Alteration in the activity of oxidative enzymes in the tissues of male Wistar albino rats exposed to cadmium.

IJOMEH 2010;23(1) 55
O R I G I N A L P A P E R S
International Journal of Occupational Medicine and Environmental Health 2010;23(1):55 – 62
DOI 10.2478/v10001-010-0002-y
ALTERATION IN THE ACTIVITY OF OXIDATIVE
ENZYMES IN THE TISSUES OF MALE WISTAR
ALBINO RATS EXPOSED TO CADMIUM
SAMUEL O. ASAGBA
Delta State University, Abraka, Nigeria
Department of Biochemistry, Faculty of Science
Abstract
Objective: The objective of the present study was to investigate the effect of cadmium (Cd) on the activities of some
oxidative enzymes [viz Aldehyde oxidase, AO (E.C. 1.2.3.1); Xanthine oxidase, XO (E.C. 1.2.3.2); Sulphite oxidase, SO
(E.C.1.8.3.1.); and Monoamine oxidase, MO (E.C. 1.4.3.4)] in the liver and kidney. Materials and methods: Male Wistar
albino rats were administered 1, 2 and 4 mg Cd2+/kg body weight for one and three months. The activities of the oxidative
enzymes were subsequently analyzed in the liver and kidney after both periods of exposure. Results: There was a dose
dependent increase in liver and kidney Cd concentration in the test rats as compared to control after both periods of treat-
ment with the liver retaining higher concentration of Cd than the kidney for each of the exposure dose. The oxidative en-
zymes were decreased in a dose dependent manner in the liver and kidney after both periods of treatment. The percentage
inhibition of these enzymes was less in the liver of rats treated with Cd for three months relative to the one month treated
rats for each of the exposure dose. Conversely, the inhibition of the activities of these enzymes in the kidney of rats in all the
treatment groups was more pronounced after three months relative to the trend in the one month treated rats. However,
the activities of the oxidative enzymes were higher in the liver as compared to the kidney in all the treatment groups after
both durations of Cd treatment. Conclusion: Based on the results obtained, it can be concluded that the inhibition of the
oxidative enzymes by Cd may disturb metabolism of bioactive endogenous substances, exogenous components of food and
some xenobiotics.
Key words:
Cadmium, Oxidative enzymes, Xenobiotics, Metabolism, Rat
Received: September 22, 2009. Accepted: January 27, 2010.
Address reprint request to S.O. Asagba, Department of Biochemistry, Faculty of Science, Delta State University, Abraka PMB 1, Abraka, Nigeria
(e-mail: asagbabch@yahoo.com).
INTRODUCTION
Cadmium is a nonessential trace element which is present
as a contaminant in the general environment. Because of its
widespread nature, cadmium can either be ingested via con-
taminated foods or inhaled. Cadmium accumulates mostly in
the liver and kidney and therefore there is a high potential
for toxicity of the metal in these organs [1]. One of the basis
of cadmium toxicity is its negative influence on enzymatic sys-
tems of cells, owing to substitution of other metal ions (main-
ly Zn2+ and Cu2+) in metalloenzymes and its very strong affin-
ity to biological structures containing SH groups [2,3].
Some of the important enzymes whose activities have been
negatively influenced by cadmium are the xenobiotics
metabolizing enzymes [4]. Foreign compounds are enzy-
matically transformed by these enzymes to less harmful
excretable compounds. This biotransformation process
occurs mostly in the hepatic tissues and to a lesser extent,
some extra hepatic tissues [3]. Oxidation reactions are
probably the most common phase I reactions in xenobi-
otic biotransformation and for these processes, a group of
non-specific, cytochrome P-450 dependent mixed function
oxidases (MFO) are required. Studies have shown that ex-
posure to cadmium decreased cytochrome P-450 activity in
rodents [5–7]. Other enzymes involved in the oxidation of
xenobiotics are aldehyde, xanthine, and sulphite oxidases,
all of which are molybdenum and haem containing soluble

O R I G I N A L P A P E R S S.O. ASAGBA
IJOMEH 2010;23(1)56
liver and kidney were quickly excised, placed on ice and
subsequently weighed. Portions of the liver and kidney
were homogenized to give 20% homogenates and centri-
fuged at 10 000 g for 15 min to obtain clear supernatants
for biochemical analysis.
