Article

Efficacy of Vanadyl Sulfate and Selenium Tetrachloride as

Anti-Diabetic Agents against Hyperglycemia and Oxidative
Stress Induced by Diabetes Mellitus in Male Rats
Fawziah A. Al-Salmi and Reham Z. Hamza *
Biology Department, College of Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
[email protected]
* Correspondence: [email protected]

Abstract: The use of metals in medicine has grown in popularity in clinical and commercial settings.
In this study, the immune-protecting effects and the hypoglycemic and antioxidant activity of
vanadyl sulfate (VOSO4 ) and/or selenium tetrachloride (Se) on oxidative injury, DNA damage,
insulin resistance, and hyperglycemia were assessed. Fifty male albino rats were divided into five
groups, and all treatments were administrated at 9:00 a.m. daily for 60 successive days: control,
STZ (Streptozotocin; 50 mg/kg of STZ was given to 6 h fasted animals in a single dose, followed by
confirmation of diabetic state occurrence after 72 h by blood glucose estimation at >280 mg/dl), STZ
(Diabetic) plus administration of VOSO4 (15 mg/kg) for 60 days, STZ (Diabetic) plus administration
of selenium tetrachloride (0.87 mg/Kg), and STZ plus VOSO4 and, after 1/2 h, administration
of selenium tetrachloride at the above doses. The test subjects’ blood glucose, insulin hormone,
HbA1C, C-peptide, antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase,



myeloperoxidase, and xanthine oxidase), markers of lipid peroxidation (MDA), and histological


sections of pancreatic tissues were evaluated, and a comet assay was performed. Histological sections
Citation: Al-Salmi, F.A.; Hamza, R.Z.
in pancreas tissues were treated as indicators of both VOSO4 and selenium tetrachloride efficacy,
Efficacy of Vanadyl Sulfate and
either alone or combined, for the alleviation of STZ toxicity. The genotoxicity of diabetes mellitus
Selenium Tetrachloride as
was assessed, and the possible therapeutic roles of VOSO4 or selenium tetrachloride, or both, on
Anti-Diabetic Agents against
antioxidant enzymes were studied. The findings show that the administration of VOSO4 with
Hyperglycemia and Oxidative Stress
Induced by Diabetes Mellitus in Male
selenium tetrachloride reduced oxidative stress to normal levels, lowered blood glucose levels, and
Rats. Curr. Issues Mol. Biol. 2022, 44, elevated insulin hormone. Additionally, VOSO4 with selenium tetrachloride had a synergistic effect
94–104. https://doi.org/10.3390/ and significantly decreased pancreatic genotoxicity. The data clearly show that both VOSO4 and
cimb44010007 selenium tetrachloride inhibit pancreatic and DNA injury and improve the oxidative state in male
rats, suggesting that the use of VOSO4 with selenium tetrachloride is a promising synergistic potential
Academic Editor: Hidayat Hussain
ameliorative agent in the diabetic animal model.
Received: 16 November 2021
Accepted: 20 December 2021 Keywords: diabetes mellitus; vanadyl sulphate; selenium tetrachloride; oxidative stress
Published: 24 December 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations. Diabetes is a metabolic disease characterized by insulin resistance and β-cell dysfunc-
tion [1]. Diabetes mellitus is caused by an insulin secretion dysfunction and the activation
of oxidative injury [2].
A major marker of type II diabetes mellitus is dysfunction of the β-cells. Type II
Copyright: © 2021 by the authors.
diabetes mellitus is a systemic, complex, chronic disease. Its prevalence has been growing
Licensee MDPI, Basel, Switzerland.
over the last decades, due to increased obesity. Type II diabetes mellitus is accompanied by
This article is an open access article
an elevated risk for several cardiovascular diseases, with heart failure as a more common
distributed under the terms and
initial presentation rather than myocardial infarction [3]. Better therapies are needed
conditions of the Creative Commons
Attribution (CC BY) license (https://
to improve insulin sensitivity and promote the survival and regeneration of pancreatic
creativecommons.org/licenses/by/
β-cells to improve the health of diabetic patients without inducing side effects such as
4.0/). hepatotoxicity, cardiotoxicity, and pancreatic tissue inflammation.

Curr. Issues Mol. Biol. 2022, 44, 94–104. https://doi.org/10.3390/cimb44010007 https://www.mdpi.com/journal/cimb

