Full text of DOI:10.1207/S15327914NC3601_7

Nutrition and Cancer
ISSN: 0163-5581 (Print) 1532-7914 (Online) Journal homepage: http://www.tandfonline.com/loi/hnuc20
Effects of Bleomycin on Liver Antioxidant Enzymes
and the Electron Transport System From Ad
Libitum-Fed and Dietary-Restricted Female and
Male Fischer 344 Rats
Varsha G. Desai , Anane Aidoo , Jing Li , Lascelles E. Lyn-Cook , Daniel A.
Casciano & Ritchie J. Feuers
To cite this article: Varsha G. Desai , Anane Aidoo , Jing Li , Lascelles E. Lyn-Cook , Daniel A.
Casciano & Ritchie J. Feuers (2000) Effects of Bleomycin on Liver Antioxidant Enzymes and
the Electron Transport System From Ad Libitum-Fed and Dietary-Restricted Female and Male
Fischer 344 Rats, Nutrition and Cancer, 36:1, 42-51, DOI: 10.1207/S15327914NC3601_7
To link to this article: http://dx.doi.org/10.1207/S15327914NC3601_7
Published online: 18 Nov 2009.
Submit your article to this journal
Article views: 29
View related articles
Citing articles: 9 View citing articles
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=hnuc20
Download by: [University of Nebraska, Lincoln]
Date: 16 October 2015, At: 06:13

NUTRITION AND CANCER, 36(1), 42–51
Copyright © 2000, Lawrence Erlbaum Associates, Inc.
Effects of Bleomycin on Liver Antioxidant Enzymes and the
Electron Transport System From Ad Libitum-Fed and
Dietary-Restricted Female and Male Fischer 344 Rats
Varsha G. Desai, Anane Aidoo, Jing Li, Lascelles E. Lyn-Cook,
Daniel A. Casciano, and Ritchie J. Feuers
Abstract: Dietary restriction (DR) is the only known inter-
vention that delays aging and age-related diseases. Mecha-
nisms proposed to explain this DR effect include a decline in
free radical production and an increase in free radical de-
toxification. In the present study the effect of bleomycin
(1). Another important source of reactive oxygen species
may result from oxidation of a variety of cellular and chemi-
cal cytotoxic agents. By damaging proteins, lipids, DNA,
and other macromolecules, these reactive oxygen species
may have a significant role in aging and age-related diseases
such as atherosclerosis, Parkinson’s disease, and Alzhei-
mer’s disease (2–4).
(
BLM) as a reactive oxygen species-generating antitumor
drug has been evaluated on antioxidant enzymes and the
electron transport system in different cellular fractions of
liver in female and male Fischer 344 rats. Animals were fed
ad libitum (AL) or 60% of the AL intake (DR) and were
given a single intraperitoneal injection of 2.5, 5, or 10 mg
BLM/kg body wt. After four weeks, BLM significantly in-
creased glutathione peroxidase and lactate dehydrogenase
activities in liver cytosol of female AL rats and increased ac-
tivity even more in male rats. Similar changes were also
noted for glutathione reductase and glucose 6-phosphate
dehydrogenase activities in BLM-treated AL rats. In liver
mitochondria, glutathione peroxidase was increased in fe-
male and male AL rats but was increased more in female
rats. Drug treatment had no significant effect on these en-
zyme activities in cytosolic or mitochondrial fractions of DR
animals. Profound effects of BLM were noted in activities of
complexes I, III, and IV of the electron transport system in
AL and DR female and male rats; however, complex II dem-
onstrated no significant diet or treatment effect. Induced an-
tioxidant enzyme activities in BLM-treated AL rats may be a
response to excessive free radical generation due to BLM
metabolism in AL animals that is mitigated by DR. Further-
more, dysfunction of the electron transport system might
suggest its role in a secondary generation of free radicals
during BLM metabolism contributing to its toxicity.
The damaging effects of free radicals are controlled to
some extent through cellular enzymatic and nonenzymatic
antioxidant defenses. Glutathione peroxidase, catalase, and
superoxide dismutase, which are considered the primary an-
tioxidant enzymes, are involved in the direct elimination of
reactive oxygen species with the help of secondary enzymes
such as glutathione reductase and glucose 6-phosphate
dehydrogenase. The secondary enzymes help maintain a
steady supply of the metabolic intermediates required by the
primary antioxidant enzymes (5). The nonenzymatic de-
fenses include reduced glutathione, uric acid, bilirubin, and
ubiquinol (4). Despite these systems, accumulation of oxida-
tive damage may result in altered tissue function and ulti-
mately affect health status.
