Keywords
brain tissues - deferoxamine - homogenates - lipid peroxidation - vanadium - vitamin
C
Introduction
Vanadium is a ubiquitous element. The use of vanadium due to its insulin-mimetic properties
has been referred to as antidiabetic agent and anabolic agent.[1] On the other hand, environmental and occupational vanadium exposure has been associated
with several deleterious health hazards linked with carcinogenic, immunotoxic, and
neurotoxic insults.[1] Among the handful of proposed mechanisms of vanadium toxicity, which include interference
with lipids, and proteins, depletion of antioxidant defense system and induces double-strand
breaks in DNA. However, among all induction of oxidative stress is of paramount importance
for biological systems.[2]
We have established that vanadium induces lipid peroxidative damage and this has been
proposed as a likely basis for its neurotoxicity.[3]
[4]
[5]
[6] Antioxidants such as vitamin E, vitamin C, selenium, and doxycycline have been used
with success against vanadium toxicity in experimental animals.[3]
[4]
[5]
[6]
[7]
The literature review provides in vitro only one such study,[1] where manganese decreased the cellular uptake of vanadium and prevented DNA damage
in hepatocytes.[1] The general view is that vanadium has the potential to induce aneuploidy, micronucleus,
and chromosomal aberrations in some cells in vitro and in vivo.[8]
Therefore, the aim of this study was to examine in vitro effects of vanadium on occurrence
of lipid peroxidation (LPO) in fresh homogenates in rat brain hypothalamus, hippocampus,
mid-brain, and medulla-pons, to identify possible beneficial effects of vitamin C
(ascorbic acid) and deferoxamine, which is a chelator class of drug for iron and aluminum
toxicity),[9] against vanadium-stimulated lipid peroxidative damage.
Methods
Chemicals
Sodium metavanadate, L-ascorbic acid, trichloroacetic acid, and 1,1,3,3 tetraethoxy-propane
were purchased from Sigma Chemical Co. (St. Louis, Missouri, United States). Deferoxamine
was obtained from Ciba-Geigy AG (Klybeckstrasse 141, Basel, 4002 Switzerland) and
potassium chloride was purchased from Merck Darmstadt (Federal Republic of Germany).
Operative and Sampling Technique
Standard anatomical technique for the dissection of multiple brain regions from a
single brain and standard biochemical colorimetric procedures were utilized in this
study.
All experiments were performed on brain hippocampus, hypothalamus, mid-brain, and
medulla-pons tissues from fed six male Sprague-Dawley rats (body weight 300–400 g)
obtained from the Central Animal House of the Faculty of Medicine, University of Benghazi.
Animals were sacrificed by decapitation without anesthesia. The brains were rapidly
excised on a petri dish placed on crushed ice, and the tissues were kept at −80°C
until used for the in vitro experiments.
Preparation of Brain Homogenates
Brain tissues, hippocampus, hypothalamus, mid-brain, and medulla pons (up to 400 mg
from each brain) were dissected on an ice plate and transferred to an ice-cold 150 mM
potassium chloride solution. The tissue was kept in the cold medium for 10 minutes
before it was homogenized in chilled 150 mM KCl, using a glass homogenizer with Teflon
pestle fitted to motor drive, and the volume was adjusted to give a 10% w/v homogenate.
In this study, brain tissue homogenates were exposed to elemental vanadium in the
form of sodium metavanadate. The chosen exposure levels (20 μM and 100μM) were referred
to as no observed adverse effect level concentration. Previously, one research group
has used the same concentration(in μM) in an oral study[10] therefore, equivalent concentration of antioxidants vitamin C as ascorbic acid (20
and 100 μM) and deferoxamine (20 and 100 μM) was added to the reaction mixture. Three
series of experiments were performed. In the first series, homogenates were equilibrated
under molecular oxygen on bench followed by addition of elemental vanadium (20 and
100μM). In the second series, homogenates were equilibrated with molecular oxygen
on bench followed by addition of vitamin C (20 and 100 μM) + vanadium (20 and 100
μM). In the third series, homogenates were equilibrated with molecular oxygen on bench
followed by addition of deferoxamine (20 and 100 μM) + vanadium (20 and 100 μM).
