53
Research report
Fluoride 39(1)53–59
January-March 2006
Lipid peroxidation and antioxidant activity in rats exposed to F and EtOH
Inkielewicz, Rogowska, Krechniak
53
LIPID PEROXIDATION AND ANTIOXIDANT ENZYME ACTIVITY IN
RATS EXPOSED TO FLUORIDE AND ETHANOL
I Inkielewicz,a,b M Rogowska,a J Krechniaka
Gdańsk, Poland
SUMMARY: The aim of this study was to investigate the impact of fluoride (F) and
ethanol (EtOH) administered separately or together on free radical mediated
parameters and on F accumulation and excretion in rats in a 4-week experiment.
Thirty adult male Wistar rats were divided into five equal groups: 1. controls drinking
tap water (0.3 mg F/L); 2. controls drinking tap water and for the last 14 days of the
experiment dosed intragastrically with 1 mL of tap water containing 0.3 mg F/L (twice/
day); 3. animals receiving 25 mg F/L in drinking water; 4. animals receiving 5 g
ethanol/kg bw/day for the last 14 days of the experiment; 5. animals receiving 25 mg
F/L in drinking water and 5 g ethanol/kg bw/day for the last 14 days of the experiment.
In rats treated only with F a significant increase in F concentration was found in liver,
kidney, brain, and serum, and the F excretion in urine increased in an exposure timedependent manner. The activity of catalase (CAT) and the concentration of sulfhydryl
(SH) groups decreased, whereas the concentration of thiobarbituric acid reactive
substances (TBARS) increased in all investigated tissues. In animals treated with
EtOH alone, the F content in liver and kidney, CAT activity, and TBARS concentration
increased, and the concentration of SH groups decreased in all investigated tissues.
In rats co-exposed to F and EtOH, the F concentration significantly increased in
brain, and the CAT activity and the concentration of SH groups increased in all
investigated tissues, whereas the TBARS concentration was lower (except in kidney)
than in animals given only F. The results of this study indicate that both F and EtOH
can induce lipid peroxidation in rats under separate or combined exposure.
Keywords: Antioxidant enzyme activity; Ethanol treatment; Fluoride in urine; Fluoride in rats;
Lipid peroxidation.
INTRODUCTION
In their natural and occupational environments, humans are frequently exposed
to many unsuspected chemical substances. Recently, increased attention has been
paid to interactions of xenobiotics with one another or with dietary factors. It is
well known that uptake, accumulation, and toxicity of many xenobiotics can be
modified by dietary factors.1-6 Particularly important are interactions between
xenobiotics to which exposure is quite common. Examples of such substances are
fluoride (F) and ethanol (EtOH). Interactions between F and EtOH are an
important problem in modern toxicology since both pose a risk to human and
animal health.
Exposure to F can occur in the workplace and in the environment because F is
utilized in a number of industrial practices and is a ubiquitous ingredient of
drinking water, foodstuffs, and dental products.7 Likewise, widespread alcoholism
is a serious problem in modern society. Excessive consumption of EtOH in the
form of alcoholic beverages may be common among industrial workers and
inhabitants of areas with elevated water fluoride.8,9 Because ethanol increases the
aDepartment
of Toxicology, Medical University of Gdańsk. bFor Correspondence: Department
of Toxicology, Medical University of Gdańsk. 80-416, Gdańsk, Al. Gen. Hallera107, Poland.
E-mail: [email protected]
54
Research report
Fluoride 39(1)53–59
January-March 2006
Lipid peroxidation and antioxidant activity in rats exposed to F and EtOH
Inkielewicz, Rogowska, Krechniak
54
permeability of biological membranes it can make alcoholics more susceptible to
the effects of other xenobiotics including ethanol compared to non-alcoholics.
