Carcinogenesis vol.19 no.6 pp.1133–1139, 1998
Ferric nitrilotriacetate promotes N-diethylnitrosamine-induced
renal tumorigenesis in the rat: implications for the involvement of
oxidative stress
Mohammad Athar1 and Mohammad Iqbal
Department of Medical Elementology and Toxicology, Hamdard University
(Jamia Hamdard), New Delhi-110062, India
1To
whom correspondence should be addressed
Ferric nitrilotriacetate (Fe-NTA) is a known complete renal
carcinogen. In this study we show that Fe-NTA is a potent
inducer of renal ornithine decarboxylase (ODC) activity
and DNA synthesis and promoter of N-diethylnitrosamine
(DEN)-induced renal tumorigenesis in rat. Fe-NTA induced
renal ODC activity several fold as compared with salinetreated rats. Renal DNA synthesis, measured as [3H]thymidine incorporation into DNA, was increased after Fe-NTA
treatment. Similar to other known tumor promoters, FeNTA also depleted the antioxidant armory of the tissue. It
depleted glutathione (GSH) levels to ~55% of saline-treated
controls. It also led to a dose-dependent decrease in the
activities of glutathione reductase and glutathione S-transferase. Similarly, activities of catalase, glutathione peroxidase and glucose 6-phosphate dehydrogenase decreased
significantly (45–65%). In contrast, γ-glutamyl transpeptidase activity showed an increase. The maximum changes in
activities of these enzymes could be observed at 12 h
following Fe-NTA treatment. In addition, Fe-NTA augmented renal microsomal lipid peroxidation .150% over
saline-treated controls, which was concomitant with the
alterations in GSH metabolizing enzymes and depletion of
the antioxidant armory. These effects were alleviated in
rats which received a pretreatment with an antioxidant,
BHA or BHT. Fe-NTA promoted DEN-induced renal tumorigenesis. In saline alone- and DEN alone-treated animals
no tumors could be recorded, whereas in Fe-NTA alonetreated animals 17% tumor incidence was observed. However, in DEN-initiated and Fe-NTA-promoted animals
tumor incidence increased to 71%. Our results show that
Fe-NTA induces oxidative stress in the kidney and decreases
antioxidant defenses, as indicated by the fall in GSH level
and in the activities of glutathione peroxidase and catalase.
Concomitantly, Fe-NTA increases ODC activity and DNA
synthesis, which may be compensatory changes following
oxidative injury to renal cells in addition to providing a
strong stimulus for renal tumor promotion. Thus oxidative
stress and impaired antioxidant defenses induced by FeNTA in the kidney may contribute to the observed nephrotoxicity and carcinogenicity.
*Abbreviations: Fe-NTA, ferric nitrilotriacetate; ROS, reactive oxygen
species; GSH, glutathione; 8-OH-dG, 8-hydroxydeoxyguanosine; ODC,
ornithine decarboxylase; DEN, N-diethylnitrosamine; PMSF, phenylmethylsulfonyl fluoride; DTNB, dithionitrobenzene; CDNB, 1-chloro-2,4-dinitrobenzene; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; PMS
post-mitochondrial supernatant; TCA, trichloroacetic acid; MDA, malonaldehyde; TBA, thiobarbituric acid; PCA, perchloric acid; RCT, renal cell
tumors; HNE, 4-hydroxy-2-nonenal.
© Oxford University Press
Introduction
Nitrilotriacetic acid is a synthetic tricarboxylic acid which
forms water-soluble chelate complexes with various metal
ions, including iron, at neutral pH and has been used as a
substitute for polyphosphates in detergents utilized both in
developed and developing countries (1). Chronic treatment to
rats with its iron complex, ferric nitrilotriacetate (Fe-NTA*),
induces diabetic conditions such as hyperglycemia, polyuria,
glucosuria and polydipsia (2). Fe-NTA-induced neoplastic
transformation of cultured rat hepatic epithelial cells has also
been demonstrated (3). Recently we showed that Fe-NTA
induces a cell proliferative response in liver (4). We have also
demonstrated that alterations in hepatic glutathione metabolizing enzymes and generation of oxidative stress following FeNTA treatment play a definite role in the causation of hepatic
injury (5).
Fe-NTA is also a potent nephrotoxic agent (2). The renal
toxicity is assumed to be caused by an elevation in serum free
iron concentration following its reduction at the luminal side
of the proximal tubule, which generates reactive oxygen
species (ROS) leading to enhanced lipid peroxidation with a
concomitant decrease in tissue glutathione (GSH) level (6,7).
Repeated i.p. administration of Fe-NTA produces acute and
subacute proximal tubular necrosis associated with a high
incidence of renal adenocarcinoma in male mice and rats (8,9).
