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Effect of Telmisartan on Arsenic-Induced (Sub-chronic) Perturbations in

Redox Homeostasis, Pro-inflammatory Cascade and Aortic Dysfunction in
Wistar Rats

Article  in  Biological Trace Element Research · April 2022

DOI: 10.1007/s12011-021-02804-0

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Biological Trace Element Research
https://doi.org/10.1007/s12011-021-02804-0

Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations

in Redox Homeostasis, Pro‑inflammatory Cascade and Aortic
Dysfunction in Wistar Rats
B. Rudresh Gowda1   · N. Prakash2   · C. R. Santhosh1   · B. H. Pavithra1   · Rashmi Rajashekaraiah1   ·
M. L. Sathyanarayana3   · Suguna Rao3   · Prashantkumar Waghe4   · K. R. Anjan Kumar3   · G. R. Shivaprasad1   ·
Y. Muralidhar1 

Received: 19 May 2021 / Accepted: 19 June 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
An experimental study was conducted in male Wistar rats to explore the antioxidant potential of telmisartan (an ­AT1 receptor
blocker) to overcome arsenic (‘As’)-induced perturbations in redox homeostasis pro-inflammatory cytokines, prostaglandin-
E2 levels and aortic dysfunction in Wistar rats. Wistar rats were randomly divided into four groups of six each. Group-I
served as untreated control, while group-II received sodium (meta) arsenite ­(NaAsO2) (10 mg/kg b.wt. p.o) for a period of
60 days. Experimental rats in group-III received treatment similar to group-II, but in addition received telmisartan (with 1%
aqueous solution of Tween 80) @ 10 mg/kg b.wt. (p.o) for a similar duration, while rats in group-IV received telmisartan
alone. Arsenic exposure resulted in significant (p < 0.05) elevation in the levels of superoxide anion ( O2 ⋅ ) radicals (control:
768.20 ± 126.77 vs group-II: 1232.75 ± 97.85 pmol of NBT reduced/min/mg protein). Telmisartan administration showed sig-
nificant (p < 0.05) reduction in O2 ⋅ generation (815.34 ± 43.41 pmol of NBT reduced/min/mg protein). Sub-chronic exposure
to ‘As’ significantly (p < 0.05) decreased the activities of SOD, CAT, GPx and GR activity and GSH levels in the aorta, thus
induced lipid peroxidation (LPO) measured as measured in terms of thiobarbituric acid reactive substances (TBARS) called
as malondialdehyde (MDA). However, the administration of telmisartan effectively countered the LPO (24.03 ± 1.18 nmol
of MDA/g) on account of restoring the levels of aforesaid antioxidant defense system. Telmisartan administration effectively
attenuated the ‘As’-induced surge in pro-inflammatory cytokines (viz., IL-1β, IL-6 and TNF-α) levels, as well as countered
the activity of cyclooxygenase (­ COX2) as indicated by a significant (p < 0.05) decrease in P­ GE2 level in the aorta. In addi-
tion to it, there was a significant (p < 0.05) decrease in plasma angiotensin II (Ang-II) levels in experimental rats receiving
telmisartan. Quantitative RT-PCR studies revealed that sub-chronic exposure to ‘As’ upregulated the Nox 2 mRNA expression,
but there was a 1.2-fold reduction in expression level upon co-administration of telmisartan. Histopathological examination
revealed marked recovery from ‘As’-induced disruption of tunica adventitia and loss of connective tissue in experimental
rats receiving telmisartan. The study concludes that telmisartan can overcome aortic dysfunction induced by sub-chronic
exposure to arsenic through drinking water in experimental rats through restoration of redox balance, attenuation of pro-
inflammatory cytokines and mediators and downregulation of Nox2 mRNA expression.

Keywords  Arsenic · Sub-chronic · Aorta · Redox homeostasis · Telmisartan · Wistar rats

Introduction

Arsenic (‘As’) contamination of natural sources of water is a

global issue today. Arsenic which exists in both organic and
inorganic forms [1], present ubiquitously and its abundance
ranks twelfth in the human body, fourteenth in seawater [2]
* N. Prakash and twentieth in the earth’s crust, occurring most often as
pnadoor@rediffmail.com sulfide in a variety of complex minerals [3].
Extended author information available on the last page of the article