This reactive oxygen species-mediated degradation of bi-
ological systems might be prevented by maintaining the bal-
ance between free radical production and its detoxification.
Dietary restriction (DR) has been shown to reduce free radi-
cals in biological systems by increasing their removal (6–8)
and/or by lowering their production (9). This effect of DR
may contribute to its ability to extend the maximum achiev-
able life span in rodents and in other organisms (10) and also
delay various age-related disorders.
Bleomycin (BLM) is a radiomimetic drug used in the
treatment of different types of cancer. BLM cytotoxicity has
been demonstrated to be a result of reactive oxygen species
generated during its metabolism, where BLM interacts with
molecular oxygen and iron, producing superoxide anion and
other oxygen metabolites (11). Genotoxic effects of BLM
include the release of free base residues and the creation of
single- and double-strand breaks in nuclear (12,13) as well
Introduction
Aerobic organisms are continuously under attack by reac-
tive oxygen species generated from cellular sources such as
neutrophils, macrophages, and oxidative phosphorylation
The authors are affiliated with the Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Food and Drug
Administration, Department of Health and Human Services, Jefferson, AR 72079.

as mitochondrial DNA (14). The genotoxic effects of BLM
have primarily been studied with regard to nuclear DNA.
Thus little information is available regarding BLM-induced
oxidative injury to mitochondrial DNA. Unlike nuclear
DNA, mitochondrial DNA lacks introns and protective his-
tones (4). Also, mitochondria are capable of limited DNA
repair (4). In addition to these characteristics, its proximity
to the respiratory chain makes mitochondrial DNA a more
likely target for free radical attack.
according to the method of Ji and co-workers (17). The buffer
was discarded, and the tissues were homogenized in five vol-
umes of ice-cold homogenization buffer with a motor-driven
Teflon pestle. The homogenates were centrifuged twice at
1,000 g for 10 minutes. The pellets were discarded, and
supernatants were recentrifuged at 12,000 g for 20 minutes.
The resultant pellets were resuspended in 0.5 ml of homoge-
nization buffer and labeled as “mitochondrial fractions.” The
supernatants were subjected to further centrifugation at
Mitochondrial DNA is a double-stranded, closed circular
molecule that encodes 11 subunits of the electron transport
system complexes. The mitochondrial electron transport sys-
tem is composed of four complexes: complex I (NADH-ubi-
quinone oxidoreductase), complex II (succinate-ubiquinone
oxidoreductase), complex III (ubiquinol-cytochrome c
oxidoreductase), and complex IV (cytochrome c oxidase).
Nuclear and mitochondrial genomes contribute to the elec-
tron transport function by interacting to affect the synthesis
and assembly of the electron transport system proteins (15).
Oxidative damage to these genomes may lead to altered pro-
tein synthesis, ultimately resulting in electron transport dys-
function and, thus, generation of more free radicals. The
present study was carried out to determine whether DR might
inhibit the toxic effects of BLM, which are believed to be me-
diated by reactive oxygen species.
1
05,000 g for 60 minutes. These final supernatants corre-
spond to the “cytosolic fractions.” All the centrifugation steps
were carried out at 4°C, and all the fractions were stored at
–
70°C for subsequent analysis.
Antioxidant enzyme assays: Enzyme activities were
measured spectrophotometrically on a Cobas FARA
autoanalyzer (Roche, Nutley, NJ). Glutathione peroxidase
and glutathione reductase activities were measured according
to Wheeler and colleagues (18). Glutathione peroxidase ac-
tivity was determined using a reaction mixture containing
phosphate buffer (100 mM, 1 mM EDTA, pH 7.0), NADPH
(2.25 mM), glutathione reductase (100 U/ml), and reduced
glutathione (37.5 mM). Activity was measured as a decrease
in absorbance at 340 nm at 30°C as H O (15 mM) was added
2
2
to the reaction mixture containing the tissue fraction.
Glutathione reductase assay was performed by adding the tis-
sue fraction to the reaction mixture containing oxidized
glutathione (3.7 mM) and NADPH (2 mM) in potassium
phosphate buffer (147 mM with 0.74 mM EDTA, pH 7.2).