It is assumed that Fe-NTA-mediated generation of free radicals
plays an important role in renal tumorigenesis. Renal DNA
damage leading to single-strand and double-strand DNA breaks
(10), DNA–protein cross-links (11) and enhanced formation
of 8-hydroxydeoxyguanosine (8-OH-dG) has been observed
following exposure of animals to Fe-NTA (12). 8-OH-dG is a
marker product of oxidative Fe-NTA treatment (12). Recently
enhanced formation of 4-hydroxy-2-nonenal-modified proteins
in the renal proximal tubules of Fe-NTA-treated rats has been
shown (13). Fe-NTA stimulates oOH production, which is
responsible for initiating many of these effects (6,7). In most
studies it has been suggested that Fe-NTA acts as a complete
renal carcinogen. However, many of the chemical compounds
capable of generating oxidative stress in tissues also act as
tumor promoters (14). To the best of our knowledge no
systematic study has been carried out to show the renal tumor
promoting potential of Fe-NTA. In this communication we
show that Fe-NTA is a potent inducer of renal ornithine
decarboxylase (ODC) activity, enhances [3H]thymidine incorporation into DNA and acts by generating oxidative stress in
the kidney. Multiple applications of Fe-NTA to N-diethylnitrosamine (DEN)-initiated animals produce tumorigenesis in
.70% of animals as compared with Fe-NTA alone-treated
animals, which developed tumors in only 17% of animals, and
DEN alone-treated animals, which developed no tumors.
Materials and methods
Chemicals
Dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), NADP, DEN, 2-mercaptoethanol, L-ornithine, pyridoxal 5-phosphate, oxidized and reduced gluta-
1133
M.Athar and M.Iqbal
Fig. 1. Effect of Fe-NTA on renal ODC. Each value represents the mean 6
SE of six animals. Dose regimens and the treatment protocol are described
in the text. (Inset) Time–response relationship of ODC induction following
Fe-NTA treatment at a dose of 9 mg Fe/kg body wt. **Significantly
different (P , 0.001) when compared with the saline-treated control group.
The values for normal saline treatment and NTA alone treatment are 185.58
6 13.91 and 201.33 6 47.87 pmol 14CO2 released/h/mg protein
respectively.
Fig. 3. Histopathology of RCT in rats initiated with DEN and promoted
with Fe-NTA. A small adenomatous growth consisting of clear and granular
cells with focal leukocytic infiltration (a); adenocarcinoma with acinar (b)
and papillary (c) patterns of growth. Arrows indicate mitotic figures.
Hematoxylin and eosin. (a) and (c) 3200; (b) 3400.
Table I. Summary of tumor data
Fig. 2. Effect of Fe-NTA on [3H]thymidine incorporation into renal DNA.
Each value represents the mean 6 SE of six animals. Dose regimens and
the treatment protocol are described in the text. **Significantly different
(P , 0.001) when compared with the saline-treated control group. The
values for normal saline treatment and NTA alone treatment groups are
26.39 6 14.68 and 27.50 6 3.97 d.p.m./µg DNA respectively.
thione, nitrilotriacetic acid, NADPH, H2O2, dithionitrobenzene (DTNB),
1-chloro-2,4-dinitrobenzene (CDNB), γ-glutamyl-p-nitroanilide, glutathione
reductase and glucose 6-phosphate were purchased from Sigma Chemical Co.
(St Louis, MO). [14C]Ornithine (sp. act. 56 mCi/mmol) and [3H]thymidine
(sp. act. 82 Ci/mmol) were purchased from Amersham Corp., UK. All other
chemicals and reagents used were of the highest purity commercially available.
Preparation of Fe-NTA
Fe-NTA solution was prepared by the method of Awai et al. (2).
Experimental protocol
Animals and treatments. Male albino rats of the Wistar strain (4–6 weeks
old), weighing 125–150 g, from the Jamia Hamdard Central Animal House
colony were used throughout this study. Animals were housed in an airconditioned room and had free access to pellet diet (Hindustan Lever Ltd,
Bombay, India). These animals were injected with Fe-NTA i.p. The dose
regimens selected for this study were usually 6 or 9 mg Fe/kg body wt as Fe-
1134
Treatment group No. of
animals
treated
No. of animals
No. of
Incidence of
studied
animals with tumors (%)
histopathologically renal cell
tumors
Saline alone
DEN alone
DEN 1 Fe-NTA
Fe-NTA alone
22
20
21
17
25
25
25
25
0
0
0
0
15 (12b, 3U) 71
3 (b)
17
Experimental details are provided in the text.
U, unilateral; b, bilateral.
