Download Changes in Activities of Free Radical Detoxifying

[CANCER RESEARCH 49, 1475-1480, March 15, 1989]
Changes in Activities of Free Radical Detoxifying Enzymes in Kidneys of Male
Syrian Hamsters Treated with Estradici'
Deodutta Roy and Joachim G. IJehr
Dépannentof Pharmacology and Toxicology, The University of Texas Medical Branch, Cohesion, Texas 77550-2774
ABSTRACT
Target organ-specific estrogen-induced DNA adducts were previously
shown to precede renal carcinogenesis in Syrian hamsters. Because
estrogens induced these DNA modifications, but were not part of the
adduct structure, free radical activation of endogenous electrophiles was
postulated as a mechanism of tumor induction by estrogens. In the present
study, the activities of enzymes which detoxify reactive intermediates
were studied in liver and kidney of hamsters treated with estradici for 1,
2, and 4 mo and in untreated controls. These studies were done to detect
oxidative stress in the target organ of carcinogenesis. In the estrogenexposed hamster kidney (1, 2, and 4 mo), activities of glutathione
peroxidases I and II were significantly increased. The activity of catalase
was decreased compared to those in untreated controls. In livers which
are not the target organ of carcinogenesis, treatment of hamsters with
estrogen for 1, 2, and 4 mo resulted in changes of activities of glutathione
peroxidases I and II and catalase, which were opposite to the pattern
found in the kidney. Activities of Superoxide dismutase, glutathione
reducÃ-ase,glucose-6-phosphate dehydrogenase, -y-glutamyl transpeptidase, and glutathione transferase in estradiol-treated hamster liver and
kidney did not differ significantly from those in either liver or kidney of
untreated age-matched controls. Fluorescent products of lipid peroxidation more than doubled in the kidney, but not in the liver of hamsters
treated with estradici for 1 mo. It is concluded that the increases in
glutathione, in the activity of glutathione peroxidase, and in products of
lipid peroxidation in the kidneys of hamsters treated chronically with
estrogen all point towards elevated levels of oxidative stress.
INTRODUCTION
Estrogens administered chronically for 6 to 8 mo induce
kidney tumors in male Syrian hamsters (1). Identical sets of
covalent DNA adducts arise in premalignant kidneys of ham
sters treated with structurally diverse estrogens (2, 3). Irrespec
tive of the stilbene or steroid estrogen used, chromatograms
showed identical mobilities of each of these adducts in 11
different Chromatographie systems (3). The Chromatographie
characteristics showed these adducts to be more lipophilic than
normal nucleotides. It has therefore been concluded (3) that
estrogens induce endogenous reactive intermediates of yet un
known structure which bind covalently to DNA. Livers, which
do not develop tumors under these conditions, are free of such
estrogen-induced DNA modification.
A free radical-based mechanism represents a possible expla
nation for the induction of endogenous DNA-reactive inter
mediates in hamster kidney. Microsome-mediated oxidationreduction cycling of estrogens (4) is known to stimulate free
radical formation (5). Moreover, Superoxide radical generation
by renal microsomes of estrogen-treated hamsters is elevated
compared to values from untreated controls (6), while with liver
microsomes, there is no change.3 It is not yet known how
Received 4/7/88; revised 9/26/88, 12/15/88; accepted 12/20/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported by grants from the National Cancer Institute, NIH
(CA43232, CA43233, and CA44069).
2To whom requests for reprints should be addressed, at Department of
Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX 77550-2774.
3 D. Roy and J. G. Udir, unpublished data.
lipophilic electrophiles responsible for endogenous DNA ad
duction in hamster kidney are formed by free radicals generated
during oxidation-reduction cycling of estrogens. This is cur
rently under investigation.
In the present study, the activities of enzymes which directly
or indirectly detoxify free radical or reactive oxygen interme
diates were investigated to assess the role of free radicals in
estrogen-induced cancer. Enzymes were assayed in kidney tissue
of hamsters treated with estradiol for 1, 2, and 4 mo and in
untreated age-matched controls. Livers which do not develop
tumors under these conditions were also examined. In addition,
the influence of chronic treatment with estradiol on glutathione
concentrations and on fluorescent products of lipid peroxida
tion was determined. Alterations in activities of catalase and
glutathione peroxidase and an increase in damage by lipid
peroxidation in response to estrogen treatment were uncovered.
MATERIALS
AND METHODS
Materials
Estradiol, cholesterol, l-chloro-2,4-dinitrobenzene,
1,2-epoxy-3-(pnitrophenoxy)propane, 3,4-dichloronitrobenzene, GSH4, GSSG, glu
tathione reducÃ-asetype III from bakers' yeast, hydrogen peroxide,
eumene hydroperoxide, NADP, NADPH, glucose-6-phosphate, L-7glutamyl-p-nitroanilide, xanthine, xanthine oxidase from buttermilk,
Superoxide dismutase from bovine liver, and ferricytochrome c from
horse heart were purchased from Sigma Chemical Co., St. Louis, MO.