One of the effects of F exposure in organisms is the impact of free radical
parameters on antioxidant defense systems, such as the activity of antioxidant
enzymes and nonenzymatic species leading to an increase in free radical mediated
tissue impairments such as lipid peroxidation.10-16 Exposure to EtOH can also
interfere with free radical parameters in the organism, especially by increasing
lipid peroxidation. Induction of oxidative stress by ethanol is well established
experimentally.17-21
The aim of this study was to investigate the impact of F and EtOH given
separately or together to rats in a short-term experiment on free radical mediated
parameters and on F accumulation in organs and excretion with urine.
MATERIALS AND METHODS
Animals and experimental design: The study was carried out for a period of 4
weeks on 30 adult male Wistar rats weighing approximately 162 ± 8.2 g at the
beginning of the experiment. The animals were kept under standard laboratory
conditions (temperature 22±2ºC in a natural light-dark cycle). All animals were
fed a standard laboratory pellet diet containing 0.7 mg water-extractable F/kg. The
animals were randomly allocated to five groups of six rats each:
I
Controls, drinking tap water containing 0.3 mg F/L
II
Controls, drinking tap water containing 0.3 mg F/L and given tap water containing 0.3 mg F/L
intragastrically (1 mL twice/day by a stomach tube) for the final 14 days of the experiment
III
Exposed, drinking water containing 25 mg F (from NaF)/L
IV
Exposed, receiving ethanol intragastrically (1 mL) in a total dose of 5 g/kg bw/day divided into two
equal doses administered at 8:00 am and 2:00 pm for the final 14 days of the experiment
V
Exposed, receiving NaF as in Group III and ethanol as in Group IV
At the end of the experiment animals were sacrificed by exsanguination under
ether narcosis, and samples of blood, kidney, liver, and brain were collected.
Blood was taken with and without anticoagulant by cardiac puncture.
Analytical procedures: In blood, kidney, liver, and brain, catalase (CAT) activity
was determined by the method of Aebi.22 The concentration of TBARS
(thiobarbituric acid reactive substances) was determined by the method of RiceEvans et al.23, and the level of SH (sulfhydryl) groups was determined according
to Ellman et al.24
Protein content in blood, kidney, liver, and brain was determined by the method
of Lowry et al.25 Hemoglobin content in the blood was determined by the method
of Drabkin.26
Each week the concentration of F in urine was determined directly after dilution
with equal volumes of TISAB buffer by a fluoride ion-specific electrode (Orion)
and Ag/AgCl reference electrode.27 Urinary creatinine was determined by the
method of Folin-Morris.28 Urinary F is reported as mg F/g creatinine.
The concentration of F in soft tissues was determined after dry combustion of
the sample according to the method described recently by us.29 The accuracy of
measurements was assessed with reference materials – in serum and soft tissues
with Serum Control (Clin Check, Munich, Germany), and in urine with Seronorm
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Lipid peroxidation and antioxidant activity in rats exposed to F and EtOH
Inkielewicz, Rogowska, Krechniak
55
Control Urine (Nycomed Pharma AC, Oslo, Norway). Mean F recovery was
99.6% from urine, 97.9% from serum, and 103% from soft tissues.
Statistical analysis: Statistical analysis was performed using the FisherSnedecor and Student’s t-test.
RESULTS
Results of water consumption and fluoride intake by the five groups of rats are
presented in Table 1.
Animals
group
treatment
I
Controls
II
Controls (i.g.)
III
F
IV
EtOH
V
F + EtOH
Table 1. Water consumption and F intake
Water consumption mL/24 hr
F intake mg F/24 hr/rat
mean±SD
mean
range
34.5±2.5
0.010
0.009–0.011
33.8±3.6
0.010
0.009–0.011
35.3±3.9
0.882
0.780–0.980
30.2±4.2
0.009
0.010–0.008
35.9±4.3
0.897
0.790–1.000
F concentrations in soft tissues, serum, and urine are given in Tables 2 and 3.