Curr. Issues Mol. Biol. 2022, 44 95

Type II diabetes mellitus patients are at high risk of cardial stroke compared to the
general population. A large meta-analysis showed kidney disease and cardiovascular
diseases, as demonstrated by hazards for cardiovascular mortality, are higher among
Type II diabetic patients throughout the whole spectrum of diseases. Such a marked
cardiovascular risk significantly modifies the outcome of diabetic patients who often do not
survive long enough to reach the natural fate of end-stage kidney disease. The importance
of a multifactorial approach in type II diabetes mellitus has been emphasized by the analysis
of Swedish National Diabetes and gender matched controls. In particular, type II diabetes
mellitus patients with five risk-factors within target range showed either a small or any
excess risk of death as compared with controls [4].
Antioxidants have been used excessively in diabetic experimental models to enhance
the effects of free radical accumulation. Diabetes mellitus is characterized by vascular
pathology that leads to vascular complications. Diabetes mellitus is also characterized by a
lack of insulin [5].
Vanadium compounds act as insulin mimetics and have, since the 1970s, been known
to be biologically important. Their physiological effects have been studied for their hypo-
glycemic action, and vanadium may act as a therapeutic agent against diabetes mellitus [6].
Vanadium has been extensively studied for its diabetes-fighting properties. Vanadium
salts have a very low oral bioavailability, which lengthens their half-life due to bidentate
coordination. This effect improves vanadium absorption and stability, resulting in a positive
antidiabetic effect [6].
Vanadium supplementation in the diet occurs mainly as V4+ . Vanadium ingested via
the stomach also exists as V4+ , which affects the absorption of the ingested vanadium. The
development of metal-based drugs with vanadium may be of interest for its pharmaco-
logical properties [7]. VOSO4 has an insulin-memetic effect and may, like insulin, have
sensitizing properties, as indicated in previous studies.
Diabetes mellitus was induced using STZ in experimental rats, as previously reported.
As Vanadyl salts possess insulin-mimicking effects in the body, vanadium may be an
alternative treatment for diabetic patients [8].
Vanadyl is able to induce hypoglycemia, as documented previously [9]. Studies of vanadyl
salts primarily focused on improving insulin sensitivity and treatment for diabetes [10].
Micronutrients are dietary minerals required by the body in a very small quantity.
They may interact with xenobiotics at several sites during absorption and excretion [11].
Selenium tetrachloride plays a vital role in human health and is a basic component of
selenoproteins, which share an important antioxidant enzyme in the body. Selenoproteins
play a vital role in redox homeostasis and protection from oxidative stress and inflamma-
tion [12]. Most foods enriched with selenium tetrachloride are natural foods, including
seafood and major crops. The level of selenium tetrachloride intake varies greatly among
individuals and populations [13].
Selenium tetrachloride is a basic nutrient for humans; it is a vital microelement of
antioxidant enzymes, including glutathione peroxidase (GPx). Selenium tetrachloride
exhibits protective effects against symptoms and side effects, and studies have verified the
essential role of selenium tetrachloride in the preservation of the metabolic and endocrine
functions of the human body. It is vital for all cellular processes [14].
Evidence suggests that many selenoproteins, which contain Se in the form of amino acid
selenocysteine, have vital enzymatic functions associated with antioxidant activity. Selenium
tetrachloride is a vital dietary element required for the maintenance of human health [15].
Selenium tetrachloride is an essential trace element for the maintenance of immune–
endocrine function. However, its effect on glucose metabolism has not been well studied.
Chronic hyperglycemia may produce reactive oxygen species (ROS) and induce oxidative
stress, leading to diabetes-related pathological complications and a decline in Se levels.
It has been suggested that patients with type II diabetes mellitus could benefit from
supplementation with selenium tetrachloride, as it may affect glucose homeostasis and
insulin sensitivity [15].
Curr. Issues Mol. Biol. 2022, 44 96

In the study presented herein, I sought to estimate the impact of vanadyl sulfate
(VOSO4 ) and/or selenium tetrachloride on alleviating oxidative stress and hyperglycemia
induced by experimental diabetes mellitus.

2. Materials and Methods

2.1. Chemicals and Analyses
I purchased all chemicals, including VOSO4 , selenium tetrachloride, and STZ, from
Sigma-Aldrich. All chemicals were used without purification.

2.2. Experimental Animals

Fifty mature male rats (two months old), weighing 170–180 g each, were kept in sensi-
tized metal cages with free access to food and water in a room maintained at a temperature
of 25 ◦ C ± 2 ◦ C with a 12 h light/dark cycle. The rats were obtained from the King Fahd
Center for Medical Research (King Abdulaziz University, Jeddah, Saudi Arabia). The ex-
perimental protocol was approved by the Deanship of Scientific Research at Taif University
Ethical Committee, encoded with approval number 40-31-0142, and in accordance with
Animal Research: Reporting of In Vivo Experiments Guidelines (Kilkenny et al., 2010) [16].
Rats were sacrificed under ketamine/xylazine anesthesia, and efforts were made to reduce
stress and pain.

2.3. Animals and Experimental Design

Fifty Wistar male Albino rats weighing 170–180 g each were divided into five groups
for 60 days. Animals that were involved in the experiment and were expected to become
diabetic rats were fed high-fat diets for about 3 weeks before the start of the experiment,
based on a previous study [17]; then, experimental diabetes mellitus was induced by a
single injection of STZ to mimic type 2 diabetes, which is affected greatly by high-fat
diets and the mode of nutrition before the incidence of diabetes mellitus disease. Group I,
the control group, was administered physiological saline by intraperitoneal (IP) injection.
Group II, the STZ group, was given a single dose of STZ (50 mg/kg) by intraperitoneal
(IP) injection [17]. Group III was a diabetic group given STZ and orally administrated
15 mg/kg of VOSO4 [18]. Group IV was a diabetic group given an oral administration of
0.87 mg/kg of selenium tetrachloride, according to the method described by Johri et al. [19].
Group V was diabetic and was treated orally with both VOSO4 and selenium tetrachloride
successively, as shown in the experimental protocol (Figure 1).

2.4. Experimental Induction of Diabetes Mellitus

Freshly prepared STZ dissolved in PBS (Ph = 4.5) was used to induce diabetes. STZ
at 50 mg/kg, freshly prepared in the early morning, was given by IP injection to animals
that had fasted for 6 h [20], following the administration of high-fat feed (composed of
66.5% commercial feed, 13.5% artificial butter, and 20% sugar) for the 3 weeks prior to the
experimental induction of diabetes mellitus by the single injection of STZ [21]. Seventy-two
hours after STZ injection, blood glucose levels were measured to evaluate the diabetic
status of the test animals. Subjects with blood glucose levels higher than 280 mg/dL were
considered to be diabetic.

2.5. Blood Collection

Using capillary tubes, blood samples were taken from the eye plexus with light
anesthesia for biochemical and physiological analyses. The rats were ethically decapitated.
Pancreatic tissue samples were kept at −25 ◦ C.
 44FOR PEER REVIEW
2022, 1,
Curr. Issues Mol. Biol. 2021, 97
4

Figure
Figure 1.
1. Experimental
Experimental protocol.
protocol.