NTA complex unless mentioned otherwise. Selection of the dose regimen is
based on our own preliminary experiments, which indicate substantial alterations in many of the biochemical parameters at these doses, and is also based
on previously published data (5,8,12). For various sets of biochemical studies
different groups of animals were used. All animals were killed 12 h after FeNTA or saline or NTA treatment within a period of 1 h by cervical dislocation
unless mentioned otherwise.
For ODC induction studies the animals were divided into two major groups,
one containing 42 rats, the other only 24 rats. To study the kinetics of ODC
induction group I was subdivided into seven subgroups. Subgroup I received
only saline and served as a control, whereas all other subgroups (II–VII)
received a single dose of 9 mg Fe/kg body wt as Fe-NTA. These animals
were killed 0, 3, 6, 12, 24, 48 and 72 h after saline or Fe-NTA treatment
Fe-NTA promotes DEN-induced renal tumorigenesis
Table II. Time-dependent effect of Fe-NTA administration on renal GSH level and on the activities of glutathione metabolizing enzymes
Enyme
Time after Fe-NTA administration (h)
0 (control)
Glutathione (mmol/g tissue)
0.56 6 0.01
Glutathione reductase
256.36 6 3.72
(nmol NADPH oxidized/min/mg protein)
Glutathione S-transferase
111.43 6 1.76
(nmol CDNB formed/min/mg protein)
Glucose 6-phosphate dehydrogenase
24.52 6 0.59
(nmol NADPH formed/min/mg protein)
Glutathione peroxidase
202.75 6 6.55
(nmol NADPH oxidized/min/mg/protein)
γ-Glutamyl transpeptidase
1686.04 6 96.14
(nmol p-nitroanilineformed/min/mg protein)
3
6
12
0.32 6 0.07a
0.32 6 0.08a
237.80 6 12.49b 167.22 6 7.81a
24
48
0.25 6 0.04a
104.59 6 3.68a
0.31 6 0.01a
170.34 6 3.67a
0.31 6 0.07a
181.42 6 1.61a
98.56 6 2.56b
72.80 6 1.83a
45.46 6 2.46a
87.30 6 4.83a
92.72 6 6.73b
19.27 6 1.19b
13.12 6 0.33a
8.88 6 0.55a
14.26 6 0.56a
17.90 6 2.06b
ND
ND
ND
ND
92.60 6 4.15a
4187.48 6
193.47a
ND
ND
ND
ND
ND, not done.
Each value represents the mean 6 SE of six animals. Saline-treated animals served as the control and are represented here as the 0 h control.
bSignificantly different (P , 0.05) when compared with the saline-treated control group.
aSignificantly different (P , 0.001) when compared with the saline-treated control group.
Table III. Dose-dependant effect of Fe-NTA administration on renal GSH level and on the activities of glutathione metabolizing enzymes
Enzyme
Glutathione (mmol/g tissue)
Glutathione reductase (nmol NADPH oxidized/min/mg protein)
Glutathione S-transferase (nmol CDNB formed/min/mg protein)
Glucose 6-phosphate dehydrogenase (nmol NADPH formed/min/mg protein)
Glutathione peroxidase (nmol NADPH oxidized/min/mg protein)
γ-Glutamyl transpeptidase (nmol p-nitroaniline formed/min/mg protein)
Dose of Fe-NTA (mg Fe/kg body wt)
Saline control
3 (dose 1)
9 (dose 2)
0.56 6 0.01
256.36 6 3.72
111.43 6 1.76
24.52 6 0.59
202.75 6 6.55
1686.04 6 96.14
0.36 6 0.02a
142.76 6 7.72a
63.03 6 6.30a
11.99 6 0.78a
109.39 6 3.68a
2292.67 6 133.76c
0.25
104.59
45.46
8.88
92.60
4187.48
6
6
6
6
6
6
0.04a1
3.68ab
2.46ab
0.55ab
4.15ab
193.47ab
Each value represents the mean 6 SE of six animals. Saline-treated animals served as the control.
cSignificantly different (P , 0.05) when compared with the saline-treated control group.
aSignificantly different (P , 0.001) when compared with the saline-treated control group.
bSignificantly different (P , 0.05) when compared with dose 1.
within a period of 1 h. The 24 animals of group II were subdivided into four
groups. The subgroup I animals received only saline, subgroup II received
only NTA and served as a control and subgroup III and IV animals received
6 and 9 mg Fe/kg body wt respectively as Fe-NTA and were killed 12 h
following treatment with saline, NTA or Fe-NTA.
For [3H]thymidine incorporation studies animals were divided into four
groups of six rats each. Groups I and II received saline and NTA alone
respectively and served as controls, whereas groups III and IV received 6 and
9 mg Fe/kg body wt as Fe-NTA respectively. All these animals received
[3H]thymidine (30 µCi/animal) as an i.p. injection 18 h after the first treatment
with saline, NTA or Fe-NTA and were killed 20 h after the first treatment.