Determination of Malondialdehyde Levels
The content of malondialdehyde (MDA) formation (a product of LPO) was estimated in
the form of thiobarbituric acid reactive material. It was estimated in four sets of
homogenate reaction mixtures, namely control, vanadium exposed, vitamin C + vanadium
tested, and vanadium + deferoxamine tested. Aliquots (200 μL), containing 300–400 mg
brain tissue homogenates (in triplicate) [11] were pipetted in test tubes followed by addition of 200 μL of 8.1 of sodium dodecyl
sulfate, 1.5 mL of 20% acetic acid, and 1.5 mL of 1% thiobarbituric acid. The contents
were mixed on a vortex mixer. The final volume of the reaction mixture was adjusted
to 4 mL with distilled water. The tubes were mixed on a vortex mixer. The samples
were heated in a hot water bath at 100°C for 60 minutes. After cooling under tap water,
the samples were added 1 mL of distilled water, extracted with 5 mL of n-butanol: pyridine (15 mL: 1 ml v/v), mixed vigorously on a vortex mixer, and centrifuged
at 4,000 rpm for 15 minutes. The MDA content in the n-butanol: pyridine layer was then spectrophoto-metrically determined at 535 nm.[12] MDA levels were calculated from the standard curve using 1, 1, 3, 3-tetra ethoxy
propane and expressed as nano-moles of MDA/g fresh tissue.
Statistical Analysis
The data was presented as means ± standard error of mean (n = 6). Data were analyzed by one-way analysis of variance. When the analysis indicated
a significant difference (p < 0.05), the fortified groups were compared with their respective controls. Statistical
analysis was performed by F-test for homogeneity of variance followed by t-test.[13]
Results
In Vitro Effect of Vanadium on Lipid Peroxidation in Homogenates from Different Regions
of Rat Brain
The results in [Table 1] demonstrate a dose–response relationship in the occurrence of LPO following exposure
to vanadium (20 and100 μM). The order of enhancement was hypothalamus (+145 and 175%),
hippocampus (+111 and 126%), mid-brain (+81 and 113%), and the medulla-pons (81 and
325%, respectively (see [Table 2]). The hypothalamus was most vulnerable region, where occurrence of LPO was highest.
[Table 2] shows that the average vanadium (20μM)-induced percent increase in occurrence of
LPO was +105% among brain regions compared with the controls. However, the average
percent increase in occurrence of LPO in total brain homogenates-induced by vanadium
(100μ) was by +185.3%. This was 80.3% faster than in homogenates exposed to 20μM vanadium.
Table 1
The effect of vitamin C and deferoxamine in the rat brain tissue homogenates against
vanadium-induced lipid peroxidation
|
In vitro test
|
Dose
(µ mM)
|
Brain regions
|
Total levels of MDA
|
|
Mid-Brain
|
Hypothalamus
|
Hippocampus
|
Medulla-pons
|
|
Lipid peroxidation (nano-mole MDA/fresh weight tissue)
|
|
Control (water)
|
0
|
0.471 ± 0.007
|
0.471 ± 0.090
|
0.515 ± 0.075
|
0.458 ± 0.009
|
1.915
|
|
Vanadium
|
20
|
0.854[a] ± 0.034
|
1.156[a] ± 0.039
|
1.090[a] ± 0.018
|
0.828[a] ± 0.006
|
3.928
|
|
Vanadium
|
100
|
1.007[a] ± 0.018
|
1.294[a] ± 0.066
|
1.166[a] ± 0.057
|
1.950[a] ± 0.030
|
5.417
|
|
Vitamin C + vanadium
|
20 + 20
|
1.317[b] ± 0.008
|
1.179 ± 0.033
|
1.164 ± 0.042
|
1.093[b] ± 0.045
|
4.753
|
|
Vitamin C + vanadium
|
100 + 100
|
1.447[c] ± 0.002
|
1.323 ± 0.024
|
1.279 ± 0.021
|
1.326[c] ± 0.043
|
5.375
|
|
Deferoxamine + vanadium
|
20 + 20
|
0.373[b] ± 0.014
|
0.398[b] ± 0.018
|
0.366[b] ± 0.060
|
0.282[b] ± 0.033
|
1.419
|
|
Deferoxamine + vanadium
|
100 + 100
|
0.494[c] ± 0.030
|
0.587[c] ± 0.024
|
0.518[c] ± 0.037
|
0.381[c] ± 0.028
|
1.980
|
Abbreviations: MDA, malondialdehyde; SD, standard deviation.