Digestion of samples
Weighed samples of the liver and kidney of each rat were
digested separately in beakers with 20 ml of acid mixture
(HNO3/HClO4; 4:1 v/v). The digestion was facilitated by
heating at 100°C after which the samples were allowed to
cool and then diluted with deionised water to give a final
volume of 100 ml. Before use, all glass and plastic utensils
were washed in dilute nitric acid and rinsed with deionised
water.
Cadmium analysis
The cadmium concentrations in the digests were measured
by atomic absorption spectrophotometry (Varian AA 1475
Spectrophotometer). The test metal was dissolved in de-
ionised water and used as standard. The analytical meth-
od was checked by using reference material V-10 (Hays)
obtained from the International Atomic Energy Agency
(IAEA).
The cadmium concentration obtained for the reference
sample (0.03±0.002 mg Cd/kg) was in agreement with the
certified value (0.03±0.001 mg Cd/kg). In all the determi-
nations, blanks were prepared to determine the effect of
reagent purity on the metal levels.
Biochemical analysis
The supernatants obtained from the kidney and liver were
used for the determination of the activities of Monoam-
ine oxidase, MO (E.C. 1.4.3.4), Aldehyde oxidase, AO
(E.C. 1.2.3.1.), Sulphite oxidase, SO (E.C. 1.8.3.1) and
Xanthine oxidase, XO (E.C. 1.2.3.2). MO activity was as-
sayed by the method of Tabor et al. [11] based on the oxi-
dative deamination of benzylamine to benzaldehyde. The
activity of the enzyme is expressed in units per gramme
tissue weight and one unit of the enzyme is defined as the
amount of enzyme that is required for the production of
one micromole of benzaldehyde per minute. The activity
enzymes that are present in the liver and other tissues
[8–10]. Monoamine oxidase is also important as it is in-
volved in the biotransformation of aromatic monoamines,
including classical neurotransmitters such as serotonin,
adrenalin, histamine and dopamine.
Despite the role played by these important oxidative en-
zymes in the biotransformation of xenobiotics, animal
studies on the effect of cadmium on these enzymes are
missing. Thus, the present study examines the effect of the
exposure to cadmium on the activities of these oxidative
enzymes (viz aldehyde, xanthine, sulphite and monoamine
oxidases) in the liver and kidney using the rat as animal
model.
MATERIALS AND METHODS
Experimental design
Eighty male albino rats (Wistar strain) with a mean
weight 104±3 g were used for this study. The rats were dis-
tributed into four groups with twenty rats per group such
that the average weight difference between the groups was
less than 0.5 g. Rats in three of these groups were injected
subcutaneously once a week with 1, 2 or 4 mg Cd/kg body
weight, respectively, using CdCl2 (May & Baker, Dange-
ham, England) in same volume of saline. Rats in the last
group served as the control and were treated with same
volume of Cd-free saline. Half of the animals in each
group were given this treatment for one month, while the
remaining half was similarly treated for three months and
the animals were allowed free access to food and water.
All the treatment procedures were approved by the Ethics
Committee for Animal Experiments of the University of
Benin Teaching Hospital, Benin-City, Nigeria.