For tumorigenesis studies using an initiation–promotion protocol the animals
were divided into four groups of 25 rats each. The animals of groups II and
III were initiated with a single i.p. injection of DEN at a dose level of 200
mg/kg body wt in saline. Ten days after initiation the animals of group III
were promoted with increasing doses of from 3.0 to 9.0 mg Fe/kg body wt
as Fe-NTA, administered i.p. 3 days a week for 16 weeks. The animals of
group IV were uninitiated but were treated with Fe-NTA in exactly the same
manner as the animals of group III. At the end of 24 weeks promotion animals
were killed by cervical dislocation and their kidneys quickly removed and
preserved in 10% neutral buffered formalin for histopathological studies.
Hematoxylin and eosin preparations of processed sections were prepared for
microscopic examination.
To study the time-dependent effect of Fe-NTA on GSH metabolizing
enzymes and antioxidant enzymes the animals were divided in two groups
consisting of 36 and 18 animals respectively. In the first group 36 rats received
a single i.p. injection of Fe-NTA (9 mg Fe/kg body wt). To study the kinetic
effects of Fe-NTA on the above-described biochemical parameters six rats
out of the 36 were killed by cervical dislocation 0, 3, 6, 12, 24 and 48 h after
Fe-NTA treatment within a period of 1 h. To demonstrate the dose–response
relationship, in a second group 18 rats divided into three groups of six in
each were used. Subgroup I animals received saline and served as controls
and subgroup II and III animals received 3 and 9 mg Fe/kg body wt as Fe-
NTA and were killed 12 h following treatment with saline or Fe-NTA. In this
experiment there was no control group treated with NTA alone as no significant
differences could be observed between the saline alone and NTA alone
treatment groups. Similar observation were made earlier (25).
To study the effect of the antioxidants butylated hydroxyanisole (BHA) and
butylated hydroxytoluene (BHT) on Fe-NTA-mediated induction of renal
ODC activity and [3H]thymidine incorporation 36 animals each were taken
and divided into six groups of six rats each. Group I received saline, group
II received 2 mg/animal/day BHA in 0.2 ml corn oil for 1 week orally by
gavage. Similarly, group III received 2 mg/animal/day BHT in 0.2 ml corn
oil for 1 week. These animals served as negative controls. Group IV animals
received Fe-NTA (9 mg Fe/kg body wt) and served as positive controls.
Group V and VI animals received a pretreatment with BHA or BHT as
described for groups II and III respectively and 24 h following the last
treatment with BHA/BHT these animals received a single i.p. injection of FeNTA (9 mg Fe/kg body wt). All these animals were killed 12 h after Fe-NTA
or saline treatment. However, for [3H]thymidine incorporation studies animals
receiving similar treatments were given an i.p. injection of [3H]thymidine
exactly as described in the preceding section.
Biochemical assays
Post-mitochondrial supernatant (PMS) and microsome preparation. Kidneys
were quickly removed, cleaned free of extraneous material and immediately
perfused with ice-cold saline (0.85% sodium chloride). The kidneys were
homogenized in chilled phosphate buffer (0.1 M, pH 7.4) containing KCl
(1.17%) using a Potter–Elvehjem homogenizer and subjected to subcellular
fractionation as described by Iqbal et al. (4,5).
Estimation of reduced glutathione. Reduced glutathione in the kidney was
determined by the method of Jollow et al. (15). An aliquot of 1.0 ml PMS
(10%) was precipitated with 1.0 ml sulfosalicylic acid (4%). The samples
were kept at 4°C for at least 1 h and then subjected to centrifugation at 1200
g for 15 min at 4°C. The assay mixture contained 0.1 ml filtered aliquot, 2.7
ml 0.1 M phosphate buffer, pH 7.4, and 0.2 ml DTNB (40 mg/10 ml 0.1 M
1135
M.Athar and M.Iqbal
(PCA) at 4°C overnight. After incubation it was centrifuged and the precipitate
washed with cold PCA (5%). The precipitate was dissolved in warm PCA
(10%) followed by incubation in a boiling water bath for 30 min and filtered
through Whatman 50 filter paper. The filtrate was used for 3H counting in a
liquid scintillation counter (LKB-Wallace 1410) by adding scintillation fluid.
The amount of DNA in the filtrate was estimated by the diphenylamine
method of Giles and Myers (18). The amount of [3H]thymidine incorporated
was expressed as d.p.m./µg DNA.