2.6. Experimental
2.4. DeterminationInduction
of the Blood Glucose Mellitus
of Diabetes Level
Blood glucose
Freshly levels
prepared STZwere evaluated
dissolved using
in PBS (Phcommercial kits (Bio-diagnostic
= 4.5) was used co.). STZ
to induce diabetes.
at 50 mg/kg, freshly prepared in the early morning, was given by IP injection to animals
2.7. Measurements of Serum Insulin, C-Peptide, and HbA1c
that had fasted for 6 h [20], following the administration of high-fat feed (composed of
66.5%Serum insulinfeed,
commercial was 13.5%
evaluated using
artificial a rat ELISA
butter, kitsugar)
and 20% (ALPCOfor Diagnostics). I used
the 3 weeks prior the
to the
C-peptide enzyme commercial immune assay (Sigma-Aldrich) and HbA1c kits according
experimental induction of diabetes mellitus by the single injection of STZ [21]. Seven-
to the manufacturers’ protocols.
ty-two hours after STZ injection, blood glucose levels were measured to evaluate the di-
abetic status of the
2.8. Preparation test animals.
of Pancreatic Subjects
Tissue with blood glucose levels higher than 280 mg/dL
Homogenates
were considered to be diabetic.
A small pancreatic tissue sample was used to estimate the antioxidant biomarkers.
Pancreatic tissue was homogenized in 5 mL of cold buffer per gram at 4 ◦ C and centrifuged
2.5. Blood Collection
at 5000 rpm for 30 min; the resulting supernatant was kept at −20 ◦ C.
Using capillary tubes, blood samples were taken from the eye plexus with light an-
esthesia for biochemical
2.9. Determination and physiological
of Oxidative Stress Markersanalyses. The rats were ethically decapitated.
Pancreatic tissue samples were
Supernatant fluids centrifugedkept at
from−25the
°C.pancreatic tissue were used to evaluate my-
loperoxidase (MPO) and xanthine oxidase (XO) [22]. Superoxide dismutase (SOD) was
2.6. Determination
determined using of
thethe Blood Glucose
method Levelet al. [23]. Pancreas MDA levels were determined
by Litwack
usingBlood glucoseby
the method levels wereetevaluated
Ohkawa using commercial
al. [24]. Catalase (CAT) waskits (Bio-diagnostic
determined co.).to the
according
method by Beers and Sizer [25].
2.7. Measurements of Serum Insulin, C-Peptide, and HbA1c
2.10.Serum
Single-Cell Gel was
insulin Electrophoresis
evaluated (SCGE)
using a (Comet Assay)
rat ELISA kit (ALPCO Diagnostics). I used the
Pancreas tissues were placed in a Petri dish with ice solution 2+ , Mg2+ free with
C-peptide enzyme commercial immune assay (Sigma-Aldrich) and(Ca
HbA1c kits accord-
EDTA),
ing and
to the the cell viability
manufacturers’ was determined.
protocols.

2.11.Preparation
2.8. HistologicalofAnalysis of the
Pancreatic Pancreas
Tissue Tissues
Homogenates
The pancreatic tissues were fixed in 10% neutral buffered formalin and were embedded
A small pancreatic tissue sample was used to estimate the antioxidant biomarkers.
in paraffin, then thin sections were cut and stained with hematoxylin and eosin (H&E) and
Pancreatic tissue was homogenized in 5 mL of cold buffer per gram at 4 °C and centri-
examined using a light microscope.
fuged at 5000 rpm for 30 min; the resulting supernatant was kept at −20 °C.

2.9. Determination of Oxidative Stress Markers

Curr. Issues Mol. Biol. 2022, 44 98

2.12. Statistical Analysis

Data were analyzed as the mean ± SEM using the SPSS v.22 program (SPSS Inc., USA)
via one-way ANOVA. The significance of mean differences was examined using the post
hoc Duncan test [26].

3. Results
3.1. Blood Glucose Level, Insulin Hormone, and Fasting C-Peptide Serum
STZ induced a highly significant and marked increase in blood glucose levels accom-
panied by a significant reduction in insulin levels and serum fasting C-peptides in the
diabetic untreated group as compared to the control group. As shown in Table 1, diabetic
rats treated with VOSO4 and/or selenium tetrachloride exhibited a non-significant increase
in blood glucose levels as compared with the control group. They also demonstrated a
significant decrease in blood glucose levels with elevated insulin hormones and serum
fasting C-peptides as compared with the STZ group.

Table 1. Blood glucose level, insulin hormone, HBA1C, and fasting serum C-peptide of male rats
treated with VOSO4 and/or selenium tetrachloride or their combinations.

Parameters
Groups Blood Glucose Insulin Hormone HbA1C Fasting Serum
(mg/dl) (uIU/mL) (mmol/mol) C-Peptide (ng/mL)
Control group 84.01 ± 3.25 e 25.36 ± 1.25 a,b 3.02 ± 0.65 d 4.18 ± 0.69 a
STZ group 371.25 ± 4.02 a 4.40 ± 0.24 d 9.51 ± 1.36 a,b 0.52 ± 0.05 d
c 2.98 ± 0.87 c
STZ plus VOSO4 group 140.36 ± 4.03 b 19.36 ± 2.15 5.02 ± 1.02 b
STZ plus selenium tetrachloride group 129.36 ± 2.75 c 20.03 ± 2.11 c
4.01 ± 0.87 c,d 3.74 ± 0.87 b
STZ plus VOSO4 and selenium
97.25 ± 4.26 d,e 23.65 ± 2.02 b 3.54 ± 0.96 d 4.00 ± 0.96 a
tetrachloride group
Means within the same column (mean ± SE) carrying different letters are significant at p ≤ 0.05 using Duncan’s mul-
tiple range test, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically.