All other solvents and chemicals used were either analytical grade or of
highest grade available. All reagents required for the assay of GSH and
GSSG were obtained from the sources identified by Fariss and Reed
(7). Waters Models 510 and 501 high-pressure liquid chromatography
solvent delivery systems, an automated gradient controller, and a Model
490 multiwavelength detector were used. Data were analyzed by a
Waters Model 740 data module. The column used was a Spherisorb 3aminopropyl (5 pm) column purchased from Custom LC, Houston,
TX.
Estrogen Treatment of Animals
Male Syrian hamsters (8 wk of age obtained from HarÃ-anSpragueDawley, Houston, TX) were used for all experiments. Each animal was
treated with one s.c. implant (25 mg of estradiol containing 10%
cholesterol) as described previously for other estrogens (1,8,9). Control
hamsters remained untreated. After 1, 2, and 4 mo of estradiol treat
ment, hamsters were killed by decapitation. Their kidneys were excised
and separated into cortex and medulla. The cortical tissue of both
kidneys was homogenized in 0.25 M sucrose containing 10 m\i EDTA.
Subcellular Fractionations
The homogenate was centrifugea for 10 min at 1,000 x g at 4°C.
The supernatant was decanted and centrifuged for 20 min at 9,000 x g
at 4"( '. The pellet was washed with homogenizing medium and consid
ered to be crude mitochondria! fraction. The supernatant was centri
fuged at 105,000 x g for 60 min at 4°C.The pellet was washed with
0.25 M sucrose containing 1.5% potassium chloride and 10 HIMEDTA.
This pellet was considered to be microsomal fraction and was suspended
in 0.25 M sucrose. The supernatant was again centrifuged at 105,000
4 The abbreviations used are: GSH, reduced form of glutathione;
oxidized glutathione.
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GSSG,
DETOXIFYING
ENZYMES IN ESTRADIOL-TREATED
x g for 60 min and considered to be cytosol. Protein concentrations
were determined by the method of Bradford (10).
HAMSTER KIDNEY
spectrophotofluorometer. Fluorescence intensity was calibrated with a
solution of quinine sulfate (l Mg/ml in 0.1 N sull'urie acid) measuring
1750 relative fluorescence units.
Enzyme Assays
Catatase. Catalase activity was assayed by the method of A. Claiborne
(11). The assay mixture consisted of 0.05 M phosphate buffer (pH 7.0),
0.019 M hydrogen peroxide, and postnuclear supernatant in a final
volume of 3.0 ml per measurement. Changes in absorbance were re
corded at 240 nm. Catalase activity was calculated in terms of nmol of
hydrogen peroxide consumed/mg of protein/min.
Superoxide Dismutase. Superoxide dismutase activity was measured
by the method of Beyer and Fridovich (12). Reaction mixtures con
tained 0.05 M phosphate buffer (pH 7.8), 0.1 HIM EDTA, 10 ^M
ferricytochrome c, 50 ^M xanthine, 6.25 nM xanthine oxidase, and
cytosol in a total volume of 3.0 ml at 25°C.Inhibition of cytochrome c
reduction was monitored at 550 nm. One unit of Superoxide dismutase
is defined as that amount which causes 50% inhibition of the initial
rate of reduction of cytochrome c.
Glutathione Peroxidase. Glutathione peroxidase was measured ac
cording to the procedure described by Mohandas et al. (13). The assay
mixture consisted of 0.05 M phosphate buffer (pH 7.0), 1 mM EDTA,
1 mM sodium azide, 1 unit of glutathione reducÃ-ase,1 mM GSH, 0.2
mM NADPH, cytosol, and either 0.25 mM hydrogen peroxide or 1.5
mM eumene hydroperoxide in a final volume of 1.0 ml. Disappearance
of NADPH at 340 nm was recorded at 25°C.The activity with hydrogen
RESULTS
Glutathione Peroxidase Activity. Since glutathione metabo
lism plays a significant role in the detoxification of cellular
reactive intermediates [reviewed by Ketterer et al. (19)], en
zymes and substrates involved in these processes were measured
in hamster kidney prior to estrogen-induced tumorigenesis. In
cortical cytosol obtained from estradiol-exposed kidney, the
target organ of hormonal carcinogenesis in hamsters, the activ
ity of glutathione peroxidase I was increased over activities in
untreated age-matched controls by approximately 45% after 1
to 2 mo of treatment and by 77% after longer exposures to
estradiol (Fig. 1). Comparable changes were observed with
glutathione peroxidase II. After 1 to 4 mo of hormone admin
istration, increases in activity of glutathione peroxidase II
ranged from 55 to 114% over those observed in age-matched
untreated kidneys (Fig. 2). At all 3 times (1, 2, and 4 mo)
measured, the changes in response to hormone treatment were
statistically significant for both peroxidases.
peroxide as substrate represented glutathione peroxidase I, and the
difference between eumene hydroperoxide- and hydrogen peroxideinitiated activities represented glutathione peroxidase II.
