
of thymidine
--in vivo
and may simply
(8),
into
diffuse
rat
Copyright All rights
@ I979
by Academic Press, Inc. in ony form reserved.
of reproduction
damage.
1302
tar-
has created
un-
and consideration
DNA represent A number
in man. (9),
DNA has been
0006-291X/79/241302-07$01.00/0
rise
has been presented
These
This
include
effect In other
for
(7).
There
is
Inhibition
up to 32 hours
is non-specific, work,
agents
alkylating
DNA.
reported
an im-
of these
and bleomycin
on PQ on intracellular
of PQ (10). cellular
PQ gives
macromolecular
however,
toxicity.
radiation
lung
that
and hydrogen
data
of intracellular
toxicity
the effects
in
(6).
produce
ionizing
published
peroxidation,
seems timely
pulmonary
administration
reflect
of lipid
anion
the major
injury
suggests
(3),
of malonaldehyde
Recently
many agents
regarding
incorporation
(5).
to date
superoxide
formation
pulmonary
parenchyma
to represent
and function
to cause
nitrosoureas
felt
causes
evidence
including
and the
role
which
of the pulmonary
species
in structure
agents
following
cells
mechanisms
mechanism
have been
into
the precise
Alterations portant
of the experimental
products
lipid
about
Most
chemical
Membrane
herbicide
George
however, and George
Vol. 91, No. 4, 1979
(11)
noted
of Vicia
that
BIOCHEMICAL
PQ causes This
faba.
uncoiling
result
of the
could
of this
cellular
DNA.
blasts
in culture.
dled, ular
well
We have This
characterized
work applied
chromatin
be explained
damage to the DNA such as strand The purpose
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
fibers
in meiotic
by some form
of direct
chromosomes PQ-induced
breakage.
is
to directly
demonstrate
the DNA alkaline
mammalian
cell
and allows
direct
line
elution was chosen
correlation
that
PQ can damage intra-
technique because between
to mouse lymphoit
is
easily
han-
DNA damage and cell-
toxicity.
METHODS: Mouse leukemia L1210 cells, grown in suspension in EPMl 1630 medium with 20% fetal calf serum were employed in all experiments. Details of tissue culture technique and the labeling of cells with radioactive thymidine have been published (12). Colony forming ability was determined using the soft agar forming technique of Chu and Fisher (13). Paraquat (Aldrich Chemicals) was dissolved in sterile water just prior to use. Superoxide dismutase (Palosein, Diagnostic Data, Inc.) and catalase (Sigma Chemicals) were dissolved in culture medium just prior to use. Cells were resuspended in fresh warm medium at 7~10~ cells/ml just before treatment with PQ. Drug treatment was for 2 hours at 37O after which the cells were washed twice with fresh cold medium and resuspended. When cells were studied for repair of the drug-induced DNA damage, they were washed free of PQ and allowed to incubate in the fresh medium at 37O for various periods of time prior to analysis. In some experiments, metabolic activation of PQ was attempted by including an aliquot of reconstituted lyophylized 9OOOg supernatant (S9) from the livers of phenobarbital-induced rats along with the co-factors NADP (2.4mg/ml) and isocitric a&d (4.5mg/ml). Lyophylized material was prepared according to the method of Leibman (14). DNA damage in the form of single strand breaks was determined by the method of alkaline elution. The theoretical basis for and technical details of DNA alkaline elution have been discussed elsewhere (12). Briefly, cells containing 14C-.labeled DNA are exposed to drug, washed, deposited on a membrane filter and lysed with a solution containing 2M NaCl, O.OlM Na2EDTA, 0.2% SDS, and proteinase K (.5mg/ml). The DNA is then eluted from the filter using tetrapropylatmnonium hydroxide at pH 12.1. Fractions are collected for 15 hours at 90 minute intervals and counted in Aquasol liquid scintillation fluid (New England Nuclear, Boston, MA). The fraction of the total counts remaining on the filter at each collection interval is calculated and plotted against the simultaneous elution of an internal standard (vide infra). The rate of DNA elution from the filter increases quantitatively with increasing frequency of strand breaks. The 3H-labeled reference cells (abscissa, Figure 2), henceforth referred to as internal standard DNA, received 150 rads irradiation prior to elution. These cells provided DNA within each experiment with a known frequency of strand breaks and consistent elution kinetics which are not influenced by the elution rate of the experimental 14C-labeled DNA. Normalization of the 14C-labeled NA to this internal standard reduces quantitative variability. The shapes of the ?4 C-labeled DNA elufion curves are the same whether plotted against time or elution of the internal standard because the elution of the 3H-labeled DNA is first order with respect to time. RESULTS: There malian
cells
is
little in culture.
information Thus,
regarding it
paraquat
was important
1303
toxicity to establish
in non-pulmonary a dose response
mamre-
Vol. 91, No. 4, 1979
BIOCHEMICAL
0.001 1
I 1.0
AND BIOPHYSICAL
I 2.0
I 3.0
I 4.0
RESEARCH COMMUNICATIONS
‘p 5.0
mM PARAQUAT Figure
1:
lationship ations. ability
Effect of paraquat Following a 2 hour absence of S9, cells untreated cells as (f S9) is 80-90X. 3 experiments.