3.2. Oxidative Stress Enzyme Biomarkers

Tables 2 and 3 show that STZ induced a significant decrease in CAT, SOD, and glu-
tathione peroxidase (GPx) enzymes while eliciting a highly significant increase in malondi-
aldehyde (MDA), myeloperoxidase (MPO), and xanthine oxidase (XO) levels. Compared
with the control group, diabetic rats treated with VOSO4 and/or selenium tetrachloride,
either alone or in combination, showed a marked increase in CAT, SOD, and GPx levels and
a decrease in MPO, XO, and MDA levels. The group treated with VOSO4 in conjunction
with selenium tetrachloride presented the best results recorded.

Table 2. Oxidative/antioxidant parameters of antioxidant enzymes in pancreatic tissues of male rats

treated with VOSO4 and/or selenium tetrachloride or their combinations.

Parameters
Pancreatic Pancreatic Pancreatic Pancreatic
Groups
Catalase SOD MDA GPx
(U/g) (U/g) (U/g) (U/g)
Control group 1.88 ± 0.21 a 22.05 ± 1.15 a,b 3.05 ± 0.48 e 34.05 ± 1.85 a
STZ group 0.26 ± 0.10 d 5.22 ± 1.35 d 81.15 ± 0.96 a 7.56 ± 1.18 e
STZ plus VOSO4 group 1.42 ± 0.36 c 19.91 ± 1.58 c 20.42 ± 1.02 b 23.15 ± 1.15 d
STZ plus selenium tetrachloride group 1.63 ± 0.48 b 20.52 ± 2.16 b 12.26 ± 1.45 c 26.41 ± 1.28 c
STZ plus VOSO4 and selenium
1.74 ± 0.22 a 21.19 ± 2.25 b 8.78 ± 1.25 d 31.58 ±1.58 b,c
tetrachloride group
Means within the same column (mean ± SE) carrying different letters are significant at p ≤ 0.05 using Duncan’s mul-
tiple range test, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically.
Curr. Issues Mol. Biol. 2022, 44 99

Table 3. MPO and XO levels in the pancreatic tissues of male rats treated with VOSO4 and/or
selenium tetrachloride or their combinations.

Parameters
Groups MPO XO
(nmol/min/mL) (U/g)
Control group 15.16 ± 1.36 e 16.25 ± 1.25 e
STZ group 25.16 ± 1.15 a 38.55 ± 1.16 a
STZ plus VOSO4 group 18.24 ± 1.25 b 20.15 ± 1.25 b
STZ plus selenium tetrachloride group 17.55 ± 1.19 c 19.65 ± 1.36 c
STZ plus VOSO4 and selenium tetrachloride group 16.48 ± 2.16 d,e 17.16 ± 1.39 d,e
Means within the same column (mean ± SE) carrying different letters are significant at p ≤ 0.05 using Duncan’s mul-
tiple range test, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically.

3.3. Histological Examination

Figure 2: Photomicrographs of the pancreas showing (A) normal pancreatic parenchyma
and normal appearance of islets of Langerhans; (B) the STZ-treated group, showing very
reduced islets of Langerhans; (C) the STZ plus VOSO4 group, showing high restoration
of detached pancreatic parenchyma with mild-sized islets of Langerhans; (D) the STZ
plus selenium tetrachloride group, showing normal pancreatic parenchyma with partially
Curr. Issues Mol. Biol. 2021, 1, FOR PEERmild islets of Langerhans; and (E) the STZ plus VOSO4 and selenium
REVIEW 7 tetrachloride group,
showing intact pancreatic parenchyma with enlarged islets of Langerhans.

Figure
Figure2. Photomicrograph of pancreas showing
2. Photomicrograph (A) normal
of pancreas pancreatic parenchyma
showing (A) normal and pancreatic
normal parenchyma and normal
appearance of Islets of Langerhans (black arrow) (H&E X400). (B) STZ treated group showing de-
appearance
tached of Islets with
pancreatic parenchyma of Langerhans
reduction of islets(black arrow)
of Langerhans (H&E
(black arrow) X400).
(H&E X400).(B) STZ treated group showing
(C) STZ plus VOSO4 group showing high restoration of the detached pancreatic parenchyma with
detached pancreatic parenchyma with reduction of islets of Langerhans (black arrow) (H&E X400).
mild sized islet of Langerhans (black star) (H&E X400). (D) STZ plus selenium tetrachloride group
(C) STZ
showing plus
normal VOSO
pancreatic 4 groupwith
parenchyma showing high
appearance veryrestoration of the (black
little islet of Langerhans detached
star) pancreatic parenchyma with
with smaller size than diabetic group treated with VOSO4 (H&E X400). (E) STZ plus VOSO4 and
mild sized
selenium isletgroup
tetrachloride of Langerhans
showing intact (black
pancreaticstar) (H&Ewith
parenchyma X400). (D)islet
enlarged STZ plus selenium tetrachloride group
of Lang-
erhans (black star)
showing which ispancreatic
normal the better effect (H&E X400).
parenchyma with appearance very little islet of Langerhans (black star)
with
3.4. smaller
Comet Assay size than diabetic group treated with VOSO4 (H&E X400). (E) STZ plus VOSO4 and
selenium
Figure 3. Comet images group
tetrachloride of cells showing intact
derived from pancreatic
the pancreatic parenchyma
tissues. with enlarged islet of Langerhans
(A) Control
group showed intact nuclei with normal round cell.
(black star) which is the better effect (H&E X400). (B) STZ group showed higher degree
of damage with the presence of a lot of apoptotic cells. (C) STZ plus VOSO4 group
showing some of the intact nuclei with some apoptotic cells. (D) STZ plus selenium tet-
rachloride group showed less DNA damage, which is confirmed by a less damaged nu-
clei. (E) STZ plus VOSO4 and selenium tetrachloride group showed less damaged DNA
strands and less damaged nuclei.
 Curr. Issues Mol. Biol. 2022, 44 100

3.4. Comet Assay

Figure 3. Comet images of cells derived from the pancreatic tissues. (A) Control group
showed intact nuclei with normal round cell. (B) STZ group showed higher degree of
damage with the presence of a lot of apoptotic cells. (C) STZ plus VOSO4 group showing
some of the intact nuclei with some apoptotic cells. (D) STZ plus selenium tetrachloride
group showed less DNA damage, which is confirmed by a less damaged nuclei. (E) STZ
urr. Issues Mol. Biol. 2021, 1, FOR PEER REVIEW 8
plus VOSO4 and selenium tetrachloride group showed less damaged DNA strands and
less damaged nuclei.