Table 2. Fluoride content in soft tissues (µg F/g) and serum (µg F/mL)
Animals
Kidney
Liver
Brain
Serum
group
treatment
mean±SD (n)
mean±SD (n)
mean±SD (n)
mean±SD (n)
I
Controls
0.848±0.037 (6)
0.708±0.028 (6)
0.632±0.033(5) 0.051±0.006 (5)
II
Controls (i.g.) 0.784±0.054 (6)
0.666±0.023 (6)
0.586±0.054(4) 0.056±0.007 (5)
III
F
5.380±0.464 (6)
3.940±0.134 (6)
3.350±0.252(5) 0.095±0.049 (4)
IV
EtOH
0.863±0.081 (6)
0.721±0.052 (6)
0.616±0.046(5) 0.053±0.007 (4)
V
F + EtOH
5.770±0.395 (6)
3.720±0.507 (5)
3.660±0.227(5) 0.090±0.017 (5)
Statistical significance:
I vs. III
II vs. IV
I vs. V
II vs. V
III vs. V
IV vs. V
n=number of animals.
p<0.001↑
p<0.05↑
p<0.01↑
p<0.001↑
n.s.
p<0.001↑
p<0.001↑
p<0.05↑
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
p<0.001↑
p<0.05↑
p<0.05↑
Table 3. Fluoride concentration in urine (mg F/g creatinine ± SD)
Animals
Exposure time in weeks
group
treatment
0
1
2
I
Controls
2.12±0.078
2.26±0.610
1.76±0.161
II
Controls (i.g.)
1.79±0.152)
1.62±0.218
III
F
4.02±0.912
5.34±0.036
IV
EtOH
2.07±0.129
1.95±-0.161
V
F + EtOH
4.51±0.250
5.58±0.611
Statistical significance:
p<0.001↑
I vs. III
n.s.
II vs. IV
p<0.001↑
I vs. V
p<0.001↑
II vs. V
n.s.
III vs. V
p<0.001↑
IV vs. V
Values are means±SD for 4 animals in each group.
p<0.0001↑
n.s.
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
3
1.83±0.009
1.95±0.092
6.62±1.720
2.21±0.349
6.21±0.318
p<0.001↑
n.s.
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
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Lipid peroxidation and antioxidant activity in rats exposed to F and EtOH
Inkielewicz, Rogowska, Krechniak
56
Catalase (CAT) activity in soft tissues and erythrocytes is presented in Table 4.
Table 4. Catalase (CAT) activity in soft tissues (k/mg protein) and erythrocytes (k/g Hb)
Animals
Liver
Kidney
Brain
Erythrocyte
group
treatment
mean±SD
mean±SD
mean±SD
mean±SD
I
Controls
0.225±0.028
5.26·10-2±4.90·10-3 1.01·10-3±8.56·10-5
32.82±3.58
II
Controls (i.g.)
0.213±0.018
4.59·10-2±1.89·10-3 9.22·10-4±1.81·10-4
29.84±3.94
III
F
0.153±0.019
3.69·10-2±1.89·10-3 6.35·10-4±7.38·10-5
36.36±2.99
-2
-3
-3
-5
39.82±3.34
40.12±1.90
IV
EtOH
0.252±0.019
7.82·10 ±7.73·10
V
F + EtOH
0.239±0.015
5.76·10-2±2.27·10-3 7.64·10-4±8.16·10-5
Statistical significance:
I vs. III
II vs. IV
I vs. V
II vs. V
III vs. V
IV vs. V
p<0.01↓
p<0.01↑
n.s.
p<0.05↑
p<0.001↑
n.s.
p<0.001↓
p<0.001↑
n.s.
p<0.05↑
p<0.001↑
p<0.001↓
1.12·10 ±4.15·10
p<0.0001↓
n.s.
p<0.01↓
n.s.
p<0.05↑
p<0.001↓
n.s.
p<0.01↑
p<0.01↑
p<0.001↑
p<0.05↑
n.s.
Values are for 6 animals in each group.