13
Vol.:(0123456789)

Groundwater contamination is the primary source of ‘As’
in man and animals. As per the recommendations of the
World Health Organization (WHO) [4] and the US Environ-
ment Protection Agency (US-EPA) [5], the current maxi-
mum contaminant level (MCL) of ‘As’ in potable water is
0.01 ppm. However, the permissible limit of ‘As’ in potable
water in the absence of an alternate source is 0.05 ppm [6]
as per the Bureau of Indian Standards (BIS).
Nearly about 108 countries [7] have been affected by arse-
nic contamination in groundwater including southeast Asian
countries like India, Bangladesh, Nepal, Vietnam and China
[8]. The exact molecular and cellular mechanisms involved
in ‘As’ toxicity have not been explained unequivocally.
Among the several hypotheses, oxidative stress appears to be
the most widely accepted and studied the mechanism of ‘As’
toxicity [9]. The mechanisms linked with these adversities
include hypomethylation [10], lipid peroxidation [11], angi-
ogenesis [12], induction of oxidative stress by the formation
of reactive oxygen species (ROS) along with reducing ROS
metabolizing enzymes [13], increased inflammatory cas-
cades including abnormal expression of inflammatory genes
[14], necrotic and apoptotic pathways [15] lead eventually
to cell death [16], increased vessel remodeling [17] and low
concentrations of arsenite induce increased glutathione lev-
els in vascular endothelial cells [18].
Natural exposure to ‘As’ (arsenicosis) can cause hepato-
cellular diseases [2], diabetes [19] neurological disorders
[20] and cardiovascular afflictions like Raynaud’s and black-
foot disease [21], aortic aneurysm [22], atherosclerosis [23],
peripheral vascular disease, hypertension and ischemic heart
disease [24].
Sub-chronic exposure to N ­ aAsO2 resulted in vascular
dysfunction due to reduction in both enzymatic (SOD, CAT,
GPx, GR) and non-enzymatic (GSH) antioxidative defense
systems [25], increase in the levels of pro-inflammatory
cytokines (IL-1β, IL-6) [26], increased Ang-II levels and
upregulation of Nox2 expression, a major precursor for ROS
production [27] in the aorta of rats. Similarly, Sarath et al.
[28] reported ‘As’-induced hypertension and dyslipidemia
leads to decrease in enzymatic antioxidants with a concomi-
tant increase in lipid peroxidation and O2 ⋅ in rat aorta. Fur-
ther, sub-chronic exposure to ­As2O3 resulted in an increased
level of TNF-α [29] in cardiac tissue.
Amelioration of oxidative stress induced by various arse-
nic compounds and recovery from vascular dysfunction can
be observed upon exogenous administration of a diverse
range of antioxidants, viz., n-acetylcysteine [30], vitamin-C,
vitamin-E [31], zinc [32], α-lipoic acid [33], arjunolic acid
[34], selenium [35], silibinin [36] and crocetin [29].
Telmisartan, a potent long-acting nonpeptide angiotensin-
II type-1 ­(AT1) receptor antagonist with an excellent safety
profile and unique pharmacological properties, viz., anti-
inflammatory, antioxidant [37], anti-apoptotic effects [38]
13
B. R. Gowda et al.
and high lipophilicity [39], possesses the longest half-life
(24 h) among the commercially available angiotensin recep-
tor blockers [40]. Telmisartan exerts its antioxidant and anti-
inflammatory effects by two metabolic pathways: activation
of PPAR-γ partially and blockade of ­AT1 receptor [41]. It
also inhibits activation of superoxide sources like NADPH
oxidase, mitochondria and xanthine oxidase which benefits
in reverting endothelial dysfunction [42].
Experimental studies have shown that telmisartan admin-
istration significantly attenuated sodium arsenite-induced
oxidative stress as shown by the decrease in TBARS, GSH
content, SOD activity [43] and structural remodeling of
vascular tissue in SH rats [44] through decreased protein
expression levels of MAPK, JNK and ERK than with those
of control group rats [45]. Telmisartan treatment prevents
transcription of the TNF-α and ­COX2 gene [46] and down-
regulates the Nox2 gene expression [42].
However, a comprehensive investigation to examine the
ameliorative role of telmisartan in arsenic (sub-chronic)-
induced oxidative injury to vascular tissue is lacking. Keep-
ing this in view, the current study was undertaken with
the following objectives to study the redox homeostasis in
vascular tissue of Wistar rats following sub-chronic expo-
sure to sodium (meta) arsenite and to study the influence of
telmisartan, an A­ T1-receptor antagonist and antioxidant on
arsenic-induced vascular perturbations in Wistar rats.
Material and Methods
Chemicals, Reagents and Assay Kits