During the treatment period, water intake, food con-
sumption and dry fecal output were measured daily, while
weight gain was recorded weekly. All these animal treat-
ments were carried out in accordance with the principles
of laboratory animal care of the NIN guide for laboratory
animal welfare as contained in the NIN guide for grants
and contracts. At the end of the specified treatment peri-
od, the animals were fasted for three hours before sacrifice
under chloroform anesthesia. While under anesthesia, the

CADMIUM EXPOSURE ALTERS OXIDATIVE ENZYMES O R I G I N A L P A P E R S
IJOMEH 2010;23(1) 57
relative to the control, although the feed consumption
was not significantly different in all experimental groups.
Conversely, there was a dose dependent increase in dry fe-
cal output of cadmium treated rats relative to the control.
This is a reflection of the inferior feed efficiency of the Cd
treated rats relative to the control.
The status of rat tissue Cd-load, organ/bodyweight ra-
tio, AO, SO, MO and SO activities at the end of one
month Cd treatment is presented in Table 2. There was
a dose dependent increase in liver and kidney Cd con-
centration in the test rats as compared to control. How-
ever, the liver retained higher concentration of Cd than
the kidney for each exposure dose. The liver/body weight
ratio of the test rats was significantly increased in a dose
dependent manner relative to the control. Similarly the
kidney/body weight ratio was significantly increased only
in rats treated with the highest dose (0.4 mg Cd2+/kg
body weight) of the metal. The oxidative enzymes in the
liver were decreased in a dose dependent manner. These
enzymes were also decreased in a dose-dependent man-
ner in the kidney of Cd treated rats as compared to con-
trol. The level of these enzymes was higher in the livers
of rats in each treatment group when compared to the
kidney.
The changes in the oxidative enzymes after three months
of Cd treatment are shown in Table 3. Like in rats treated
with Cd for one month, there was a dose dependent de-
crease in the activities of the oxidative enzymes in the liver
of the test rats relative to the control. However, the per-
centage inhibition was less in rats treated with Cd for three
months relative to the one month treated rats for each of
of AO was monitored by the method of Johns [12] which
is based on the oxidation of benzaldehyde to benzoate
using 2,6-dichloroindolephenol (DCIP) as the electron
acceptor. The activity of the enzyme is given in units per
gramme tissue weight and one unit is the amount of en-
zyme that produces one micromole of benzoate per min-
ute. The activity of XO was determined by the method of
Stirpe and Della Corte [13] using xanthine as the substrate
and oxygen as electron acceptor. The enzyme activity is ex-
pressed in XO units per gramme tissue and each XO unit
is the amount of the enzyme that produces one micromole
uric acid. SO activity was determined by the method of
Macleod et al. [14]. The principle is based on the oxida-
tion of sulphite to sulphate by the enzyme using ferricya-
nide as electron acceptor. The activity of the enzyme is
also expressed in units per gramme tissue and one unit
represents the amount of the enzyme that reduces one mi-
cromole of ferricyanide in one minute.
Statistical analysis
The values are reported as means ±SD. Statistical dif-
ferences for the biochemical values were determined us-
ing analysis of variance (ANOVA) and differences in the
means were tested by Duncan’s multiple range test [15].
RESULTS
Table 1 shows the feed consumption, weight gain, feed
efficiency and dry fecal output of rats exposed for three
months to varying doses of Cd. There was a dose depen-
dent decrease in weight gain of cadmium treated rats
Table 1. Values of food consumption, weight gains, feed efficiency and dry fecal output of rats treated with cadmium for three months
Parameter
Dose (mg Cd/kg b.w.)
Control +Cd (1.0) +Cd (2.0) +Cd (4.0)
Food intake (g/rat/day) 26.8±4.5a 25.7±5.4a 26.5±5.7a 25.2±4.8a
Weight gain (g/rat/day) 1.8±0.2a 1.6±0.3a 1.2±0.3b 0.7±0.04c
Feed efficiency (g b.w. / g feed) 0.07 0.06 0.05 0.03
Dry fecal output (g/day/rat) 1.3±0.04a 1.6±0.04a 2.4 ±0.05b 2.7±0.05c
b.w. — body weight.