Glutathione ReducÃ-ase.Glutathione reducÃ-asewas estimaled by ihe
method of Carlberg and Mannervik (14) as modified by Mohandas et
al. (13). The assay mixlure consisted of 0.1 M phosphate buffer (pH
7.6), 0.1 mM NADPH, 0.5 mM EDTA, 1 mM GSSG, and cytosol in a
final volume of 1.0 ml. The enzyme activity was quantitaled at 25°Cby
measuring the disappearance of NADPH at 340 nm and was expressed
as nmol of NADPH oxidized/mg of protein/min.
Glutathione Transferase. Glutathione transferase activity was meas
ured by the method of Habig et al. ( 15). The reaction mixture consisted
of 0.1 M phosphate buffer (pH 6.5), 1 mM GSH, 1 mM l-chloro-2,4dinitrobenzene, and cytosol in a final volume of 1.0 ml. A similar assay
method was used for the assay with 1 mM 3,4-dichloronitrobenzene as
substrate in 0.1 M phosphate buffer (pH 7.5) with 5 mM GSH and
sample. Using l,2-epoxy-3-(/>-nitrophenoxy)propane as substrate, the
reaction was carried out similarly in 0. l M phosphate buffer (pH 6.5)
with 5 mM GSH, cytosol, and 0.5 mM epoxide. The enzyme activity
was expressed as nmol of product formed/mg of protein/min.
•¿Y-Glutamyl
Transpeptidase. 7-GlutamyI transpeptidase was meas
ured by the method of Tate and Meister (16) as modified by Mohandas
et al. (13). The assay mixture contained 0.05 M Tris-HCl (pH 8.0), 20
mM glycyl glycine, 75 mM sodium chloride, mitochondria! or microsomal extract, and 2.5 mM L-7-glutamyl-p-nitroanilide in a final volume
of 1.0 ml. Increases in absorbance at 410 nm were recorded, and enzyme
activity was expressed as nmol of product formed/mg of protein/min.
Glucose-6-phosphate Dehydrogenase. This enzyme was measured by
the method of Baquer and McLean (17). The assay mixture contained
0.1 M glycyl glycine (pH 7.6), 0.1 M magnesium chloride, 2 mg/ml of
NADP, 0.05 M glucose-6-phosphate, and cytosol. Changes in absorb
ance were recorded at 340 nm, and the enzyme activity was calculated
as nmol of NADP reduced/mg of protein/min.
GSH and GSSG. GSH and GSSG were measured by the method of
Fariss and Reed (7). Freshly obtained hamster liver or kidney tissues
(100 mg each) were homogenized in 10% perchloric acid containing 1
mM bathophenanthrolinedisulfonic acid. The acid extracts were derivatized as described (7) and analyzed by high-pressure liquid chromatography.
Fluorescent Lipid Peroxidation Products. Fluorescent damage prod
ucts of lipid peroxidation were measured by the assay of Dillard and
Tappel (18). Livers and kidneys of control hamsters and of animals
treated with estradiol for 1 mo were extracted with chloroform:methanol (2:1, v:v). Fluorescence intensity was determined from
spectra recorded in the uncorrected mode using an Aminco-Bowman
***11Ii*^!_L\
400-E
Olo
l
M
LJ
LJU
PEROXIDASE
oGLUTATHION
o
protein/minM
nmoles/mcUl
O
Ulo
or
1
2
TREATMENT WITH E2
4
Fig. 1. Activity of glutathione peroxidase I in the kidney of male Syrian
hamsters treated with estradiol. Male Syrian hamsters were treated with s.c.
implants of estradiol for 1, 2, or 4 mo (values along abscissa). Age-matched
untreated animals served as controls. Hamster kidneys were excised immediately
after decapitation and separated into cortical and medullary tissue. Glutathione
peroxidase I activity with hydrogen peroxide as substrate was measured in cortical
cytosol by the method of Mohandas et al. (13). Q, control values; D. values
obtained from renal cortical cytosol exposed to estradiol (EJ (n = 8 to 12). *, P
< 0.02; **, P < 0.005.
200 T
ASin
15°-X
8\
cGLUTATHIONE
nmoles/mg
proteilcn
/l
PERO
oD
0 pLT*•T'ifÌJL/
1
2
TREATMENT WITH E2
4
Fig. 2. Activities of glutathione peroxidase II in the kidney of male Syrian
hamsters treated with estradiol. Renal cortical cytosol obtained as described in
Fig. 1 was examined for glutathione peroxidase II activity by the method of
Mohandas et al. (13). Enzyme activity was measured using eumene hydroperoxide
as substrate. The difference between eumene hydroperoxide- and hydrogen per
oxide-initiated activities was taken as that of glutathione peroxidase U.U. control
values; G, values obtained from renal cortical cytosol exposed to estradiol (/•.';)
(n
= 8 to 12). Values along abscissa, months. *, P < 0.001; **, P < 0.03.
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DETOXIFYING
ENZYMES IN ESTRADIOL-TREATED
In contrast to changes in kidney, cytosolic enzyme activities
in liver, which is not a target organ under these experimental
conditions, were lowered as a result of hormone administration.