Activity was measured as the rate of oxidation of NADPH at
Material and Methods
Experimental Design
3
40 nm at 30°C. Glucose 6-phosphate dehydrogenase analy-
Animals: Forty female and 40 male Fischer 344 rats
were obtained from the breeding colony of the National Cen-
ter for Toxicological Research. The maintenance and han-
dling of the animals were based on the Guide for the Care and
Use of Laboratory Animals (16).
sis was carried out according to Bergmeyer (19). The reaction
mixture contained glucose 6-phosphate (6 mM) and NADP
(1.6 mM) in triethanolamine·HCl buffer (172 mM, with 13.8
mM MgCl , pH 7.6). The activity was determined as an in-
2
crease in absorbance at 340 nm at 30°C by addition of cyto-
solic fraction to the mixture. Cytosolic lactate dehydro-
genase activity was measured spectrophotometrically (20)
as oxidation of NADH (1.6 mM) by pyruvic acid (2.39 mM)
in 0.2 M potassium phosphate buffer (pH 7.4) at 30°C. Activ-
ity was expressed in terms of the decrease in absorbance at
Treatment regimen: All 80 animals were weaned at 3
weeks of age and fed the standard NIH-31 diet (containing
.315% vitamin mixture and 0.185% mineral mixture) until
0
they reached 14 weeks of age. The rats were then randomly
assigned to a control group that was fed the standard NIH-31
diet ad libitum (AL) or a group that received 60% of the AL
intake of the NIH-31 diet supplemented with vitamins and a
mineral mixture at 1.67 times that in the standard diet (DR).
AL and DR animals received a single dose of 2.5, 5, or 10 mg
BLM/kg body wt (Sigma Chemical, St. Louis, MO) or the
BLM vehicle, phosphate-buffered saline, pH 7.4, at 18 weeks
of age. Each group consisted of five animals. All animals
340 nm.
Mitochondrial electron transport system enzyme as-
says: Activities of all four electron transport system com-
plexes were determined spectrophotometrically on a Cobas
FARA autoanalyzer by modifications of the method of Ragan
and co-workers (21). Complex I (NADH-ubiquinone oxi-
doreductase) activity was measured by monitoring the oxida-
tion of NADH by ubiquinone-1 at 30°C. The reaction mixture
contained potassium phosphate buffer (10 mM, pH 8.0),
NADH (5 mM), lecithin (15 mg/ml), and the mitochondrial
fraction. The reaction was started by addition of ubi-
quinone-1 (10 mM) to the reaction mixture, and the decrease
in absorbance at 340 nm was monitored. Complex II (suc-
cinate-ubiquinone oxidoreductase) activity was determined
were sacrificed by CO asphyxiation four weeks after
2
mutagen treatment. The livers were aseptically removed and
stored at –80°C.
Tissue preparation: The livers were thawed, weighed,
minced into small pieces, and rinsed twice with ice-cold ho-
mogenization buffer [250 mM sucrose, 5 mM tris(hydroxy-
methyl)aminomethane·HCl (pH 7.4), 1 mM EDTA] at 4°C
Vol. 36, No. 1
43

as the rate of reduction of ubiquinone-2 at 30°C followed by
the secondary reduction of 2,6-dichlorophenolindophenol by
the ubiquinol formed. The reaction mixture contained potas-
sium phosphate buffer (1 mM, pH 7.4), sodium succinate (1
M, pH 7.4), and EDTA (10 mM, pH 7.3). The decrease in
absorbance was measured at 600 nm after addition of a mix-
ture of ubiquinone-2 (2.5 mM), 2,6-dichlorophenolindo-
phenol (4.65 mM), and the mitochondrial fraction to the
mixture described above. Complex III (ubiquinol-cyto-
chrome c oxidoreductase) activity was assayed by following
the rate of reduction of cytochrome c by ubiquinol-2 at 30°C.
The reaction was started by addition of ubiquinol-2 (1 mM) to
the reaction mixture containing potassium phosphate buffer
one peroxidase activity was higher in cytosolic and mito-
chondrial fractions of DR male rats than AL male rats. A sta-
tistically significant (p < 0.001) diet × gender interaction
was noted in mitochondrial glutathione peroxidase.
Similar to glutathione peroxidase, a gender-related sig-
nificant difference in glutathione reductase activity was ob-
served in cytosol, with male rats showing higher activity
than female rats in both diet groups, either treated or un-
treated. Glutathione reductase (Figures 1 and 2) was higher
in all DR animals in both cellular fractions than in their AL
counterparts, with a significant (p < 0.05) increase noted at
2.5 (15%) and 10 (30%) mg BLM/kg in cytosol and in mito-
chondria, respectively, in females. Glutathione reductase ac-
tivity rose significantly in mitochondria of male DR control
rats and male rats treated with 2.5 mg BLM/kg.