Values are given as mean ±SD.
a-c Means with matching superscripts in each row are not significantly different at p < 0.05.

O R I G I N A L P A P E R S S.O. ASAGBA
IJOMEH 2010;23(1)58
relative to the control. There was a dose dependent in-
crease in tissue Cd-load of test rats relative to control.
As observed in rats treated with Cd for one month, the
liver Cd concentration for each exposure dose remained
higher than that of the kidney. The Cd concentration in
the tissues of the three months treated rats was higher as
compared to that of the one month exposed rats for each
exposure concentration.
DISCUSSION
This study provides evidence for the pattern of accumula-
tion of cadmium over time and its effect on some oxidative
enzymes in the tissues of the rat.
the exposure doses. Again, the activity of these enzymes
in the kidney was also similarly decreased in a dose de-
pendent manner. Unlike in the liver, the inhibition of the
activities of these enzymes in the kidney of rats in all treat-
ment groups was more pronounced after three months
relative to the trend in the one month treated rats. How-
ever, as was observed in the one month exposed rats, the
activities of the oxidative enzymes were higher in the liver
as compared to the kidney in all the treatment groups.
The changes in other parameters under study and
the Cd-load in the organs after three months Cd treatment
are also presented in Table 3. The organ/body weight ratio
for the liver and kidney of the test rats were not signifi-
cantly different, but were increased significantly (p < 0.05)
Table 2. Status of rat tissue Cd-load, organ/body weight ratio, AO, SO, MO and XO activities at the end of one month
cadmium treatment
Organ/parameter
Dose (mg Cd/kg b.w.)
Control +Cd (1.0) +Cd (2.0) +Cd (4.0)
Liver
Cadmium load (μg/g tissue) 0.005±0.0007a 25.7±2.5b 55.4±5.2c 115.8±12.6d
Organ/b.w. ratio (× 10-2) 2.9±0.3a 3.6±0.4b 4.8±0.4c 5.8±0.8d
Aldehyde oxidase (AO) 85.7±8.5a 70.2±5.2b
(18.1)
56.8±6.5c
(33.7)
40.6±5.5d
(52.7)
Xanthine oxidase (XO) 75.50±4.5a 60.4±5.4b
(20)
49.3±4.7c
(34.7)
28.6±5.0d
(62.1)
Monoamine oxidase (MO) 157.0±10.5a 138.2±8.5b
(12)
87.0±8.2c
(44.9)
63.0±5.5d
(60)
Sulphite oxidase (SO) 685.4±45.8a 583.9±40.7b
(14.8)
424.9±34.6c
(38)
322.0±36.5d
(53)
Kidney
Cadmium load (μg/g tissue) 0.007±0.0004a 13.6±2.3b 28.3±4.6c 50.4±9.2d
Organ/b.w. ratio (× 10-2) 0.42±0.04a 0.47±0.04a 0.45±0.05a 0.56±0.05b
Aldehyde Oxidase (AO) 18.4±2.1a 16.7±2.6a 15.6±2.2b
(15.2)
14.3±2.2b
(22.3)
Xanthine Oxidase (AO) 50.4±7.5a 38.6±10.4b
(23.4)
37.5±7.6b
(25.6)
22.6±5.8c
(55.2)
Monoamine oxidase (MO) 52.0±5.4a 44.4±5.5b
(14.6)
37.5±4.4c
(27.9)
30.6±5.2d
(41.2)
Sulphite Oxidase (SO) 254.6±25.4a 239.5±30.5a 215.8±32.5b
(15.2)
205±30.5b
(16.4)
Abbreviations as in Table 1.
The activities of the oxidative enzymes are in units/g tissue.
Figures in parenthesis represent percentage inhibition relative to control.