Hepatic glutathione peroxidase I was decreased 62% after ex
posure for 1 mo (Fig. 3). After an extended treatment period
(2 and 4 mo), glutathione peroxidase I activity recovered to a
range of 600 to 690 nmol/mg of protein/min, but was still
lower than that in untreated controls by approximately 25%. A
similar trend in activity changes was observed for hepatic glu
tathione peroxidase II (Fig. 4). The initial decrease in enzyme
activity in liver cytosol after a short estradiol exposure (1 mo)
was 57% but recovered to 33% after 2 mo and 24% below
control after 4 mo of chronic estrogen treatment, respectively.
As was observed for the kidney, changes in activity of glutathi
one peroxidases I and II as a result of estrogen treatment were
statistically significant at all time points when compared to
untreated controls. Hepatic enzyme activities in untreated ham
sters were in good agreement with values found previously (20).
When comparing cytosolic enzyme activities in liver with
those in kidney, it must be noted that, in untreated and in
estrogenized animals, hepatic activities of glutathione peroxi
dases I and II were markedly higher than those of the renal
enzymes. For instance, glutathione peroxidase I in untreated
hamster kidneys ranged from 117 to 196 nmol/mg of protein/
min which underwent an increase in response to estradiol
1000 T
800
i{
"I
2
600
«"
400°£
HAMSTER KIDNEY
treatment to 165 to 347 nmol/mg of protein/min (Fig. 1). In
contrast, hepatic glutathione peroxidase I decreased from a
level of 783 to 865 nmol/mg of protein/min to 331 nmol/mg
of protein/min (1 mo of estradiol), but then recovered to levels
of 602 to 688 nmol/mg of protein/min after prolonged treat
ment (Fig. 3). Differences between renal and hepatic activities
of glutathione peroxidase II were even more pronounced (see
Figs. 2 and 4).
Activities of Glutathione Metabolism Enzymes. The extraor
dinary changes in glutathione peroxidase activity preceding
estrogen-induced renal tumor formation in the hamster
prompted an expanded investigation of other enzymes involved
in glutathione metabolism. Activities of glutathione reducÃ-ase,
7-gIutamyl transpeptidase, glutathione transferase, and glucose-6-phosphate dehdyrogenase were assayed in the kidney
(Table 1) and liver (Table 2). Activities of these enzymes in
untreated control liver (Table 2) were comparable with those
published previously for the hamster (20). Estradiol treatment
for 1 mo (Tables 1 and 2) and longer time periods (data not
shown) did not result in any significant changes in enzyme
activities when compared to controls. It was thus demonstrated
that treatment of hamsters with estradiol did not result in
changes in activities of glutathione metabolism enzymes except
glutatione peroxidases I and II as shown above.
Levels of Glutathione. Because of the marked changes in
glutathione peroxidase activities in response to chronic admin
istration of estradiol, levels of GSH and GSSG were measured
in livers and kidneys of estrogen-exposed and control hamsters.
The method of Fariss and Reed (7) was used which allows
simultaneous determination of GSH and GSSG. GSH concen
trations in control liver (3.09 ^mol/g of wet tissue in 4-mo-old
animals), obtained by this procedure (Table 3), were slightly
lower than those determined previously (5.43 ^mol/g of liver)
by Igarashi et al. (20). The product isolation and analysis by
high-pressure liquid chromatography (7) used here likely re-
200-
Table1 Activities
ofenzymesregulatingGSHmetabolismin thekidneyof
TREATMENT WITH E2
Fig. 3. Activities of glutathione peroxidase I in the liver of male Syrian
hamsters treated with estradiol. Liver cytosol of hamsters treated as described in
Fig. 1 was examined for glutathione peroxidase I activity using hydrogen peroxide
as substrate by the method of Mohandas et al. (13). D, control values; Ü,values
obtained from hepatic cytosol exposed to estradiol (/•/•)
(n = 8 to 12). Values
along abscissa, months. *,P< 0.001; **, P < 0.04.
hamsters treated with estradiol for I mo
Male Syrian hamsters (10 to 12 animals/group) were treated with one s.c.
implant of estradiol for 1 mo. The treated and age-matched untreated hamsters
were decapitated, and their kidneys were separated into cortical and medullary
tissue. Enzyme activities were measured in the subcellular fractions of the cortical
tissue. Enzyme assays were performed as described in "Materials and Methods."
7-Glutamyl transpeptidase was determined in microsomes, and all other enzyme
activities were assayed in cytosol.
Activity (nmol/mg of pro
tein/min)
1000
Enzyme
Glutathione reducÃ-ase
Glucose-6-phosphate dehydrogenase
1-Glutamyl transpeptidase
Glutathione transferase
•¿
Mean ±SD (n = 6).
Lu
<I
800
|
IE
600 •¿â€¢
400-
Control
14.0 ±3°
19.4 ±5
1266 ±85
120 ±8
Estradiol
treated
14.5 ±4
21.5 ±2.5
1200 ±61
150 ±12
Table 2 Activities of enzymes regulating GSH metabolism in the liver of
hamsters treated with estradiol for 1 mo
Livers of hamsters described in Table 1 were excised, and microsomal (yglutamyl transpeptidase) or cytosolic enzyme activities were assayed as described
in "Materials and Methods."