Fig. 4. Effect of Fe-NTA on renal microsomal lipid peroxidation and GSH
levels. Each value represents the mean 6 SE of six animals. Dose regimens
and the treatment protocol are described in the text.
Ornithine decarboxylase activity. ODC activity was determined utilizing 0.4
ml renal 105 000 g supernatant fraction per assay tube by measuring release
of 14CO2 from DL-[1-14C]ornithine by the method of O’Brien et al. (19) as
described by Athar et al. (20). The kidneys were homogenized in 50 mM
Tris–HCl buffer, pH 7.5, containing 0.1 mM EDTA, 0.1 mM pyridoxal
phosphate, 1.0 mM PMSF, 1.0 mM 2-mercaptoethanol, 0.1 mM dithiothreitol
and 0.1% Tween-80 at 4°C using a polytron homogenizer (Kinematica AGPT
3000). In brief, the reaction mixture contained 400 µl enzyme and 0.095 ml
cofactor mixture containing 0.32 mM pyridoxal phosphate, 0.4 mM EDTA,
4.0 mM dithiothreitol, 0.4 mM ornithine, 0.02% Brig 35 and 0.05 µCi
[14C]ornithine in a total volume of 0.495 ml. After adding buffer and cofactor
mixture to the blank and test tube the tubes were covered immediately with
a rubber cork containing 0.2 ml ethanolamine and methoxyethanol mixture in
the central well and kept in a water bath at 37°C. After 1 h incubation enzyme
activity was arrested by injecting 1.0 ml 2.0 M citric acid solution along the
sides of glass tubes and incubation continued for 1 h to ensure complete
absorption of 14CO2. Finally, the central well was transferred to a vial
containing 2 ml ethanol and 10 ml toluene-based scintillation fluid were
added, followed by counting of radioactivity in a liquid scintillation counter
(LKB-Wallace 1410). ODC activity was expressed as pmol 14CO2 released/
h/mg protein.
Glutathione peroxidase activity. Glutathione peroxidase activity was measured
according to the procedure described by Mohandas et al. (21). The reaction
mixture consisted of 1.44 ml 0.05 M phosphate buffer, pH 7.0, 0.1 ml 1 mM
EDTA, 0.10 ml 1 mM sodium azide, 0.05 ml 1 U/ml glutathione reductase,
0.10 ml 1 mM glutathione, 0.10 ml 2 mM NADPH, 0.01 ml 0.25 mM
hydrogen peroxide and 0.10 ml 10% PMS in a total volume of 2.0 ml.
Disappearance of NADPH at 340 nm was recorded at 25°C. Enzyme activity
was calculated as nmol NADPH oxidized/min/mg protein using a molar
extinction coefficient of 6.223103/M/cm.
Glucose 6-phosphate dehydrogenase activity. The activity of glucose 6phosphate dehydrogenase was assayed by the method of Zaheer et al. (22).
The reaction mixture in a total volume of 3.0 ml consisted of 0.3 ml 0.05 M
Tris–HCl buffer, pH 7.6, 0.1 ml 0.1 mM NADP, 0.1 ml 0.8 mM glucose 6phosphate, 0.1 ml 8 mM MgCl2, 0.3 ml enzyme and 2.1 ml distilled water.
The changes in absorbance were recorded at 340 nm and enzyme activity was
calculated as nmol NADP reduced/min/mg protein using a molar extinction
coefficient of 6.223103/M/cm.
Fig. 5. Effect of Fe-NTA on renal antioxidant enzymes. Each value
represents the mean 6 SE of six animals. Dose regimens and the treatment
protocol are described in the text.
phosphate buffer, pH 7.4) in a total volume of 3.0 ml. The yellow color
developed was read immediately at 412 nm in a spectrophotometer (Milton
Roy Model-21D).
Lipid peroxidation. The assay for microsomal lipid peroxidation followed the
method of Wright et al. (16). The reaction mixture in a total volume of 1.0
ml contained 0.58 ml 0.1 M phosphate buffer, pH 7.4, 0.2 ml microsomes,
0.2 ml 100 mM ascorbic acid and 0.02 ml 100 mM ferric chloride. The
reaction mixture was incubated at 37°C in a shaking water bath for 1 h. The
reaction was stopped by addition of 1 ml 10% trichloroacetic acid (TCA).
Following addition of 1.0 ml 0.67% thiobarbituric acid (TBA) all tubes were
placed in a boiling water bath for a period of 20 min. After this time the
tubes were shifted to a crushed ice bath and then centrifuged at 2500 g for
10 min. The amount of malonaldehyde (MDA) formed in each sample was
assessed by measuring the optical density of the supernatant at 535 nm using
a spectrophotometer (Milton Roy 21 D) against a reagent blank. The results
were expressed as nmol MDA formed/h/g tissue at 37°C using a molar
extinction coefficient of 1.563105/M/cm.