In female AL rats, cytosolic glucose 6-phosphate
dehydrogenase activity was not affected by BLM treatment
(
50 mM), EDTA (100 mM), cytochrome c (1 mM), potassium
cyanide (50 mM), and the mitochondrial fraction diluted 1:10
with sucrose (250 mM) and tris(hydroxymethyl)amino-
methane·HCl (10 mM) buffer (pH 7.8). The reduced
cytochrome c was measured as an increase in absorbance at
(Figure 1). However, a significant (p < 0.05) decrease of
>
45% in glucose 6-phosphate dehydrogenase activity was
5
50 nm. Complex IV (cytochrome c oxidase; EC 1.9.3.1) ac-
observed in DR female rats compared with their AL counter-
parts. Enzyme activity was also significantly decreased in
male DR rats treated with 2.5 and 10 mg BLM/kg compared
with their AL counterparts and with untreated DR controls.
However, BLM significantly induced glucose 6-phosphate
dehydrogenase activity at the highest dose in AL male rats
compared with untreated controls.
tivity was measured as the rate of oxidation of reduced
cytochrome c (10 mM) by the enzyme at 550 nm. Total protein
contents of the cytosolic and mitochondrial fractions were
measured by using a kit supplied by Cobas FARA, with bo-
vine serum albumin as a standard. The enzyme activities were
expressedasnanomolesperminute permilligram ofprotein.
Lactate dehydrogenase activity was altered (Figure 3) in
a manner similar to cytosolic glutathione peroxidase in
BLM-treated AL rats of both genders. Diet, dose, and gender
interaction was also noted in lactate dehydrogenase activity
(p < 0.05) in cytosol. Enzyme activity was induced by BLM
in AL male rats by >35% of controls, with the highest two-
fold increase at 5 mg/kg, but not in female rats. No BLM
treatment effect was noted in female or male DR rats. All
DR animals had lower lactate dehydrogenase activity than
their AL counterparts. The decrease was statistically signifi-
cant at all BLM doses tested in male rats, whereas in female
rats the decrease was significant only in untreated DR rats
and in DR rats treated with 5 mg BLM/kg.
Statistical Analysis
A three-way analysis of variance was used to test the
main effect of gender, diet, and dose as well as their interac-
tions. When interactions were significant, main effects were
tested separately using two-way analysis of variance with
post hoc multiple-comparison (Bonferroni) tests between
the control and treated groups. Differences were considered
significant at p < 0.05. Values are means ± SEM.
Results
BLM produced significant (p < 0.05) effects on the activ-
ities of the electron transport system complexes I, III, and
IV, but not complex II, in DR and AL rats (Figure 4). The
diet × gender interaction was statistically significant in com-
plex I. Complex I activity was significantly higher in female
AL rats than in male AL rats. Complex I showed a tendency
toward a dose-dependent increase in its activity in female
and male AL rats due to BLM compared with controls.
However, this increase was significant at the highest dose
only in female rats. DR animals also showed changes similar
to AL animals in female rats, and their activities were lower
than those of their AL counterparts. In contrast, in male rats,
complex I activity was significantly higher in DR rats than in
their AL counterparts. In the case of complex II activity, fe-
male and male rats had a nonsignificant diet × treatment ef-
fect in AL as well as in DR animals. All controls and treated
male rats had significantly (p < 0.001) higher complex II ac-
tivity than female rats.
The results were subjected to three-way analysis of vari-
ance for statistical significance at the 95% confidence level,
and the data are represented graphically in Figures 1–6. A
significant diet, dose, and gender interaction was noted in
cytosolic glutathione peroxidase activity. All male AL and
DR rats treated or untreated with BLM had significantly
higher (p < 0.05) glutathione peroxidase activity than fe-
male rats. Glutathione peroxidase showed tendencies toward
increased activity in BLM-treated AL animals in both gen-
ders compared with controls. However, significant induction
was noted only in cytosol of male rats treated with 5 mg
BLM/kg. Glutathione peroxidase activity was higher in the
cytosolic fraction from AL female rats than in that from DR
female rats. Conversely, glutathione peroxidase activity was
higher in the mitochondrial fraction from DR female rats
than in that from AL female rats. However, these changes
were statistically nonsignificant (Figures 1 and 2). Glutathi-
4
4
Nutrition and Cancer 2000

Vol. 36, No. 1
45

Figure 2. Glutathione peroxidase and glutathione reductase activities in liver mitochondria of AL and DR female and male rats treated with different doses of
BLM. c, p < 0.05 compared with respective AL counterparts.