5ll I 200o c
Activity (nmol/mg of pro
tein/min)EnzymeGlutathione
TREATMENT WITH E2
Fig. 4. Activities of glutathione peroxidase II in the liver of male Syrian
hamsters treated with estradiol. Liver cytosol of hamsters treated as described in
Fig. I was examined for glutathione peroxidase II activity using eumene hydroperoxide as substrate (13). The difference between eumene hydroperoxide- and
hydrogen peroxide-initiated activities was taken as that of glutathione peroxidase
II. D, control values; W, values obtained from hepatic cytosol exposed to estradiol
8 to 12). Values along abscissa, months. r.P< 0.002; *', P < 0.01.
reducÃ-ase
Glucose-6-phosphate dehydrogenase
•¿y-Glutamyl
transpeptidase
221°
Glutathione transferaseControl38.5
Mean ±SD (n = 6).
treated38.0
±6a
±4
117 + 21
123 ±17
130± 12
150 ±29
2308 ±159Estradiol
2418 ±
1477
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DETOXIFYING
ENZYMES IN ESTRADIOL-TREATED
suited in values slightly below those obtained by the fluorometric method used by Igarashi et al. (20). Liver GSH concen
trations were lower in younger animals (Table 3). Estrogen
treatment for 1 and 4 mo significantly increased renal GSH
and GSSG levels. Hepatic GSH concentrations increased by
38% after 1 mo of estradiol over those in age-matched untreated
controls, but were unchanged after longer hormone treatment
(Table 3). Hepatic GSSG levels were unchanged after 1 mo of
estradiol, but were 25% less than the levels in untreated agematched controls during prolonged hormone exposure. The
differences observed in the liver were not statistically signifi
cant.
Activities of Catalase and Superoxide Dismutase. Toxic con
centrations of hydrogen peroxide may be metabolized by glutathione peroxidase I or by catalase. The activity of the latter
enzyme was investigated to obtain a more detailed understand
ing of oxidative stress in estrogen-exposed tissues prior to
tumor formation. Catalase activity in untreated Syrian hamster
kidney was found to be in the range of 280 to 325 nmol/mg of
protein/min (Fig. 5). In response to estradiol treatment, enzyme
activity was decreased by 55% after 1 mo. After 2 and 4 mo of
chronic hormone administration, catalase activity recovered
(34% and 24% below control, respectively) but did not reach
levels prevailing in untreated kidney.
In contrast, catalase activities in livers (Fig. 6) of untreated
animals were much higher (716 to 918 nmol/mg of protein/
min) than those found in kidney and increased even further in
response to estrogen treatment. After 1 mo of hormone expo-
IBOOi
c 1600Ë1^1400p Õ12001000-ÜJ(g
< S
800-^
1"
> 600l—
•¿>
< o
400-c
200n._i_*\—J—**\l,»1
1
tissueOrganKidneyLiverTreatment1
mo of estradiol
1 mo of control
4 mo of estradiol
4control1
mo of
»imol/gofwet
±0.24"-*
0.70 ±0.10
1.16 ±0.07e
±0.100.95
0.82
mo of estradiol
1 mo of control
0.69
2.9
4 mo of estradiol
4 mo of controlGSH1.28 3.1
" Mean ±SD (n = 4 to 6).
*/><0.01.
±0.01Ie
0.052 ±0.01 1
0.079 ±0.01 6C
110.0370
0.046 ±0.0
±0.15
±0.006
±0.13
0.037 ±0.006
±0.80
0.1 10 ±0.029
±0.9GSSG0.0720.146 ±0.035
fP<0.05.
350-•
JL
_L
Ã
o. 200 •¿
!< I
0 E
i
y,
10°
50
1
2
TREATMENT WITH E2
4
Fig. 5. Activities of catalase in the kidney of male Syrian hamsters treated
with estradiol. Postnuclear supernatant (1000 x g) from renal cortex obtained as
described in Fig. 1 was examined for catalase activity by the method of Claiborne
(11). G, control values; Ü,values obtained from renal cortical cytosol exposed to
estradiol (E2) (n = 8 to 12). Values along abscissa, months. *, P < 0.001 ; **, P <
0.02.
2
TREATMENT WITH E2
4
Fig. 6. Activities of catalase in the liver of male Syrian hamsters treated with
estradiol. Hepatic postnuclear supernatant (1000 X />) of hamsters treated as
described in Fig. 1 was examined for catalase activity by the method of Claiborne
(11). D. control values; D, values obtained from hepatic cytosol exposed to
estradiol (£2)(n = 8-12). Values along abscissa, months. *,P< 0.001; **, P <
0.04.