Renal DNA synthesis. The isolation of renal DNA and incorporation of
[3H]thymidine into DNA were as described by Smart et al. (17). The kidneys
were quickly removed, cleaned free of extraneous material and a homogenate
(10% w/v) prepared in ice-cold water. The precipitate thus obtained was
washed with cold TCA (5%) and incubated with cold 10% perchloric acid
1136
Catalase activity. Catalase activity was assayed by the method of Claiborne
(23). Briefly, the assay mixture consisted of 1.95 ml 0.05 M phosphate buffer,
pH 7.0, 1.0 ml 0.019 M hydrogen peroxide and 0.05 ml 10% PMS in a final
volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase
activity was calculated in terms of nmol H2O2 consumed/min/mg protein.
Glutathione reductase activity. Glutathione reductase activity was assayed by
the method of Carlberg and Mannervik (24) as modified by Mohandas et al.
(21). The assay system consisted of 1.65 ml 0.1 M phosphate buffer, pH 7.6,
0.1 ml 0.5 mM EDTA, 0.05 ml 1 mM oxidized glutathione, 0.1 ml 0.1 mM
NADPH and 0.1 ml 10% PMS in a total volume of 2.0 ml. The enzyme
activity was quantitated at 25°C by measuring the disappearance of NADPH
at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein
using a molar extinction coefficient of 6.223103/M/cm.
Glutathione S-transferase activity. This was measured by the method of Habig
et al. (25) as described by Athar et al. (26). The reaction mixture consisted
of 1.425 ml 0.1 M phosphate buffer, pH 6.5, 0.2 ml 1 mM reduced glutathione,
0.025 ml 1 mM CDNB and 0.30 ml 10% PMS in a total volume of 2.0 ml.
The changes in absorbance were recorded at 340 nm and enzyme activity was
calculated as nmol CDNB conjugate formed/min/mg protein using a molar
extinction coefficient of 9.63103/M/cm.
γ-Glutamyl transpeptidase activity. γ-Glutamyl transpeptidase activity was
determined by the method of Orlowski and Meister (27) using γ-glutamyl pnitroanilide as substrate. The reaction mixture in a total volume of 1.0 ml
contained 0.2 ml 10% homogenate which was incubated with 0.8 ml substrate
mixture (containing 4 mM γ-glutamyl p-nitroanilide, 40 mM glycylglycine
and 11 mM MgCl2 in 185 mM Tris–HCl buffer, pH 8.25) at 37°C. Ten
minutes after initiation of the reaction 1.0 ml 25% TCA was added and mixed
to terminate the reaction. The solution was centrifuged and the supernatant
Fe-NTA promotes DEN-induced renal tumorigenesis
Table IV. Effect of pretreatment of rats with the antioxidants BHA and BHT on Fe-NTA-induced renal ODC activity and [3H]thymidine incorporation
Treatment group
pmol14CO2/h/mg protein
Control
BHA
BHT
Fe-NTA
BHA 1 Fe-NTA
BHT 1 Fe-NTA
[3H]Thymidine incorporation
ODC activity
208.03
179.58
200.54
1090.55
599.75
424.89
6
6
6
6
6
6
31.57
17.98a
19.72a
46.45a
50.11a
38.62a
Percent of control
d.p.m./µg DNA
100
86
96
524
288
204
35.28
33.27
28.57
151.82
79.56
72.19
6
6
6
6
6
6
4.13
2.48a
3.20a
14.70a
4.78a
6.02a
Percent of control
100
94
80
430
225
204
Each value represents the mean 6 SE of six animals. Saline-treated animals served as the control. Dose of Fe-NTA administered was 9 mg Fe/kg body wt.
Doses of both BHA and BHT were 2 mg/animal/day given orally every day for 1 week before treatment with Fe-NTA.
aSignificantly different (P , 0.001) when compared with the Fe-NTA group.
fraction read at 405 nm. Enzyme activity was calculated as nmol p-nitroaniline
formed/min/mg protein using a molar extinction coefficient of 1.743103/M/
cm. Protein in all samples was determined by the method of Lowry et al.
(28) using bovine serum albumin as the standard.
Statistical analysis
The levels of significance between different groups are based on Dunnett’s ttest followed by the analysis of variance test.
Results
The effect of Fe-NTA treatment on renal ODC activity is
shown in Figure 1. Fe-NTA treatment induced renal ODC
activity at various times. A maximum of 5-fold induction was
observed 12 h after Fe-NTA treatment (cf. inset to Figure 1).
At a dose of 6 mg Fe/kg body wt induction was ~361% of
the saline-treated control, whereas at a dose of 9 mg of Fe/kg
body wt as Fe-NTA induction could be recorded up to 524%.