In vitro test homogenates were incubated in test tubes in hot water bath for 1 hour
at 100°C.
Each value represents the mean ± SD from six independent experiments.
a
p < 0.001 versus control group.
b
p < 0.001 versus (20 μM) vanadium group.
c
p < 0.001 versus (100 μM) vanadium group.
Table 2
The percentage changes in the rat brain tissue homogenates following exposure to vitamin
C and deferoxamine against vanadium-induced lipid peroxidation
|
In vitro test
|
Dose (μM)
|
Brain regions
|
Total % change in brain regions
|
|
Mid-brain
|
Hypothalamus
|
Hippocampus
|
Medulla-pons
|
|
% change from control
|
% change from vanadium alone
|
% change from control
|
% change from vanadium alone
|
% change from vanadium control
|
% change from vanadium alone
|
% change from control
|
% change from vanadium
|
% change from control
|
% change from vanadium alone
|
|
Control (water)
|
0
|
0
|
–
|
–
|
–
|
–
|
–
|
–
|
–
|
|
|
|
Vanadium
|
20
|
81
|
–
|
145
|
–
|
112
|
–
|
81
|
–
|
105.1
|
|
|
Vanadium
|
100
|
114
|
–
|
175
|
–
|
126
|
–
|
326
|
–
|
185
|
|
|
Ascorbic acid + vanadium
|
20 + 20
|
180
|
54
|
150
|
2
|
126
|
7
|
139
|
32
|
148.0
|
21.0
|
|
Ascorbic acid + vanadium
|
100 + 100
|
207
|
43.7
|
181
|
2.24
|
148
|
10
|
190
|
21
|
180.7
|
17.8
|
|
Deferoxamine + vanadium
|
20 + 20
|
−21
|
−56
|
−15
|
−66
|
−29
|
−66
|
−38
|
−66
|
−25.9
|
−64
|
|
Deferoxamine + vanadium
|
100 + 100
|
5
|
−51
|
25
|
−55
|
1
|
−56
|
−17
|
−65
|
3.4
|
−56.6
|
In Vitro Effect of Coexposure to Vitamin C and Vanadium on Lipid Peroxidation in Homogenates
from Different Regions of Rat Brain
[Table 1] and [2] demonstrate that the combined exposure to vitamin C (20 and 100 μM) and vanadium
(20 and 100 μM) resulted in dose-dependent provocation in the occurrence of LPO. The
average percent increase in LPO in total brain region homogenates was +23.7% compared
with homogenate exposed to 20μM vanadium only. This provocation was significant in
the brain stem (+54%) followed by medulla pons (+32%). There were non-significant
increases in LPO in both the hippocampus (+ 7%) and hypothalamus (+ 2%), respectively.
On the other hand, the average percent increase in LPO in total brain homogenates
was faster (+17.8%) when compared with brain tissue homogenates exposed to 100 μM
vanadium only. The order of provocation was significant both in brain stem (+21%)
and medulla-pons (+ 21%), respectively. However, there was nonsignificant increase
in occurrence of LPO in both hypothalamus (+12%) and hippocampus (10%).
In Vitro Effect of Coexposure to Deferoxamine and Vanadium on Lipid Peroxidation in
Homogenates from Different Regions of Rat Brain
[Table 1] and [2] demonstrate that the combined exposure to deferoxamine (20 and 100μM) and vanadium
(20 and 100 μM) to rat brain tissue homogenates resulted in significantly dose-dependent
inhibition in the occurrence of LPO. The average percent inhibition in total brain
region homogenates was by −64% when compared with brain homogenates exposed to vanadium
(20μM) only. The sequence of inhibition was alike in hypothalamus (−66%), hippocampus
(−66%), and medulla-pons (−66%) followed by mid-brain (−56%). On the other hand, exposure
of fresh homogenates from different regions of the brain to deferoxamine (100 μM)
and vanadium (100 μM) also exhibited significantly inhibited occurrence of LPO when
compared with the brain tissue homogenates incubated to vanadium (100μM) vanadium
only. The following was the sequence of inhibition: medulla-pons (−60%), hippocampus
(−56%), hypothalamus (−55%), and brain-stem (−51%). The mean percent inhibition of
LPO in total brain region homogenates was −56%.