Figure 3. Lactate dehydrogenase activity in liver cytosol of AL and DR female and male rats treated with different doses of BLM. a, p < 0.05 compared with
AL at 0 mg BLM/kg; c, p < 0.05 compared with respective AL counterparts.
4
6
Nutrition and Cancer 2000

Vol. 36, No. 1
47

Figure 5. Body weights of AL and DR female and male rats treated with different doses of BLM. c, p < 0.05 compared with respective AL counterparts.
Figure 6. Liver weights of AL and DR female and male rats treated with different doses of BLM. c, p < 0.05 compared with respective AL counterparts.
In contrast to complex I activity, a severe decline was ob-
served in the activities of complexes III and IV in
BLM-treated AL and DR rats compared with untreated con-
trols of both genders. This effect was more prominent in fe-
male rats, where the decrease was >45% (p < 0.05) for all
doses of BLM, in contrast to a <40% decrease in male
rats (Figure 4). In female rats, the percent decrease in com-
plex III and IV activities was less in DR than AL rats, in
which activities were reduced to approximately 50% of the
controls. In male rats, complex III activity appeared to be
more severely affected than complex IV activity. All fe-
male DR animals had higher complex III and IV activities
than their AL counterparts, whereas male DR rats had lower
enzyme activities than their respective AL counterparts. All
BLM-treated DR male rats had significantly lower complex
III activity than untreated DR controls. Complex III and IV
activities were significantly higher in male than in female
AL and DR animals. Diet × gender and dose × gender inter-
actions were significant for both of these complexes.
Female and male rats showed a significant diet effect in
their body weights as well as liver weights compared with
their respective AL counterparts (Figures 5 and 6). Body and
liver weights were significantly decreased (p < 0.05) in all
DR animals compared with their AL counterparts. A signifi-
cant gender × diet interaction (p < 0.05) was also noted in
body weights and in liver weights, with male rats showing
larger body as well as liver weights than female rats.
Discussion
BLM is a chemotherapeutic drug used in the treatment of
various types of cancers. Its mode of action appears to be
through the generation of reactive oxygen species in the
presence of iron and oxygen, yielding oxidative damage to
DNA (13). Various approaches have been attempted to re-
duce reactive oxygen species-mediated cytotoxicity in nor-
mal tissues by supplementation of antioxidants (22–24).
4
8
Nutrition and Cancer 2000

However, this strategy has met with little success. DR seems
to limit free radical production, improve detoxification
dehydrogenase has been reported to be higher in AL than in
DR animals when activity is measured at saturated substrate
concentrations (30). This allosteric enzyme does not fully
activate at low endogenous substrate levels in AL tissues,
whereas in DR animals full activation occurs. This seems to
result in excess synthesis of this enzyme as a compensatory
mechanism in AL, whereas DR animals seem to maintain
low levels of total enzyme with higher activity at low sub-
strate concentration.
(
6,25), and improve repair of oxidative damage (7). Addi-
tionally, DR seems to improve xenobiotic- and drug-metab-
olizing systems (26). These actions of DR may contribute
not only to the extension of life span, but also to the reduc-
tion in appearance and degree of numerous pathologies
(
27,28).
Our data demonstrate higher cytosolic glutathione
peroxidase activity in BLM-treated female and male rats.
However, in a previous study we observed a nonsignificant
decrease in glutathione peroxidase activity in cytosol of
BLM-treated male AL rats (24). In this experiment, rats
were kept on a 12:12-hour light-dark cycle, and BLM was
injected 2 hours after lights-off, whereas in the previous
study a light-dark cycle was not maintained. The light-dark
cycle might have changed the physiological condition of the
rats, such that BLM metabolism was altered, thus culminat-
ing in changes in antioxidant enzyme activities. Higher
cytosolic glutathione peroxidase activity in this study could
be the result of higher steady-state levels of reactive oxygen
species due to BLM metabolism within the cytosol of AL ro-
dent liver, leading to enzyme induction. We previously
noted induction of antioxidant activities associated with in-
creased reactive oxygen species during aging in AL animals
Like glucose 6-phosphate dehydrogenase, cytosolic
glutathione reductase activity was not affected by BLM in
AL female rats but was increased in BLM-treated AL male
rats. However, in mitochondria, glutathione reductase
showed a tendency toward an increase in activity in female
and male AL animals. Glutathione reductase levels were
higher in tissues of DR animals than in their AL counter-
parts. Glutathione reductase is responsible for reduction of
the oxidized form of glutathione (GSSG) to its reduced form
(GSH), which is required by glutathione peroxidase. This
glutathione peroxidase-glutathione reductase-catalyzed
GSH-GSSG cycle plays a key role in detoxification of H O
and various organic hydroperoxides, which are generated
primarily in mitochondria (17). Thus higher glutathione
peroxidase and glutathione reductase activities within mito-
chondria of DR animals may provide an advantage in detox-
ification of reactive oxygen species.