--in vitro For
this
which purpose,
of mouse leukemia
of untreated
cells
is
forming
ability
is
little
increase
in
is
an increase
is
important
to note
provide
we have cells
80-90%.
of colony
in drug
would
on colony forming ability by L1210 cells. exposure to paraquat in the presence or are seeded in soft agar. Survival of determined by colony forming efficiency Each point represents the mean of at least
a basis
studied
in soft
effect that
agar
(Fig.
In the absence
When S9 is and the biphasic
S9 without
subsequent
the effect
1304
1).
Colony
increased
present quality
PQ has no effect
biochemical
correl-
of PQ on the colony
of S9, there
as the dose of PQ is toxicity.
for
is
forming
during
efficiency
a progressive
to 3mM after drug
on survival.
which
incubation
of the curve
is
forming
loss there there
lost.
It
BIOCHEMICAL
Vol. 91, No. 4, 1979
The possibility explored
using
on the
the
presence
a dose-related
strand
breaks
The presence
PQ although
it
in elution biphasic
shape
or regions tion
the
which
of the elution
can also
ed by the method
is
curves
of Ewig
order
are
(15)
cells
which colony
forming 2B) by
strand
breaks
result
to time
(12).
The
a subpopulation
to drug
effect.
of cells
Biphasic
of DNA crosslinking.
and no crosslinking
pro-
(Fig.
distributed
resistant
with
indicating
formation
respect
remains
treated
decreased
PQ suggests
by the presence
and Kohn
cells
the filter
break
with
following
DNA which
be caused
which
Randomly first
DNA damage was
The PQ concentrations
increased
by itself.
filter
from
to those
of SY significantly
of intracellular
curves
2A).
comparable
are
DNA from
in elution
(Fig.
with
DNA from untreated
By contrast,
breaks
had no effect
from
technique.
increase
strand
RESEARCH COMMUNICATIONS
of PQ may be associated
the elution.
of single
duce single
toxicity
elution
throughout
exhibits
ability.
the
the alkaline
filter
paraquat
that
AND BIOPHYSICAL
This
was detected
elu-
was test(data
not
shown). Potent induced
mechanisms
DNA strand
lowing
a 6 hour
exist
in mammalian
breaks.
However,
post-treatment
cells
for
the repair
when cellular
incubation,
of x-ray
DNA was examined
there
was no evidence
and drug-
by elution
fol-
of damage repair
(Fig.2B). Montgomery H202 in
(4)
the presence
strand
breaks
vented
by superoxide
ation
it
of rat
that
lung
was of interest
mixture.
strand
has shown
dismutase
Inclusion
breakage
(data
the
microsomes.
Since
to determine
if
(300Ug/ml)
of these not
PQ stimulates
formation either
enzymes
of these
the effects
and catalase during
of superoxide
may cause DNA
of PQ could
(750Ounits/ml)
PQ exposure
and
be
pre-
in the
failed
incubi
to prevent
shown).
DISCUSSION: Using
an --in vitro
cells
to PQ results
These
breaks
following while
drug higher
system, in significant
increase removal. than
those
in a dose
we have demonstrated
exposure
damage to DNA in the form dependent
fashion
The PQ concentrations typically
that
described
1305
and are not required
in vivo,
of mammalian
of strand rapidly
to produce correlate
well
breaks. repaired
strand with
breaks, the
con-
; : c
)
i I ,
i 0 1
f
A
06 3H DNA
06
+s9
bearing 14C-labeled DNA are 2 ho&s, washed, and analyzed 150 rads x-irradiation and
ON FILTER
FRACTION
L121O cells (B) of S9 for DNA are given
0.8 RETAINED
0.2
04
Figure 2: DNA strand breakage by paraquat. to paraquat in the absence (A) or presence alkaline elution. Cells bearing 3H-labeled in each filter as an internal standard.