Since no significant differences could be recorded between the
saline alone- and NTA alone-treated controls, comparison has
been made with the saline treatment data only.
The effect of Fe-NTA on [3H]thymidine incorporation into
renal DNA is shown in Figure 2. Fe-NTA administration
stimulated [3H]thymidine incorporation into renal DNA. At a
dose of 6 mg Fe/kg body wt [3H]thymidine incorporation
increased to ~335% of the control value, whereas at a dose of
9 mg Fe/kg body wt as Fe-NTA it reached a value 4.5-fold
that of the saline-treated control.
The data given in Table I are a summary of percent incidence
of renal cell tumors (RCT) in saline- and Fe-NTA-treated
animals. Treatment with Fe-NTA of uninitiated animals led to
development of RCT in ~17% of animals studied. However,
treatment with Fe-NTA of DEN-initiated animals enhanced
development of RCT by ~71%. Saline alone- or DEN alonetreated controls did not show any incidence of tumor. Histopathological studies of the renal tissue of tumor-bearing animals
revealed adenocarcinoma of the kidney with either an acinar
or papillary type of growth pattern, which were noticed in
kidneys of 40% of animals. Figure 3 shows a representative
histopathology of RCT initiated with DEN and promoted with
Fe-NTA.
Fe-NTA administration resulted in a dose-dependent increase
in renal microsomal lipid peroxidation. At a dose of 9 mg Fe/
kg body wt there was a dramatic increase of ~163% in
peroxidation of microsomal membrane lipids. In parallel with
the increase in lipid peroxidation, a concomitant decrease in
renal GSH level could be observed. GSH was depleted to
~55% of the control value at a dose of 9 mg Fe/kg body wt,
as shown in Figure 4.
The data in Table II show time-dependant effects of Fe-
NTA (9 mg Fe/kg body wt) administration on renal GSH level
and on activities of various GSH metabolizing enzymes,
namely glutathione reductase, γ-glutamyl transpeptidase and
glutathione S-transferase. The level of GSH and the activities
of glutathione reductase and glutathione S-transferase were
found to decrease at all times studied. The maximum decrease
was recorded 12 h following Fe-NTA treatment. In contrast,
γ-glutamyl transpeptidase activity was increased at this time.
The dose-dependent effects of Fe-NTA are shown in Table III.
The increase in activity of γ-glutamyl transpeptidase and
decrease in activities of glutathione reductase and glutathione
S-transferase were found to be dependent on the dose of FeNTA administered (Table III). Similarly, GSH level also
decreased with increasing dose of Fe-NTA. Since maximum
changes in GSH and GSH metabolizing enzymes occurred 12
h following Fe-NTA treatment, the dose–response relationship
was studied at this time only.
The effect of Fe-NTA on renal antioxidant enzymes is
shown in Figure 5. Although the effect of Fe-NTA was studied
at various doses, the results shown here represent the effects
of 9 mg Fe/kg body wt as Fe-NTA. The antioxidant enzymes
glutathione peroxidase, catalase and glucose 6-phosphate dehydrogenase were depleted to 50% or less than the control value
following Fe-NTA treatment.
The effects of pretreatment of animals with the antioxidants
BHA and BHT on Fe-NTA-mediated induction of ODC activity
and enhancement of [3H]thymidine incorporation into DNA
are shown in Table IV. Pretreatment of animals with the
antioxidants BHA and BHT significantly decreased Fe-NTAmediated induction of renal ODC activity and enhancement
of incorporation of [3H]thymidine into renal DNA. The
decreases in ODC activity and [3H]thymidine incorporation
were 45 and 48% respectively in the case of BHA-treated and
60 and 53% in BHT-treated animals. These results suggest a
higher effectiveness of BHT as compared with BHA in
alleviating the toxic effects of Fe-NTA.
Discussion
Involvement of ROS in skin tumor initiation, promotion and
progression has been studied in great detail (29–31), however,
in other organs it remains rather elusive. In renal carcinogenesis
the involvement of ROS is indicated by the fact that potent
oxidizing agents, such as potassium bromate (KBrO3) (32)
and Fe-NTA (12), cause renal cancer. However, few studies
have been carried out to determine the involvement of ROS
in each stage of renal carcinogenesis. The observation in the
present study that Fe-NTA induced ODC activity in the kidney
1137
M.Athar and M.Iqbal
of rats implies that Fe-NTA may act as a renal tumor promoter.
ODC is the first and rate limiting enzyme in polyamine
biosynthesis and is induced in response to a large number of
tumor promoters (33). In several experimental models ODC
has been shown to act as a marker of cell proliferation (33).