Concentrations of thiobarbituric acid reactive substances (TBARS) and
sulfhydryl (SH) groups in soft tissues and plasma are given in Tables 5 and 6,
respectively.
Table 5. TBARS concentration in soft tissues and plasma (nM/g protein)
Animals
Liver
Kidney
Brain
group
treatment
mean±SD
mean±SD
mean±SD
I
Controls
409.8±19.23
692.2±12.7
781.0±24.9
II
Controls (i.g.)
350.7±18.02
621.5±3.4
744.7±7.0
III
F
588.8±15.94
795.0±23.3
889.8±13.6
IV
EtOH
417.3±10.11
811.5±15.1
804.8±8.9
V
F + EtOH
539.2±8.86
858.8±19.4
873.2±20.1
Statistical significance:
I vs. III
II vs. IV
I vs. V
II vs. V
III vs. V
IV vs. V
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↓
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
n.s.
p<0.001↑
Plasma
mean±SD
305.8±11.7
244.7±5.9
410.7±13.0
320.2±24.1
363.3±18.6
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↑
p<0.001↓
p<0.001↑
Values are for 6 animals in each group.
Table 6. Concentration of sulfhydryl (SH) groups in liver and kidney (mM/g protein)
and in brain and plasma (µM/g protein)
Animals
Liver
Kidney
Brain
Plasma
group
treatment
mean±SD
mean±SD
mean±SD
mean±SD
I
Controls
0.183±0.051
0.129±0.022
3.29±0.26
7.23±0.64
II
Controls (i.g.)
0.192±0.019
0.126±0.015
2.48±0.25
7.06±0.71
III
F
0.116±0.020
0.079±0.013
1.80±0.18
4.72±0.62
IV
EtOH
0.158±0.036
0.113±0.014
2.10±0.16
6.12±1.27
V
F + EtOH
0.146±0.046
0.110±0.006
2.01±0.10
5.89±0.51
Statistical significance:
I vs. III
II vs. IV
I vs. V
II vs. V
III vs. V
IV vs. V
p<0.001↓
p<0.05↓
p<0.05↓
p<0.001↓
p<0.01↑
n.s.
Values are for 6 animals in each group.
p<0.001↓
n.s.
p<0.05↓
p<0.05↓
p<0.001↑
n.s.
p<0.001↓
p<0.05↓
p<0.001↓
p<0.001↓
p<0.05↑
n.s.
p<0.001↓
p<0.05↓
p<0.001↓
p<0.001↓
p<0.001↑
n.s.
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Lipid peroxidation and antioxidant activity in rats exposed to F and EtOH
Inkielewicz, Rogowska, Krechniak
57
DISCUSSION
The present study was undertaken to assess the oxidative status in liver, kidney,
brain, and blood of rats during co-exposure to F and EtOH. For this purpose, the
activity of the antioxidant enzyme CAT, concentration of TBARS as an indicator
of lipid peroxidation, and the concentration of SH groups that protect the cell from
oxidative stress were determined in these tissues. The exposure of rats to 25 mg F/
L corresponds to human environmental exposure in areas with high F level in
drinking water or in occupational conditions.7 Intragastric administration of 5 g
ethanol/kg bw/24 hr corresponds to ethanol abuse in man.21
Decreased consumption of water was noticed only in the EtOH group. In the F
group water consumption was lower, but not insignificantly lower. The intake of
water in the F and the F + EtOH groups was at the same level.
As expected, exposure to F resulted in a marked increase of F concentration in
kidney, liver, brain, and serum.30 Ethanol alone increased the F content in kidney
and liver, but had no effect on other tissues. This may be due to increased
permeability of cellular membranes caused by ethanol. In rats co-exposed to F and
EtOH the F concentration increased only in brain compared to animals treated
with F alone.
Also, a significant increase in F excretion in urine occurred following
administration of NaF in the drinking water.30 The F increase changed in an
exposure-time dependent manner. In animals exposed only to EtOH, urinary F
excretion increased insignificantly. Co-exposure to F and EtOH did not
significantly affect the rate urinary F excretion.