08
3.6
I i 0
04
0.6
0
,
I.C
I
-s9
exposed by DNA included
04
25mM 5mM + 6 hr recar
J
1
BIOCHEMICAL
Vol. 91, No. 4, 1979
centrations within
necessary
the limits
our data
to inhibit
a fresh
The mechanism to participate system
(e.g.,
02,
Although
effects
unlikely
tance.
were that
intracellular
that
PQ treatment
strand
to the lung
This via
have
peroxidation that
for
mine)
protected
lung
demonstrating warrants
further
of free
doses
unknown
proposed
peroxidation lipid
of PQ to rats. rats
with
them a high microsomes
from
the association investigation
controversial cast
peroxidation
of PQ-induced
It
is
following with
is
DNA also
toxic
at present.
While
microsomes
--in vivo
following
Shu
on the lipid confirming in vitro,
they
administration
lethality
was not
(N,N1-diphenyl-p-phenylene
dia-
although
both
of these
by paraquat
DNA damage with
as an alternative
it
cells
some doubt (5).
to lung
an anti-oxidant
lipid
by
by causing
(17).
paraquat-induced
diet
curves
growth
by Bus et al
products
carbohydrate
it
of impor-
however,
of L1210
radicals
which
Further,
or H202 is
regard,
cell
is
when added
peroxidation
Thus,
(7).
observations
originally
by pre-treating or feeding
generation
published
lipid
demonstrate
of supralethal
inhibits
peroxides.
of involvement
treatment
antibiotic
is
anion
and survival
after
radicals,
and DNA damage,
likelihood
of the elution
shown
of a NADPH regener-
dismutase.
In this
pulmonary toxicity
PQ-induced
mechanism
not
reduced
antitumor
has been
lethality
of superoxide
system,
on DNA by free
and superoxide
observed
the mechanism
PQ stimulates
could
to those
recently
both
peroxides.
shape
intracellular
although
The basis (6)
or lipid
The drug
that
cells.
such as H202 or lipid
us to comment on the
the biphasic
(16). breaks
et al
radicals
--in vitro
the presence
increased
by catalase
we believe
mammalian
of attack
species
generation
allow
are similar
bleomycin
chemical
prevented
do not
in
the possibility
of the S9 mixture
not
free
of interest
toxic
extracellular
Our data
reactions
suggests
*OH) or other
the presence
these is
This
on how PQ may injure
transfer
basis,
by the use of this
PQ damages DNA is unclear.
electron
(5).
RESEARCH COMMUNICATIONS
On this
formation.
imposed
perspective
by which
in
ating
colony
of interpretation
provide
AND BIOPHYSICAL
mechanism
maneuvers
in vitro. cellular for
lung
Our data, toxicity,
injury.
REFERENCES 1. 2.
Bullivant, Kimbrough,
C.J. R.D.
(1966) Br. and Gaines,
J. Med. 1: 1272-1273. T.B. (1970) Toxicol.
1307
Appl.
Pharmacol.
17:
679-690.
Vol. 91, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13. 14. 15. 16.
Rose, M.S., Smith, L.L., Wyatt, I. (1974) Nature 252: 314-315. Montgomery, M.R. (1976) Toxicol. Appl. Pharmacol. 36: 543. Bus, J.S., Aust, S-D., Gibson, J.E. (1974) Biochem. Biophys. Res. Comm. 58: 749-755. Shu, H., Talcott, R.E., Rice, S.A., Wei, E.T. (1979) Biochem. Pharmacol. 28: 327-331. Green, M.R.(1977) Western J. Med. 127: 292-298. Durant, J.R., Norgard, M.J., Murad, T.M., Bartolucci, A.A., Langford, K.H. (1979) Ann. Int. Med. 90: 191-194. Rubin, P. and Casarett, G.W. (1968) Clinical Radiation Pathology, pp. 423-470 W.B. Saunders, Philadelphia. Van Osten, G.K. and Gibson, J.E. (1975) Food Cosmet. Toxicol. 13: 47-54. George, K., George, M., Indian, .J.I (1977) Biochem. Biophys. 14: 7. L.C., Ewig, R.A.G., Friedman, C.A. (1976) Biochemistry Kohn, K.W., Erickson, 15: 4629-4737. Chu, M.Y., Fischer, G.A. (1968) Biochem. Pharmacol. 17: 753-767. Leibman, K.C. (1965) Mol. Pharmacol. 1: 239-246. Kohn, K.W. (1977) Cancer Res. 37: 2122-2144. Ewig, R.A.G., Ighal, Z.M., Kohn, K.W., Ewig, R.A.G., Fornace, A.J. (1978) Cancer Res. 36:
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Oberly,
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