25 T
ç
20-S
°-15o<¿f
Tl*T1l1
10-c35-
Table3 ContentofGSHand GSSGin theliverandkidneyofhamsterstreated
withestradiol
Hamsters were treated with estradiol for 1 and 4 mo. After decapitation of the
animals, livers or kidneys were immediately homogenized in 10% perchloric acid
containing 1 HIM bathophenanthrolinedisulfonic
acid. The homogenates were
derivatized and analyzed by the method of Fariss and Reed (7).
HAMSTER KIDNEY
0-_LÃŒIT
2
TREATMENT WITH E2
4
Fig. 7. Activities of Superoxide dismutase in the kidney of male Syrian ham
sters treated with estradiol. Renal cortical cytosol obtained as described in Fig. 1
was examined for Superoxide dismutase (SOD) activity by the method of Beyer
and Fridovich (12). D, control values; D. values obtained from renal cortical
cytosol exposed to estradiol (£2)(n = 8 to 12). Values along abscissa, months. *,
P < 0.01.
sure, hepatic catalase activity was elevated to a level of 64%
over that found in untreated animals. After longer treatment
periods, hepatic catalase activities remained elevated by about
17 to 21% over those found in untreated control liver. With
these measurements it was demonstrated that catalase activities
in the liver and kidney were altered in a profound manner in
response to estradiol treatment. After prolonged exposure to
hormone (2 to 4 mo), enzyme activities approached levels found
in the unexposed control organ, but activity differences re
mained statistically significant.
Superoxide dismutase activity was also assayed in hamster
kidney and liver. This enzyme activity in the kidney increased
with the age of the animals [from 8 to 16 units/mg of protein
(Fig. 7)]. Estrogen treatment of the animals did not cause any
significant differences compared to untreated control activity.
Hepatic Superoxide dismutase activity in untreated hamsters
increased from 72 ±8 to 85 ±8 units/mg of protein over the
same time period. As was observed in the kidney, no significant
changes were observed in response to estrogen treatment (79 ±
11 units/mg of protein after 1 mo and 95 ±5 units/mg of
protein after 4 mo of chronic estradiol administration).
Fluorescent Products of Lipid Peroxidation. Lipid peroxidation, assayed by reaction of thiobarbituric acid with malondialdehyde (21 ), was not changed in livers or kidneys of hamsters
in response to estrogen treatment (data not shown). However,
the more stable fluorescent products of lipid peroxidation (18)
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DETOXIFYING
ENZYMES IN ESTRADIOL-TREATED
increased 111% in the kidney of estrogen-treated hamsters over
control values. The increase in livers, which do not develop
tumors under these conditions, was 12% over control values.
DISCUSSION
Changes in Enzyme Activities. Treatment of hamsters with
estrogen alters activities of hepatic and renal catalase and
glutathione peroxidase, but not Superoxide dismutase. These
results indicate an estradiol-induced imbalance of enzymatic
defenses against oxidative damage. An effective organismal
defense against oxidant damage has been postulated (22, 23) to
require balanced increments of Superoxide dismutase, glutathi
one peroxidase, and catalase activities together rather than
increases in the activity of single enzymes. Increased cell death
was observed (22) in Superoxide dismutase-rich bacteria under
oxidant stress. This cell death may be a result of increased
hydrogen peroxide concentrations. As discussed above, the
concept of balance of antioxidant enzymes also is useful when
applied to biochemical changes preceding estrogen-induced
renal carcinogenesis in the hamsters. Estradici treatment alters
the activity of glutathione peroxidase and catalase in both liver
and kidney in reciprocal fashion. The exact reason for this
differential induction/activation
or inhibition/inactivation
of
glutathione peroxidase or catalase activities remains to be
understood. Previously, it has been suggested that acute gen
eration of oxidant stress leads to induction/inhibition of an
tioxidant enzymes (24-26). Moreover, significant increases in
cardiac glutathione peroxidase activity were observed in re
sponse to chronic Adriamycin treatment in rats and were taken
as an indicator of increased oxidant stress in the target organ
of Adriamycin toxicity (27). Thus, an elevated activity of renal
glutathione peroxidase is likewise considered to be an indication
of increased oxidant stress in the hamster kidney in response
to estrogen treatment.
Changes in Lipid Peroxidation. The increased oxidative stress
in hamster kidney in response to estradici as postulated above
is illustrated by the increase in fluorescent products of lipid
peroxidation (Table 4). The absence of elevated concentrations
of malondialdehyde in tissues of estrogen-treated hamsters
merely indicates that this substance is an unstable intermediate
in the peroxidation sequence of unsaturated fatty acids and that
it may be metabolized and/or exported (28). The presence of
fluorescent products has previously been taken as an indicator
of oxidative stress induced by chronic treatment with Adria
mycin (27). Changes in malondialdehyde levels as measured by
a thiobarbituric acid assay were likewise not observed in that
study (27). Fluorescent lipid peroxidation products have previ
ously been shown to be similar to lipofuscin pigments (29).
Lipofuscin is considered to be an index of peroxidative damage
(30). Lipofuscin-like pigment accumulates in proximal tubules
in kidneys of rats treated with estradiol (31).