Enhanced peroxidation of lipids in intracellular and extracellular membranes is
known to cause damage to tissues and organs. Antioxidant enzymes (SOD, CAT,
GPx) are very important in protecting organisms from reactive oxygen species
(ROS). CAT is a hemoprotein found in the peroxisomes of eukaryotic cells that
catalyses the conversion of hydrogen peroxide to water and oxygen. The activity
of this enzyme also can be induced in response to cellular hydrogen peroxide
exposure.31
In the rats treated with sodium fluoride, CAT activity was significantly
depressed in liver, kidney, and brain. On the other hand, EtOH caused a significant
increase in the activity of this antioxidant enzyme in the investigated tissues. Coexposure to F and EtOH resulted in intermediate activity. In co-exposed animals
catalase activity was higher than in animals exposed only to F, whereas it was
lower in rats treated only with EtOH.
As noted by various authors, F can stimulate lipid peroxidation in membranous
structures.15,32-34 However, the mechanism of fluoride-induced lipid peroxidation
is not fully clarified. Available data indicate that the mechanism is multidirectional
and may involve a decrease in the level of glutathione and the total pool of SH
groups and changes in the activity of antioxidant enzymes. These can induce a
peroxidative state in biological systems and, in turn, lead to peroxidation of
polyunsaturated fatty acids.35
SH groups occur in living organisms as constituents of enzymes, tissue and
serum proteins, peptides (glutathione), coenzymes, and single amino acids. SH
58
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January-March 2006
Lipid peroxidation and antioxidant activity in rats exposed to F and EtOH
Inkielewicz, Rogowska, Krechniak
58
groups easily react with free radicals, notably with hydroxyl radicals, and protect
the cell against oxidative attack by chemicals. Apart from antioxidant defense
enzymes and other low-molecular-antioxidants, SH groups are important
components of the antioxidant defense system of organisms.35
In the present study, exposure of the rats to F decreased the concentration of SH
groups in all investigated tissues. A similar pattern of changes was observed in the
ethanol group. In animals co-exposed to F and EtOH, however, the level of SH
groups increased compared to those treated only with F, but the level decreased
significantly compared to animals exposed only to EtOH.
TBARS concentration increased significantly in all investigated tissues of
animals exposed to F, thereby indicating an enhancement of lipid peroxidation.
The EtOH induced escalation of lipid peroxidation might be the consequence of
increased formation of free radicals as well as the inhibition of CAT and SOD
(superoxide dismutase). The present study confirmed the involvement of EtOH in
lipid peroxidation. Ethanol is able to induce the formation of free radicals. During
EtOH biotransformation occurring with the involvement of the microsomal
ethanol-oxidizing system (MEOS), xanthine oxidase and aldehyde oxidase,
reactive oxygen species (ROS) and hydroxyethyl radicals are generated.17-19
EtOH, apart from being a source of free radicals, can also disturb the antioxidant
defense system.36
Although significant, the increase of TBARS in rats treated only with EtOH is
lower than in animals exposed to F. In animals co-exposed to F and EtOH the
concentration of TBARS was in intermediate level. Only in the kidney, probably
due to a less effective antioxidant defense system compared to the liver, the
TBARS concentration increased very distinctly indicating a synergistic effect.
A very important finding of this study is that, despite the ability of F and EtOH
to induce oxidative stress, the effect is not intensified (except in the kidney) by
simultaneous exposure to both substances. The disturbances in the oxidative status
observed in our study indicate a risk of organ damage during exposure to F and
EtOH via free radical mechanism.
The results concerning the activity of CAT, the concentration of SH groups, and
concentration of TBARS in the investigated tissues clearly indicate that F and
EtOH are able to induce the oxidative stress during repeated administration singly
or together during co-exposure.
ACKNOWLEDGEMENT
This study was supported by Grant 21 from the Medical University of Gdañsk.
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