Discussion
Effect of Vanadium (20 and 100μM) on Occurrence of Lipid Peroxidation in Brain Tissues
Homogenates
The treatment of brain tissue homogenates with vanadium exhibited dose-dependent significantly
enhanced occurrence of LPO compared with water controls. The present results are in
perfect congruence with a previous research finding[14] with the trends of enhanced LPO in brain microsomes exposed to 100μM vanadium. It
is thus likely that pentavalent (V5+) salt of vanadium, used by us, was reduced to tetravalent (V4+) vanadium by a one electron transfer reaction; therefore, O2 is reduced to superoxide anion radical (O2
-
.).[15] Henceforth, LPO was enhanced in total brain region tissue homogenates.
Effect of Coexposure to Vitamin C (20 and 100 μM) and Vanadium (20 and 100μM) on Occurrence
of Lipid Peroxidation in Brain Tissues Homogenates
Our results established that treatment of brain tissue homogenates with a combination
of vitamin C (20 or 100 μM) and vanadium (20 or 100 μM) significantly provoked acceleration
in the occurrence of LPO compared with exposure of brain tissues with 20 or 100 μM
vanadium, respectively. There is an extensive evidence that vitamin C, chemically
known as ascorbic acid, has pro-oxidative abilities in the presence of oxygen and
transition metal ions under conditions of high millimolar concentration in vitro.[16] Our results can be explained by the reactions that might have triggered the reduction
in pentavalent (V5+) to tetravalent vanadium (V
4+
) along with ascorbate. This process may have increased the generation of reactive
oxygen species in Fenton-like redox cycling reaction in the brain homogenates, and
enhanced occurrence of LPO.
AH2 → AH- + H+
Ascorbic Acid Ascorbate anion
AH- + V5+ → A · + V4+ + H+
Ascorbate anion Pentavalent vanadium Ascorbyl radical Tetravalent vanadium
H2O2 + V4+ → V5+ + OH · + OH- (Fenton Reaction)
Hydrogen peroxide Tetravalent vanadium Hydroxyl radical Hydroxyl anionl
V4+ + O2 (oxidation) → V5+ + O2
.-
This is one electron transfer reaction, where molecular oxygen is reduced to superoxide
anion (O2−.) and tetravalent vanadium (V4+) is oxidized to pentavalent vanadium (V5+).
Effect of Coexposure to Deferoxamine (20 and 100μM) and Vanadium (20 and 100μM) on
Occurrence of Lipid Peroxidation in Rat Brain Tissues Homogenates
It has been evaluated by thiobarbituric acid colorimetric procedure that deferoxamine
has the ability to revert LPO.[17] The present results also demonstrated that exposure of brain tissue homogenates
to a combination of deferoxamine (20 or 100 μM) and vanadium (20 or 100 μM) significantly
inhibited the occurrence of LPO. A similar report by a group of researchers demonstrated
that deferoxamine protected against generation of reactive oxygen species following
methyl mercury intoxication in rat brain. Our results are in congruence with these
authors.[17] Henceforth, in discussing the present results, it seems justified that deferoxamine
protected against vanadium-induced occurrence of LPO in brain tissue homogenates via
potentiating a very strong affinity chelation with vanadium metal.
Further experiments are required to lend a better insight into the etiopathogenesis
of vanadium by using various fortifiers and evaluation of antioxidant enzymes in brain
tissue homogenates in rats.
Conclusion
In vitro results demonstrated significant acceleration of LPO in brain tissue homogenates
following vanadium exposure. Exposure to vitamin C + vanadium provoked LPO, while
deferoxamine + vanadium inhibited the LPO. The brain regional heterogeneity in occurrence
of LPO is because the glial cells in various central nervous system sites are not
same.[18]