CADMIUM EXPOSURE ALTERS OXIDATIVE ENZYMES O R I G I N A L P A P E R S
IJOMEH 2010;23(1) 59
subcutaneously, more was deposited in the liver than in
the kidneys [16,23,24]. Experimental evidence also indi-
cates that the transport and distribution of Cd to tissues
is aided by metallothionein (MT) [25,26]. MTs are a fam-
ily of low molecular weight heavy metal binding proteins,
unique in their high cysteine (Cys) content. These pro-
teins are widespread in eukaryotes and plants and are
also found in prokaryotes [27].
The increased organ/body weight ratio for liver and kid-
ney of Cd treated rats could be attributed to the toxicity
of Cd in these organs. Changes in organ/body weight ratio
in rats administered Cd have also been observed by many
workers. Horiguchi et al. [16] observed hepatosplenomeg-
aly and kidney swelling after subcutaneous administration
of Cd in rats. Similarly, a Cd-induced increase in liver and
spleen weight has also been observed by Tu et al. [28].
The decrease in weight gain of the rats is in conso-
nance with previous reports on the effect of cadmium
on weight gain of rats [16,17]. The decrease in weight
gain and the corresponding increase in dry fecal out-
put of the Cd trea ted rats is also consistent with previ-
ous reports on the inhibitory effect of Cd on digestive
and absorption enzymes in rats [18–20]. The inhibitory
effect of Cd on the digestive and absorption enzymes
may also account for the decreased feed efficiency ob-
served in the Cd treated rats. The increased accumula-
tion of Cd observed in the liver and kidney of Cd exposed
rats is in consonance with earlier reports [16,21,22]. Ex-
amination of the cadmium levels in the liver of rats after
both periods of exposure to cadmium shows that it was
consistently higher than the level in kidney. Available re-
ports indicate that when Cd was administered orally and
Table 3. Status of rat tissue Cd-load, organ/body weight ratio, AO, SO, MO and XO activities at the end of three month cadmium
treatment.
Organ/parameter
Cadmium dose (mg/kg b.w.)
Control +Cd (1.0) +Cd (2.0) +Cd (4.0)
Liver
Cadmium load (μg/g tissue) 0.02±0.003a 78.5±10.2b 156.6±12.5c 308±15.4d
Organ/b.w. ratio (× 10–2) 3.4±0.3a 4.9±0.5b 4.6±0.5b 4.8±7.6b
Aldehyde oxidase (AO) 95.4±10.5a 80.5±8.2b
(15.6)
68.5±7.0c
(28.2)
53.7±8.5d
(43.7)
Xanthine oxidase (XO) 79.7±5.6a 68.5±5.2b
(18.8)
60.6±4.7c
(31.9)
48.4±4.8d
(52.4)
Monoamine oxidase (MO) 120.6±12.5a
(10.4)
108.0±10.6b
(29.5)
85.0±7.5c
(49.6)
60.8±7.8d
Sulphite oxidase 858.8±54.6a 794.5±40.5b
(7.5)
625.4±30.5c
(27.2)
586.5±36.7d
(31.7)
Kidney
Cadmium load (μg/g tissue) 0.03±0.002a 30.0±5.4b 56.4±6.5c 76.8±8.0d
Organ/b.w. ratio (× 10-2) 0.45±0.03a 0.52±0.04b 0.58±0.06b 0.56±0.05b
Aldehyde oxidase (AO) 40.8±5.5a 30.5±4.8b
(25.2)
26.4±4.2b
(35.3)
18.0±3.4c
(55.9)
Xanthine oxidase (XO) 60.5±7.4a 42.4±5.6b
(29.9)
32.6±5.5c
(46.1)
24.4±4.2d
(59.7)
Monoamine oxidase (MO) 70.6±8.6a 58.0±7.5b
(17.8)
40.6±8.5c
(42.5)
27.8±3.6d
(60.6)
Sulphite oxidase (SO) 176.4±12.0a 150.5±15.6b
(14.7)
130.4±10.4c
(26.1)
112.0±14.5d
(36.5)
Abbreviations as in Table 1 and 2.