Figure 3. Comet
Figureimages of cells
3. Comet derived
images fromderived
of cells the pancreatic
from thetissues. (A) Control
pancreatic tissues.group showedgroup
(A) Control in- showed
tact nuclei intact
with normal round cell. (B) STZ group showed appearance of a lot of apoptotic cells
nuclei with normal round cell. (B) STZ group showed appearance of a lot of apoptotic cells
(white head arrow) with large tail shadow in the form a comet-like shape. (C) STZ plus VOSO4
(white head arrow) with large tail shadow in the form a comet-like shape. (C) STZ plus VOSO4 group
group showing restoration of more intact nuclei (white star). (D) STZ plus selenium tetrachloride
showing restoration of more intact nuclei (white star). (D) STZ plus selenium tetrachloride group
group clarified less damaged DNA strands (white arrow). (E) STZ plus VOSO4 and selenium tet-
clarified
rachloride group less damaged
which DNAdamaged
showed less strands (white
DNA arrow).
strands (E)
andSTZ
lessplus VOSO4nuclei
damaged and selenium
and more tetrachloride
intact nucleigroup
(whitewhich showed less damaged DNA strands and less damaged nuclei and more intact nuclei
arrow).
(white arrow).
4. Discussion
I sought to compare the benefits of VOSO4 and selenium tetrachloride administra-
tion on STZ tissue injury by examining VOSO4 and/or selenium tetrachloride treated di-
abetic groups. To my knowledge, research on the co-effect of VOSO4 and selenium tet-
rachloride on STZ toxicity at the biochemical and histopathological levels in the pancre-
atic tissue of rats has not previously been conducted.
The ability of VOSO4 in combination with selenium tetrachloride to counteract
Curr. Issues Mol. Biol. 2022, 44 101

4. Discussion
I sought to compare the benefits of VOSO4 and selenium tetrachloride administration
on STZ tissue injury by examining VOSO4 and/or selenium tetrachloride treated diabetic
groups. To my knowledge, research on the co-effect of VOSO4 and selenium tetrachloride
on STZ toxicity at the biochemical and histopathological levels in the pancreatic tissue of
rats has not previously been conducted.
The ability of VOSO4 in combination with selenium tetrachloride to counteract STZ-
induced pancreatic toxicity in male rats was investigated in this study. Exposure to VOSO4
and/or selenium tetrachloride in the STZ diabetic groups led to altered endocrine function
and biochemical changes.
Concomitant administration of VOSO4 and/or selenium tetrachloride with STZ sig-
nificantly protected most of the biochemical variables altered by STZ, which suggests
its efficacy as a protectant. The structure of the pancreatic tissues induced by STZ also
improved due to these two antioxidant compounds and metals.
The untreated diabetic rats presented a marked increase in blood glucose levels. The
results are in complete agreement with the previous literature [27] stating that STZ action is
accompanied by severe hyperglycemia in β-cells. Hyperglycemia results in reduced insulin
levels [28], and these changes include β-cell abnormalities.
Meanwhile, the diabetic groups treated with VOSO4 exhibited hypoglycemia with
insulin levels similar to those in previous studies [29]. This confirmed that the oral admin-
istration of vanadyl sulfate improved blood glucose levels and increased insulin sensitivity
in type II diabetes, reducing (HbA1c) glycosylated hemoglobin while hepatic glucose was
suppressed.
Vanadium compounds have been studied as potential therapeutic agents for the
treatment of various major diseases. The translation of vanadium-related complexes and
compounds into treatments for diseases, especially diabetes mellitus, remains unconfirmed
due to the absence of a basic pharmacological understanding of these compounds [30].
VOSO4 can be explained by examining vanadium’s effect on insulin in three critical
areas [31]: (a) in the insulin-signaling network, where insulin receptors and insulin receptor
substrates are affected by protein tyrosine phosphatases as +ve modulation and -ve regula-
tors; (b) phosphoinositide protein kinases and mTOR as a +ve signal to protein kinases and
as a -ve modulator; and (c) insulin receptors and mitogen-activated protein kinase with its
own -ve modulations. Signaling loss implies a disturbance in the biological action of the
insulin hormone. These findings were confirmed by previous studies on the administration
of vanadium and other salts, which showed similar expression of insulin receptors when
only using vanadium in low dosages.
Vanadium complexes can impact the healthy and the ill; therefore, vanadium salts
may have multiple applications. The similarity between vanadate and phosphate allows
for ease of phosphorylation in many signaling pathways. Additionally, vanadium salts are
reported to mimic or enhance insulin [32]; these findings were confirmed in this study.
Oxidative stress is related to the decline in insulin secretion, and pancreatic tissues are
more susceptible to oxidative stress because of their low antioxidant enzyme levels [33]. The
treatment applied in our study elevated the antioxidant enzyme levels significantly when
compared with those in STZ-treated groups, indicating that the current results support the
previously obtained results. VOSO4 is able to improve insulin resistance and restore high
blood glucose levels to normal levels, as reported by Pepato et al. [32].
Oxidative stress and the accumulation of oxidative stress markers related to free
radicals are the most deleterious effects of diabetes on cellular activities. The results
showed that the combination of VOSO4 and selenium tetrachloride successfully scavenged
excess free radicals. As a result, lipid peroxidation marker (MDA) levels declined and the
antioxidant enzyme capacities of CAT, SOD, and GPx increased, which improved hepatic
function and improved blood glucose levels [30].
The current findings are in complete agreement with the findings of Treviño et al. [30],
who confirmed that treatment of diabetic rats with vanadyl complexes in high doses elicited
Curr. Issues Mol. Biol. 2022, 44 102