Some BLM toxicity may be manifested through mito-
chondrial effects, inasmuch as BLM treatment had signifi-
cant effects on electron transport activity in AL and DR
animals. BLM induced an increase in complex I activity in
AL and DR female and male rats. The mechanism by which
BLM causes an increase in complex I activity is unclear. If
BLM reduced ATP production due to a severely altered
electron transport chain, which is a prime site of ATP gener-
ation, the glycolytic pathway might be induced as a second-
ary source of ATP. This could ultimately lead to more
NADH production, causing substrate-induced higher com-
plex I activity.
In contrast to complex I, BLM caused a significant de-
cline in the activities of complexes III and IV in AL and DR
animals compared with untreated controls of both genders.
However, activities were higher in DR female rats and lower
in DR male rats than in their AL counterparts. The decrease
in activities of complexes III and IV due to BLM exposure in
AL rats indicates that complexes were more severely altered
in female than in male rats. The greater induction of
glutathione peroxidase within mitochondria of female than
of male rats might be in response to higher levels of reactive
oxygen species, which in turn would provide protection
against these mitochondrial effects of BLM. Complex II,
which is totally nuclear in origin, was not affected by BLM
in AL or DR animals. Thus it might be argued that BLM pro-
duces free radical damage at the level of mitochondrial
DNA. Enzyme analyses were carried out four weeks after
treatment; thus it seems unlikely that damage at the protein
or membrane level would yield these responses.
2
2
(
25).
BLM affected cytosolic lactate dehydrogenase in a man-
ner similar to cytosolic glutathione peroxidase in AL rats.
Induction of lactate dehydrogenase activity was >40% in
BLM-treated male AL rats (with a maximum of 91% at 5 mg
BLM/kg) as opposed to only 20% in BLM-treated female
AL rats. This provides evidence that BLM was more toxic in
male than in female rats and suggests a gender-specific ef-
fect of BLM exposure. Supporting this conclusion is the re-
sult of a concurrent mutagenicity experiment with BLM at
the Hprt locus (data not shown) that indicated an increase in
mutant frequency in male compared with female AL rats.
The toxicity was modulated by diet restriction, inasmuch as
lactate dehydrogenase activity was maintained at the control
level in female and male DR rats. Higher cytosolic glutathi-
one peroxidase activities in male DR rats could be responsi-
ble for limiting reactive oxygen species level, consequently
reducing BLM toxicity. A significant decrease was also ob-
served in body and liver weights of DR animals compared
with AL animals, irrespective of BLM treatment. The proc-
ess of cellular attrition might have contributed to the reduc-
tion in body and liver weights of the DR animals. It has been
indicated that the rate of apoptotic cell death is increased in
tissues of DR animals relative to their AL counterparts (29).
However, irrespective of dietary intake, the male rats ap-
peared larger than the female rats.
Cytosolic glucose 6-phosphate dehydrogenase activity
was unaffected by BLM in AL or DR female rats, whereas
activity was induced in male AL rats. However, significantly
lower enzyme activity in all female DR rats than in their AL
counterparts was not unexpected, since glucose 6-phosphate
Vol. 36, No. 1
49

4
5
. Adams, JD, Jr, and Odunze, IN: Oxygen free radicals and Parkinson’s
disease. Free Radic Biol Med 10, 161–169, 1991.
. Rao, J, and Jagadeesan, V: Lipid peroxidation and activities of antioxi-
dant enzymes in iron deficiency and effect of carcinogen feeding. Free
Radic Biol Med 21, 103–108, 1996.
As mentioned earlier, animals in this study also showed
dose-dependent mutant frequency in the Hprt locus. Direct
effects on the mutant frequency at this gene locus might be
the result of oxidative damage. The changes in activity asso-
ciated with complexes I, III, and IV suggest additional BLM
toxicity involving reactive oxygen species-induced mito-
chondrial DNA damage. Because subunits of complexes I,
III, and IV are also encoded by nuclear DNA, the idea that
the responses to BLM seen for complexes I, III, and IV may
involve reactive oxygen species-induced nuclear DNA dam-
age cannot be ruled out. Nevertheless, the fact that no
change occurred in complex II activity at any dose of BLM
strongly suggests some mitochondrial DNA involvement.