Table 4 Fluorescent lipid peroxidation products in kidneys and livers of hamsters
treated with estradiol far I mo
Fluorescent lipid peroxidation products were measured in kidneys and livers
by the method of Dillard and Tappel (18). A quinine sulfate standard (1 fig/ml of
0.1 N sulfuric acid) measured 1750 relative fluorescence units.
Relative fluorescence units/g of
wet tissue
OrganKidney
(65-85)'
LiverControl76° 354 (330-370)Estradiol
" Mean of four animals.
* Numbers in parentheses, range of means.
treated160(150-166)
398 (382-420)
HAMSTER KIDNEY
Changes in Glutathione. Levels of GSH and GSSG in liver
and kidney were measured in the expectation of uncovering
indications of increased stress on the pool of reduced glutathi
one specifically in kidneys of estrogenized hamsters. The con
centrations of GSSG specifically in the kidney were signifi
cantly elevated over those found in age-matched untreated
controls. Increases in GSSG concentrations have previously
been used as an indicator of acute oxidant stress (32-34). At
both time points examined, levels of GSH were also elevated
over control concentrations. Increases of total glutathione have
been observed previously (21, 27, 35) after chronic treatment
with Adriamycin and have been taken as an indicator of in
creased oxidative stress. In the liver, GSH and GSSG levels in
estradiol-treated animals were not significantly different from
controls. These observations support the conclusions derived
from enzyme activity measurements that, during estrogen treat
ment, hepatic cells remain sufficiently protected from any oxi
dant stress by both enzymatic and nonenzymatic defenses. In
the kidney, however, the increases in glutathione levels, in
activity of glutathione peroxidase, and in concentrations of
products of lipid peroxidation all point towards elevated levels
of oxidant-induced stress. Whether these events play a causative
role in tumor induction by estrogen or represent a defensive
response to an oxidative challenge remains to be ascertained.
ACKNOWLEDGMENTS
The authors thank Ewa Paszkiewicz for the determination of GSH
and GSSG concentrations, Dimitrios Dogramatzis for supplying the
hamsters with estradiol implants, and Dr. Donald J. Reed, Oregon
State University, Corvallis, OR, for valuable discussions.
REFERENCES
1. Kirkman, H. Estrogen-induced tumors of the kidney. Growth characteristics
in the Syrian hamster. Nati. Cancer Inst. Monogr., 1:1-57, 1959.
2. Mehr. J. C... Randerath, K.. and Randerath, E. Target organ-specific covalent
DNA damage preceding diethylstilbestrol-induced carcinogenesis. Carcino
genesis (Lond.), 6: 1067-1069, 1985.
3. Liehr, J. G., Avitts, T. A., Randerath, E., and Randerath. K. Estrogeninduced endogenous DNA adduction: possible mechanism of hormonal can
cer. Proc. Nati. Acad. Sci. USA, 83: 5301-5305, 1986.
4. Liehr,J.G.,Ulubelen,A.A.,andStrobel,H.W.CytochromeP-450-mediated
redox cycling of estrogens. J. Biol. Chem., 261: 16865-16870, 1986.
5. Roy, 1).. and Liehr, J. G. Temporary decrease in renal quinone reducÃ-ase
activity induced by chronic administration of estradiol to male Syrian ham
sters: increased Superoxide formation by redox cycling of estrogen. J. Biol.
Chem., 263: 3646-3651, 1988.
6. Ulubelen, A. A., Liehr, J. G., and Strobel, H. W. Microsomal target organspecific redox cycling of estrogens. Proc. Am. Assoc. Cancer Res., 28: 134,
1987.
7. Fariss, M. W., and Reed, D. J. High performance liquid chromatography of
thiols and disulfides: dinitrophenol derivatives. Methods Enzymol., 143:
101-109, 1987.
8. Liehr, J. G., and Wheeler, W. J. Inhibition of estrogen-induced renal carci
noma in Syrian hamsters by vitamin C. Cancer Res., 43: 4638-4642, 1983.
9. Liehr, J. G. 2-Fluoroestradiol: separation of estrogenicity from carcinogenicity. Mol. Pharmacol., 23: 278-281, 1983.
10. Bradford, M. M. A rapid and sensitive method for the quantification of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem., 72: 248-254, 1976.
11. Claiborne, A. Catalase activity. In: R. A. Greenwald (ed.), CRC Handbook
of Methods for Oxygen Radical Research, pp. 283-284. Boca Raton, FL:
CRC Press, 1985.
12. Beyer, W. F., and Fridovich, I. Assaying for Superoxide dismutase activity:
some large consequences of minor changes in conditions. Anal. Biochem.,
161: 559-566, 1987.
13. Mohandas, J., Marshall, J. J., Duggin, G.G., Horvath, J. S., and Tiller, D.
D. Differential distribution of glutathione and glutathione-related enzymes
in rabbit kidney: possible implications in analgesic nephropathy. Cancer Res.,
«.•5086-5091,1984.
14. Carlberg, I., and Mannervik, B. Glutathione reducÃ-aselevels in rat brain. J.
Biol. Chem., 250: 5475-5480, 1975.