a significant decline in MDA levels and resulted in significant improvement in antioxidant

enzyme capacities in pancreatic tissue homogenates.
I observed that increased lipid peroxidation in the STZ (untreated diabetic) group
could include oxidative stress [33]. It was found that VOSO4 combined with selenium
tetrachloride protects pancreatic tissues from lipid peroxidation generation and from any
changes in CAT, SOD, and MDA (a marker of peroxidation) levels.
STZ impacts the role of oxidative stress on pancreatic β-cells [34]. β-cells have a great
affinity for free radicals, causing damage to β-cells and antioxidant enzymes. This effect
was confirmed by the deterioration of the antioxidants in the pancreatic tissues [35]. The
successively increasing insulin and selenium tetrachloride levels in the VOSO4 treatment
group and amelioration of pancreatic histological structures demonstrated that the β-cell
structures under VOSO4 enriched with selenium tetrachloride have antioxidant properties.
Combining selenium tetrachloride with VOSO4 mitigated the harmful effects of STZ
in all measured parameters. The observed levels were closely related to the normal control
values in the nondiabetic group. These results were in accordance with those of Ithayarasi
and Devi [36] and El-Demerdash [37], who found that selenium tetrachloride maintained
antioxidant levels and antioxidant enzymes at near-normal levels, which enhanced their
effects as antioxidants [38].
An important marker for the detection of DNA damage is the comet assay, which
can detect lesions on DNA strands to show oxidative injury [39]. I used the comet assay
to evaluate the incidence of DNA impairment [40,41]. The assay confirmed that STZ is
highly genotoxic to pancreatic tissues. Greater amounts of DNA damage were found in
the untreated STZ diabetic group, which may be due to the generation of reactive oxygen
species that caused DNA strand delineation. The ROS declined in the diabetic group treated
with VOSO4 and selenium tetrachloride together. Thus, the comet assay confirmed high
genotoxicity induced by STZ in the pancreatic tissue homogenates and the appearance of
the tail comet of DNA in the STZ diabetic untreated group, whereas the group treated with
STZ and VOSO4 combined with selenium tetrachloride showed intact DNA strands with
markedly alleviated DNA damage.
The current findings demonstrate that VOSO4 and selenium tetrachloride increase
insulin concentrations when β-cells have been damaged by STZ. This effect can be partially
explained by the vital effect of selenium tetrachloride on insulin signaling. Selenium
tetrachloride, as a major component of antioxidant enzymes, can prevent oxidative cellular
damage by scavenging ROS. It may also cause a euglycemic balance in ROS production that
maintains insulin sensitivity because ROS are also essential for insulin sensitization. There
is growing evidence from previous studies on selenium tetrachloride’s potential benefits
that should be balanced with its potential harmful effects [42].

5. Conclusions
In this study, experimental diabetes mellitus was induced in the islets of Langerhans;
the results demonstrated that VOSO4 and selenium tetrachloride may be helpful in the
treatment of diabetes. The use of metals in enhancing insulin action and reducing cellular
inflammation may have beneficial effects. The treatment of diabetes mellitus is challenged
by the need to find safer compounds that have antidiabetic potency and can activate
the β-cells. The results herein confirmed that the combination of VOSO4 and selenium
tetrachloride is more effective, that it is the best and safest treatment for hyperglycemia
without genotoxicity, and that it may alleviate oxidative injury. With further study, this
treatment may lead to a novel antidiabetic combination.

Author Contributions: Conceptualization, F.A.A.-S. and R.Z.H.; methodology, F.A.A.-S. and R.Z.H.;
validation, F.A.A.-S.; formal analysis, R.Z.H.; investigation, R.Z.H.; resources, R.Z.H. and F.A.A.-S.;
data curation, R.Z.H. and F.A.A.-S.; writing—original draft preparation, R.Z.H. and F.A.A.-S.;
writing—review and editing, R.Z.H. and F.A.A.-S.; visualization, R.Z.H. and F.A.A.-S.; supervi-
sion, R.Z.H. and F.A.A.-S.; project administration, R.Z.H. and F.A.A.-S.; funding acquisition, R.Z.H.
and F.A.A.-S. All authors have read and agreed to the published version of the manuscript.
Curr. Issues Mol. Biol. 2022, 44 103