The deleterious effects of BLM on complex III and IV ac-
tivity may produce a situation where additional reactive oxy-
gen species are generated from a dysfunctional electron
transport system. We previously reported similar responses
of these respiratory chain complexes during aging, where al-
tered kinetic properties (9) yield an impedance against elec-
tron flow with a concomitant increase in free radical
production during oxidative phosphorylation. If this is the
case, BLM produces genetic damage with multiple and even
long-term consequences. These arguments are further sup-
ported by various in vitro studies that have demonstrated
BLM-induced oxidative damage to mitochondrial DNA,
causing single-strand breaks at multiple sites on dou-
ble-stranded DNA (14) in addition to damage at bases and
abasic sites (31). Unlike nuclear DNA, mitochondrial DNA
has very limited DNA repair (32). Hence, damage persists
longer and stimulates secondary production of reactive oxy-
gen species due to altered electron transport function (33).
In conclusion, moderate restriction of energy intake (40%
less than the ad libitum intake) in BLM-treated rats signifi-
cantly modulated the activities of free radical-scavenging
enzymes as well as the activities of the electron transport
system complexes. This suggests the role of free radical gen-
eration in the metabolism of BLM.
6. Rao, G, Xia, E, Nadakavukaren, MJ, and Richardson, A: Effect of di-
etary restriction on the age-dependent changes in the expression of an-
tioxidant enzymes in rat liver. J Nutr 120, 602–609, 1990.
7
. Koizumi, A, Weindruch, R, and Walford, RL: Influences of dietary re-
striction and age on liver enzyme activities and lipid peroxidation in
mice. J Nutr 117, 361–367, 1987.
8. Feuers, RJ, Weindruch, R, and Hart, RW: Caloric restriction, aging,
and antioxidant enzymes. Mutat Res 295, 191–200, 1993.
9
. Desai, VG, Weindruch, R, Hart, RW, and Feuers, RJ: Influences of age
and dietary restriction on gastrocnemius electron transport system ac-
tivities in mice. Arch Biochem Biophys 333, 145–157, 1996.
1
1
0. Weindruch, R, and Walford, RL: The Retardation of Aging and Dis-
eases by Dietary Restriction. Springfield, IL: Thomas, 1988.
1. Sausville, EA, Peisach, J, and Horwitz, SB: Effect of chelating agents
and metal ions on the degradation of DNA by bleomycin. Biochemistry
7, 2740–2746, 1978.
2. Vig, BK, and Lewis, R: Genetic toxicology of bleomycin. Mutat Res
5, 121–145, 1978.
13. Povirk, LF, and Austin, MJF: Genotoxicity of bleomycin. Mutat Res
57, 127–143, 1991.
4. Lim, LO, and Neims, AH: Mitochondrial DNA damage by bleomycin.
Biochem Pharmacol 36, 2769–2974, 1987.
5. Poyton, RO, and McEwen, JE: Cross talk between nuclear and mito-
chondrial genomes. Annu Rev Biochem 65, 563–607, 1996.
16. US Department of Health and Human Services: Guide for the Care and
Use of Laboratory Animals. Washington, DC: US Govt Printing Of-
fice, 1986. [DHHS (NIH) Publ 8]
7. Ji, LL, Dillon, D, and Wu, E: Alteration of antioxidant enzymes with
aging in rat skeletal muscle and liver. Am J Physiol Regulatory Inte-
grative Comp Physiol 258, R918–R923, 1990.
1
1
5
2
1
1
1
18. Wheeler, CR, Salzman, JA, Elsayed, NM, Omaye, ST, and Korte, DW,
Jr: Automated assays for superoxide dismutase, catalase, glutathione
peroxidase and glutathione reductase activity. Anal Biochem 184,
1
93–199, 1990.
1
9. Bergmeyer, HU: Glucose 6-phosphate dehydrogenase from yeast. In
Methods of Enzymatic Analysis, 2nd ed, HU Bergmeyer (ed). New
York: Academic, 1974, vol 1, pp 458–459.
20. Tietz, TW: Lactate dehydrogenase. In Fundamentals of Clinical
Chemistry. Philadelphia, PA: Saunders, 1970, pp 438.
1. Ragan, CI, Wilson, MT, Darley-Usmar, VM, and Lowe, PN:
Sub-fractionation of mitochondria and isolation of the proteins of oxi-
dative phosphorylation. In Mitochondria: A Practical Approach, VM
Darley-Usmar, D Rickwood, and MT Wilson (eds). Oxford, UK: IRL,
2
Acknowledgments and Notes
1
987, pp 79–112.