Recently it has been reported that overexpression of ODC
plays an important role in carcinogenesis (34). The degree of
ODC induction has also been correlated with the tumor
promoting potency of an agent (33,34). Several fold induction
of renal ODC activity by Fe-NTA suggests that it may be a
potent renal tumor promoter. Further, the observed effect of
Fe-NTA on renal DNA synthesis, which is similar to the effect
of other known tumor promoters in various other organs, also
suggests a proliferative potential of Fe-NTA. The time of the
highest increase in oxidant generation and fall in activities of
antioxidant enzymes after Fe-NTA treatment corresponded
with the period of maximum ODC induction, suggesting a
role of oxidant generation in induction of ODC activity.
Analogous to 12-O-tetradecanoylphorbol-13-acetate in skin,
Fe-NTA also depletes the enzymatic and non-enzymatic antioxidant armory in the kidney, including catalase, glucose 6phosphate dehydrogenase, glutathione peroxidase and GSH
levels (35,36). The turnover of GSH depends on the activities
of various GSH metabolizing enzymes. The activities of these
enzymes may, therefore, be one of the factors detrimental to
the tissue level of GSH and in turn the level of oxidants
present in the cell (35–37). Thus the decreased level of GSH
following Fe-NTA treatment due to decreased reduction of
oxidized glutathione and increased activity of γ-glutamyl
transpeptidase may lead to accumulation of peroxides, leading
to oxidative stress (35–37). As observed in the present study,
increased γ-glutamyl transpeptidase activity fixes degradation
of GSH, which may lead to higher accumulation of cysteinyl
glycine and cysteine. High levels of both cysteine and cysteinyl
glycine have been suggested to enhance reduction of Fe-NTA
to its ferrous complex, which in turn enhances peroxidative
damage to the membrane/tissue (10). Further, the observed
enhancement in renal tumor incidence by Fe-NTA may be due
to the promoting potential of Fe-NTA-generated renal oxidative
stress. Similar to oxidant tumor promoters (38), Fe-NTA
potentiates lipid peroxidation in the kidney, which may be the
result of diminished activities of antioxidant enzymes and a
low level of GSH. Precisely these effects of Fe-NTA are
comparable with the effects of the oxidant tumor promoters
benzoyl peroxide, cumene hydroperoxide and t-butyl hydroperoxide in skin, which enhance both lipid peroxidation and
generate free radicals (39). The mechanism by which Fe-NTA
may act as a promoter and may induce ODC activity remains
undefined. However, many oxidants, including benzoyl peroxide, induce protein kinase C, which may be involved in
induction of ODC activity and in inhibition of metabolic
cooperation (40). Another possible mechanism involved in
Fe-NTA-mediated renal carcinogenesis relates to enhanced
production of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation
product. HNE exhibits various genotoxic and mutagenic effects
due to its facile reactivity with biological molecules, including
proteins (41). HNE-modified proteins have been observed
mainly in the renal proximal tubules of Fe-NTA-treated animals
(41). Additionally, it has been shown that lipid peroxidation
induces 8-hydroxyguanosine formation (42) and, as Fe-NTA
enhances 8-hydroxyguanosine formation only in the kidney,
this leaves as an open possibility that HNE formation may
provide a link between lipid peroxidation and oxidative DNA
1138
damage (13). Pretreatment of rats with the antioxidants BHA
and BHT in the present study diminished Fe-NTA-induced
ODC activity and [3H]thymidine incorporation into DNA,
further suggesting a role of oxidative stress in Fe-NTAmediated induction of ODC activity and enhanced [3H]thymidine incorporation.
Our results show that Fe-NTA induces oxidative stress in
the kidney and decreases antioxidant defenses, as indicated by
a fall in GSH level and in the activities of glutathione
peroxidase and catalase. Concomitantly, Fe-NTA increases
ODC activity and DNA synthesis, which may be compensatory
changes following oxidative injury to renal cells. Thus oxidative stress due to impaired antioxidant defenses and enhanced
oxidant generation as observed in the present study could
provide a mitogenic stimulus in the tissue contributing to
promotion of DEN-initiated renal tumors. In summary, our
data suggest that Fe-NTA may be a potent renal tumor promoter
and generation of oxidative stress in the kidney may play a
role in Fe-NTA-mediated induction of renal ODC activity,
enhancement of DNA synthesis and promotion of DENinitiated renal tumors in rats.
Acknowledgements
The authors are thankful to Prof. Alauddin Ahmad, Vice Chancellor, Jamia
Hamdard, for providing necessary facilities. M.I. is also thankful to the
Hamdard National Foundation for providing a research fellowship.
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Received on April 16, 1997; revised on December 17, 1997; accepted on
January 13, 1998
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