Funding: Taif University Researcher supporting project number (TURSP-2020/113), Taif University,
Taif, Saudi Arabia.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Ethics Committee of Taif University: 40-31-0142.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data analyzed or generated during this study are included in this
manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Wang, M.; Song, L.; Strange, C.; Dong, X.; Wang, H. Therapeutic Effects of Adipose Stem Cells from Diabetic Mice for the
Treatment of Type 2 Diabetes. Am. Soc. Gene Cell Ther. 2018, 26, 1921–1930. [CrossRef]
2. Fukunaka, A.; Fujitani, Y. Role of Zinc Homeostasis in the Pathogenesis of Diabetes and Obesity. Int. J. Mol. Sci. 2018, 19, 476.
[CrossRef]
3. Palmiero, G.; Cesaro, A.; Vetrano, E.; Pafundi, P.; Galiero, R.; Caturano, A.; Moscarella, E.; Gragnano, F.; Salvatore, T.; Rinaldi,
L.; et al. Impact of SGLT2 Inhibitors on Heart Failure: From Pathophysiology to Clinical Effects. Int. J. Mol. Sci. 2021, 22, 5863.
[CrossRef]
4. Sasso, F.C.; Pafundi, P.C.; Simeon, V.; De Nicola, L.; Chiodini, P.; Galiero, R.; Rinaldi, L.; Nevola, R.; Salvatore, T.; Sardo, C.; et al.
Efcacy and durability of multifactorial intervention on mortality and MACEs: A randomized clinical trial in type-2 diabetic
kidney disease. Sasso et al. Cardiovasc. Diabetol. 2021, 20, 145. [CrossRef]
5. Hamza, R.Z.; EL-Megharbel, S.M.; Altalhi, T.; Gobouri, A.A.; Alrogi, A.A. Hypolipidemic and hepatoprotective synergistic effects
of selenium nanoparticles and vitamin. E against acrylamide induced hepatic alterations in male albino mice. Appl. Organomet.
Chem. 2020, 34, e5458. [CrossRef]
6. Pessoa, J.C. Thirty years through vanadium chemistry. J. Inorg. Biochem. 2015, 147, 4–24. [CrossRef] [PubMed]
7. Crans, D.C. Antidiabetic, Chemical, and Physical Properties of Organic Vanadates as Presumed Transition-State Inhibitors for
Phosphatases. J. Org. Chem. 2015, 80, 11899–11915. [CrossRef]
8. Domingo, J.L.; Gómez, M. Vanadium compounds for the treatment of human diabetes mellitus: A scientific curiosity? A review
of thirty years of research. Food Chem. Toxicol. 2016, 95, 137–141. [CrossRef] [PubMed]
9. Ghasemi, F.; Rezvani, A.R.; Ghasemi, K.; Graiff, C. Glycine and metformin as new counter ions for mono and dinuclear vanadium
(V)-dipicolinic acid complexes based on the insulin-enhancing anions: Synthesis, spectroscopic characterization, and crystal
structure. J. Mol. Struct. 2018, 1154, 319. [CrossRef]
10. Carpéné, C.; Garcia-Vicente, S.; Serrano, M.; Marti, L.; Belles, C.; Royo, M.; Galitzky, J.; Zorzano, A.; Testar, X. Insulin-mimetic
compound hexaquis (benzylammonium) decavanadate is antilipolytic in human fat cells. World J. Diabetes 2017, 8, 143. [CrossRef]
11. Al-Harbi, M.S.; Hamza, R.Z. Potential Ameliorative Effects of Selenium and Chromium Supplementation Against Toxicity and
Oxidative Stress in Streptozotocin Diabetic Rats. Int. J. Pharmacol. 2016, 12, 483–495. [CrossRef]
12. Abuelzahab, H.; Hamza, R.; Montaser, M.; El-Mahdi, M.M.; Al-Harthi, W.A. Antioxidant, antiapoptotic, antigenotoxic, and
hepatic ameliorative effects of L-carnitine and selenium on cadmium-induced hepatotoxicity and alterations in liver cell structure
in male mice. Ecotoxicol. Environ. Saf. 2019, 173, 419–428. [CrossRef] [PubMed]
13. Wei, J.; Zeng, C.; Gong, Q.Y.; Yang, H.B.; Li, X.X.; Lei, G.H.; Yang, T.B. The association between dietary selenium intake and
diabetes: A cross-sectional study among middle-aged and older adults. Nutr. J. 2015, 14, 18. [CrossRef]
14. Tsave, O.; Yavropoulou, M.; Kafantari, M.; Gabriel, C.; Yovos, J.; Salifoglou, A. Comparative assessment of metal-specific
adipogenic activity in zinc and vanadium-citrates through associated gene expression. J. Inorg. Biochem. 2018, 186, 217–227.
[CrossRef]
15. Hamza, R.Z.; Al-Motaan, S.E.; Malik, N. Protective and Antioxidant Role of Selenium Nanoparticles and Vitamin C Against
Acrylamide Induced Hepatotoxicity in Male Mice. Int. J. Pharmacol. 2019, 15, 664–674. [CrossRef]
16. Kilkenny, C.; Browne, W.J.; Cuthill, I.C.; Emerson, M.; Altman, D.G. Improving bioscience research reporting: The ARRIVE
guidelines for reporting animal research. PLOS Biol. 2010, 8, e1000412. [CrossRef]
17. El-Megharbel, S.M.; Hamza, R.Z.; Gobouri, A.A.; Refat, M.S. Synthesis of new antidiabetic agent by complexity between vanadyl
(II) sulfate and vitamin B1: Structural, characterization, anti-DNA damage, structural alterations and antioxidative damage
studies. Appl. Organomet. Chem. 2019, 33, e4892. [CrossRef]
18. El-Megharbel, S.M.; Hamza, R.Z.; Refat, M.S. Synthesis, spectroscopic, structural and thermal characterizations of vanadyl (IV)
adenine complex prospective as antidiabetic drug agent Spectrochim. Acta Part A 2015, 135, 850. [CrossRef]
19. Johri, S.; Shukla, S.; Sharma, P. Role of chelating agents and antioxidants in beryllium induced toxicity. Indian J. Exp. Boil. 2002, 40,
575–582.
20. Litwack, G.; Bothwell, J.W.; Williams, J.N.; Elvehjem, C.A. A Colorimetric Assay for Xanthine Oxidase in Rat Liver Homogenates.
J. Biol. Chem. 1953, 200, 303. [CrossRef]
21. Furman, B.L. Streptozotocin-induced diabetic models in mice and rats. Curr. Protoc. Pharmacol. 2015, 70, 5–47. [CrossRef]
Curr. Issues Mol. Biol. 2022, 44 104