15. Habig, W. H., Pabst, M. J., and Jakoby, W. B. Glutathione 5-transferases.
The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249:
7130-7139, 1974.
1479
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research.
DETOXIFYING
ENZYMES IN ESTRADIOL-TREATED
16. Täte,S. S., and Meister, A. -y-Glutamyl transpeptidase from kidney. Methods
Enzymol., 113: 400-419, 1974.
17. Baquer, N. I., and McLean, P. Evidences for the existence and functional
activity of pentose phosphate pathway enzymes in the large panicle fraction
isolated from rat tissues. Biochem. Biophys. Res. Commun., 46: 167-174,
1972.
18. Dillard, C. .1., and Tappel, A. !.. Fluorescent damage products of lipid
peroxidation. Methods Enzymol., 105: 337-341, 1984.
19. Ketterer, B., Coles, B., and Meyer, D. J. The role of glutathione in detoxication. Environ. Health Perspect., 49: 59-69, 1983.
20. Igarashi, T., Saloli. T., Ueno, K., and Kitagawa, H. Species difference in
glutathione level and glutathione related enzyme activities in rats, mice,
guinea pigs, and hamsters. J. Pharm. Dyn., 6: 941-949, 1983.
21. Jackson, J. A., Reeves, J. P., Muntz, K. H., Kruk, D., Prough, R. A.,
Willerson, J. T., and Buja, L. M. Evaluation of free radical effects and
catecholamine alterations in adriamycin cardiotoxicity. Am. J. Pathol., 117:
140-153, 1984.
22. Scott, M. D., Meshnick, S. R., and Eaton, J. W. Superoxide dismutase-rich
bacteria: paradoxical increase in oxidant toxicity. J. Bini. Chem., 262:36403645, 1987.
23. Nagata, ( '.. Kodama, M., loki, Y., and Kimura, T. Free radicals produced
from chemical carcinogens and their significance in carcinogenesis. In: R. A.
Floyd (ed.), Free Radicals and Cancer, pp. 1-62. New York: Marcel Dekker,
1982.
24. Perchellet, E. M., Maatta, E. A., Abney, N. L., and Perchellet, J. P. Effects
of diverse intracellular thiol delivery agents on glutathione peroxidase activ
ity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase
induction in isolated mouse epidermal cells treated with 12-O-tetradecanoylphorbol-13-acetate. J. Cell. Physiol., 131: 64-73, 1987.
HAMSTER KIDNEY
25. Chow, C. K., and Tappel, A. L. Activities of pentose shunt and glycolytic
enzymes in lungs of ozone-exposed rats. Arch. Environ. Health, 26: 205208, 1973.
26. Whiteside, C., and Hassan, H. M. Induction and inactivation of catatase and
Superoxide dismutase of Escherichia coli by ozone. Arch. Biochem. Biophys.,
257:464-471,1987.
27. Thayer, S. W. Evaluation of tissue indicators of oxidative stress in rats treated
chronically with Adriamycin. Biochem. Pharmacol., 37: 2189-2194, 1988.
28. Leibovitz, B. £.,and Siegel, B. V. Aspects of free radical reactions in
biological systems: aging. J. Gerontol., 35:45-56, 1980.
29. Tappel, A. L. Measurement of and protection from in vivo lipid peroxidation.
In: W. A. Pryor (ed.). Free Radicals in Biology, Vol. 4, pp. 1-47. New York:
Academic Press, 1980.
30. Chance, B., Sies, II, and Boveris, A. Hydroperoxide metabolism in mam
malian organs. Physiol. Rev., 59: 527-603, 1979.
31. Harris, C. A lipofuscin-like pigment in the kidneys of estrogen treated rats.
Arch. Pathol., 82: 353-355, 1966.
32. DiMonte, D., Ross, D., Bellomo, G., Eklow, L., and Orrenius, S. Alterations
in intracellular thiol homeostasis during the metabolism of menadione by
isolated rat hepatocytes. Arch. Biochem. Biophys., 235: 334-342, 1984.
33. Reed, D. J. Nitrosoureas. In: H. Sies (ed.), Oxidative Stress, pp. 115-130.
London: Academic Press, 1985.
34. Sies, H., Brigelius, R., and Akerboom, T. P. M. Intrahepatic glutathione
status. In: A. Larsson, S. Orrenius, A. Holmgren, and B. Mannervik (eds.),
Functions of Glutathione, Biochemical, Physiological, Toxicological and
Clinical Aspects, pp. 51-64. New York: Raven Press, 1983.
35. Tomlinson, C. W., Godin, D. V., and Rabkin, S. W. Adriamycin cardiomyopathy: implications of cellular changes in a canine model with mild impair
ment of left ventricular function. Biochem. Pharmacol., 34: 4033-4041,
1985.
1480
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Changes in Activities of Free Radical Detoxifying Enzymes in
Kidneys of Male Syrian Hamsters Treated with Estradiol
Deodutta Roy and Joachim G. Liehr
Cancer Res 1989;49:1475-1480.
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