The authors thank Drs. Barbara Parsons and Zbigniew Binienda for as-
sistance in the preparation of the manuscript and Jessie B. Collins for ex-
cellent technical assistance. Address correspondence to Dr. Ritchie
Feuers, NCTR/DGRT/HFT-120, Jefferson, AR 72079. Phone: (870)
22. Byers, T, and Geraldine, P: Dietary carotenes, vitamin C and vitamin E
as protective antioxidants in human cancer. Annu Rev Nutr 12,
139–159, 1992.
23. Sarkar, A, Mukharjee, B, and Chaterjee, M: Inhibitory effect of b-caro-
tene on chronic 2-acetylaminofluorene-induced hepatocarcinogenesis
in rat: reflection in hepatic drug metabolism. Carcinogenesis 15,
5
43-7437. FAX: (870) 543-7393. E-mail: rfeuers@nctr.fda.gov.
1
055–1060, 1994.
Submitted 3 June 1999; accepted in final form 1 October 1999.
2
2
4. Desai, VG, Lyn-Cook, LE, Aidoo, A, Casciano, DA, and Feuers, RJ:
Modulation of antioxidant enzymes in bleomycin-treated rats by vita-
min C and b-carotene. Nutr Cancer 29, 127–132, 1997.
5. Luhtala, TA, Roecker, EB, Pugh, T, Feuers, RJ, and Weindruch, R: Di-
etary restriction attenuates age-related increases in rat skeletal muscle
antioxidant enzyme activities. J Gerontol 49, B231–B238, 1994.
References
1
2
. Janssen, YM, Van Houten, B, Borm, PJ, and Mossman, BT: Cell and
tissue responses to oxidative damage. Lab Invest 69, 261–274, 1993.
. Ames, BN, Shigenaka, MK, and Hagen, TK: Oxidants, antioxidants,
and the degenerative diseases of aging. Proc Natl Acad Sci USA 90,
26. Leakey, JEA, Cunny, HC, Bazare, J, Jr, Webb, PJ, Lipscomb, JC, et al.:
Effect of aging and caloric restriction on hepatic drug metabolizing en-
zymes in the Fischer 344 rat. II. Effect on conjugating enzymes. Mech
Ageing Dev 48, 157–166, 1989.
7
915–7922, 1993.
3
. Knight, JA: Reactive oxygen species and the neurodegenerative disor-
ders. Ann Clin Lab Sci 27, 11–25, 1997.
27. Keenan, KP, Ballam, GC, Dixit, R, Soper, KA, Laroque, P, et al.: The
effects of diet, overfeeding and moderate dietary restriction on
5
0
Nutrition and Cancer 2000

Sprague-Dawley rat survival, disease and toxicology. J Nutr 127,
51S–856S, 1997.
8. Verdery, RB, Ingram, DK, Roth, GS, and Lane, MA: Caloric restric-
tion increases HDL2 levels in rhesus monkeys (Macaca mulata). Am J
Physiol Endocrinol Metab 273, E714–E719, 1997.
9. Makinodan, T, and Jill James, S: Possible role of apoptosis in immune
enhancement and disease retardation with dietary restriction and/or
very low doses of ionizing radiation. In Dietary Restriction: Implica-
tion for the Design and Interpretation of Toxicity and Carcinogenicity,
RW Hart, DA Neumann, and RT Robertson (eds). Washington, DC:
ILSI, 1995.
plication for the Design and Interpretation of Toxicity and Carcino-
genicity, RW Hart, DA Neumann, and RT Robertson (eds). Washing-
ton, DC: ILSI, 1995.
8
2
2
31. Shen, CC, Wertelecki, W, Driggers, WJ, Ledour, SP, and Wilson, GL:
Repair of mitochondrial DNA damage induced by bleomycin in human
cells. Mutat Res 337, 19–23, 1995.
32. Johns, DR: Seminars in Medicine of the Beth Israel Hospital, Boston:
mitochondrial DNA and disease. N Engl J Med 333, 638–644, 1995.
33. Yakes, FM, and Houten, BV: Mitochondrial DNA damage is more
extensive and persists longer than nuclear DNA damage in human cells
following oxidative stress. Proc Natl Acad Sci USA 94, 514–519,
1997.
3
0. Feuers, RJ, Duffy, PH, Chen, F, Desai, VG, Oriaku, E, et al.: Interme-
diary metabolism and antioxidant systems. In Dietary Restriction: Im-
Vol. 36, No. 1
51