22. Suzuki, H.; Ota, S.; Sasagawa, T.; Sakatani, T. Fujikura, Assay method for myeloperoxidase in human polymorphonuclear
leukocytes. Anal. Biochem. 1983, 132, 345. [CrossRef]
23. Litwack, G.; Bothwell, J.W.; Williams, J.N.; Elvehjem, C.A. Assay for lipid peroxides in animal tissues by thiobarbituric acid
reaction. J. Biol. Chem. 1953, 200, 303. [CrossRef]
24. Ohkawa, H.; Ohishi, N.; Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 1979,
95, 351. [CrossRef]
25. Beers, J.R.; Sizer, I.W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem.
1952, 195, 133. [CrossRef]
26. Snedecor, G.W.; Cochran, W.G. Statistical Methods, 6th ed.; Oxford & IBH Co.: Bombay/New Delhi, India, 1967.
27. Hamza, R.Z.; Al-Baqami, N.M.; Khojah, E.; Mansour, A.M.A.; Al-Motaani, S.E.; Al-Salmi, F.A.; El-Megharbel, S.M. Possible
Antioxidant and Antidiabetic Effects of Combretum Molle Extract in a Diabetes Mellitus Experimental Model in Male Rats. Nat.
Prod. Commun. 2021, 16, 1–10.
28. West, E.; Simon, O.R.; Morrison, E.Y. Streptozotocin alters pancreatic beta-cell responsiveness to glucose within six hours of
injection into rats. West Indian Med. J. 1996, 45, 60.
29. Treviño, S.; Diaz, A. Vanadium and insulin: Partners in metabolic regulation. J. Inorg. Biochem. 2020, 208, 111094. [CrossRef]
30. El-Megharbel, S.M.; Al-Salmi, F.A.; Al-Harthi, S.; Alsolami, K.; Hamza, R.Z. Chitosan/Selenium Nanoparticles Attenuate
Diclofenac Sodium-Induced Testicular Toxicity in Male Rats. Crystals 2021, 11, 1477. [CrossRef]
31. Evans, J.L.; Goldfine, I.D.; Maddux, B.A.; Grodsky, G.M. Are Oxidative Stress−Activated Signaling Pathways Mediators of
Insulin Resistance and β-Cell Dysfunction? Diabetes 2003, 52, 1. [CrossRef]
32. Pepato, M.T.; Khalil, N.M.; Giocondo, M.P.; Brunetti, I.L. Vanadium and its Complexes: The Renewed Interest in its Biochemistry.
Lat. Am. J. Pharm. 2008, 27, 468–476.
33. El-Megharbel, S.M.; Al-Salmi, F.A.; Refat, M.S.; Hamza, R.Z. Selenium/Chitosan-Folic Acid Metal Complex Ameliorates Hepatic
Damage and Oxidative Injury in Male Rats Exposed to Sodium Fluoride. Crystals 2021, 11, 1354. [CrossRef]
34. Steinbrenner, H.; Sies, H. Protection against reactive oxygen species by selenoproteins. Biochim. Biophys. Acta 2009, 1790,
1478–1485. [CrossRef]
35. Refat, M.S.; Hamza, R.Z.; Adam, A.M.A.; Saad, H.A.; Gobouri, A.A.; Al-Harbi, F.S.; Al-Salmi, F.A.; Altalhi, T.; El-Megharbel, S.M.
Quercetin/Zinc complex and stem cells: A new drug therapy to ameliorate glycometabolic control and pulmonary dysfunction
in diabetes mellitus: Structural characterization and genetic studies. PLoS ONE 2021, 16, e0246265. [CrossRef]
36. Ithayarasi, A.P.; Devi, C.S. Effect of alpha-tocopherol on lipid peroxidation in iso-proterenol induced myocardial infarction in rats.
Indian J. Physiol. Pharmacol. 1997, 41, 369–376.
37. El-Demerdash, F.M. Effects of selenium and mercury on the enzymatic activitiesand lipid peroxidation in brain, liver, and blood
of rats. J. Env. Sci. Health B 2001, 36, 489–499. [CrossRef]
38. Thompson, K.H.; Orvig, C. Vanadium in diabetes: 100 years from phase 0 to phaseI. J. Inorg. Biochem. 2006, 100, 1925–1935.
[CrossRef] [PubMed]
39. Stróżyk, A.; Osica, Z.; Przybylak, J.D.; Kołodziej, M.; Zalewski, B.M. Effectiveness and safety of selenium supplementation for
type 2 diabetes mellitus in adults: A systematic review of randomised controlled trials. J. Hum. Nutr. Diet. 2019, 32, 635–645.
[CrossRef] [PubMed]
40. Hamza, R.Z.; Diab, A.E.A.A. Testicular protective and antioxidant effects of selenium nanoparticles on Monosodium glutamate-
induced testicular structure alterations in male mice. Toxicol. Rep. 2020, 7, 254–260. [CrossRef] [PubMed]
41. Kida, K.; Utsuyama, M.; Takiwwa, T.; Thurlbeck, W.M. Changes in lung morphologic features and elasticity caused by
streptozotocin-induced diabetes mellitus in growing rats. Am. Rev. Respir. Dis. 1983, 128, 125–131. [CrossRef]
42. Parkman, J.K.; Sklioutovskaya-Lopez, K.; Menikdiwela, K.R.; Freeman, L.; Moustaid-Moussa, N.; Kim, J.H. Effects of high fat
diets and supplemental tart cherry and fish oil on obesity and type 2 diabetes in male and female C57BL/6J and TALLYHO/Jng
mice. J. Nutr. Biochem. 2021, 94, 108644. [CrossRef] [PubMed]