Sodium (meta) arsenite (­NaAsO 2; M.W: 129.91; Ms.
Sigma-Aldrich, St. Louis, MO, USA) and Telmisartan
­(C33H30N4O2; M.W: 514.617  g/mol; Ms. Merck KGaA,
Germany), respectively, were procured commercially.
Experimental Animals
Male Wistar rats (N = 24) of 7–8 weeks of age and weigh-
ing ~ 155–165 g were obtained from an authorized vendor
(Ms. Sri Raghavendra Enterprises, Kenchanapura, Ben-
galuru). They were housed in Small Animal House facility,
Veterinary College, Hebbal, Bengaluru (Registration No.
493/GO/ReBiBt-S/Re-L/01/CPCSEA) in polypropylene
cages and maintained under standard management practices
including light: dark cycle of 12 h each. Experimental rats
were fed with standard rodent chow (Amrut®, Ms. Pranav
Agro Industries Ltd, M.H, India, supplied by a local vendor)
and given free access to water ad libitum. Prior approval
of the Institutional Animal Ethics Committee (IAEC) was
obtained (vide No. VCH/IAEC/2019/125 dated 03/12/2019)
to carry out the current investigation as per the guidelines of
Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations in Redox Homeostasis,…
the Committee for Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), New Delhi.
Experimental Design
After 1 week of acclimatization, the experimental rats were
randomly divided into four groups as detailed below: group-I
(n = 6): Served as control group and received ‘As’-free drink-
ing water throughout the experimental period ad libitum and
gavaged with vehicle (1% aqueous solution of Tween-80).
Group-II (n = 6): received sodium (meta) arsenite ­(NaAsO2)
@ 10 mg/kg given as oral gavage [rat feeding needle (14G);
Ms. Vihaan Techno Services, Hyderabad, India] daily dur-
ing morning hours for a period of 60 days. Group-III (n = 6):
all the animals in this group received treatment similar to
group-II for a period of 60 days but in addition received
telmisartan (1% aqueous solution made by using Tween-
80) @ 10 mg/kg; per os) daily 6 h after the administration
of arsenic. Group-IV (n = 6): received telmisartan alone @
10 mg/kg as above throughout the experimental period.
General Observation: Terminal Body Weight
All the animals were observed for general clinical signs if
any during the course of the study period twice daily. At
term (day 60) the terminal body weight of experimental ani-
mals was recorded, and following overnight fasting (but had
free access to drinking water ad libtum), they were sacrificed
[i.e., day 61 (morning hours)] under anaesthesia (Ketamine
hydrochloride @ 40 mg/kg; i.p and Xylazine hydrochloride
@ 10 mg/kg; i.p) and subjected to further studies.
Preparation of Aorta Tissue Homogenate
Aorta from each of the experimental rats was minced with
scissors, transferred into a centrifuge tube and homogenized
in a homogenizer (Ika® T10 basic ultra turrax®; Pune,
M.H; India) by using about 1 ml (ten fold) extraction buffer
(ice-cold 50 mM potassium phosphate buffer, pH 7.4) at
3–5 °C. The homogenate was then centrifuged at 10,000 × g
for 10 min, and the supernatant was stored at − 80 °C until
assayed for enzymatic or non-enzymatic biomarkers to
assess the oxidative stress.
Biochemical Biomarkers
Estimation of Superoxide Anion Radical ( O2 ⋅ ) Formation
The O2 ⋅ generation was estimated indirectly in terms of
formazan (blue colour) formed due to the reduction of
nitroblue tetrazolium (NBT) as an index of superoxide
anion generation and measured (formazan) by using a spec-
trophotometer at a wavelength of 540 nm [47]. To 900 μl of
tissue homogenate, 100 μl of 1 mM NBT was added (final
NBT concentration: 100 μmol/L), and the reaction mixture
was incubated at 37 °C for 90 min. The NBT reduction
was stopped by adding one ml of 0.5 N HCl. The amount
of O2 ⋅ generated was quantified spectrophotometrically
by measuring the absorbance of blue formazan at 540 nm
against blank. The superoxide radical anion in the samples
was measured as the amount of NBT reduced (quantity of
formazan), and it was calculated and expressed as picomol
of NBT reduced/min/mg protein.
Assessment of Peroxidative Damage
Peroxidative damage of the aorta was assessed by evaluat-
ing lipid peroxidation (LPO) in terms of malondialdehyde
(MDA) production as described by Paula et al. [48]. In brief,
1 ml of the aorta homogenate was mixed with 1 ml of 2%
thiobarbituric acid, 1 ml of 25% HCl and 90 µl butylated
hydroxytoluene. The mixture was kept in a boiling water
bath for 10 min at 95 °C and then centrifuged. The superna-
tant was transferred to a quartz cuvette, and the absorbance
was read at 535 nm. Results have been expressed as nmol
MDA formed/g of aorta.
Determination of Superoxide Dismutase (SOD) Activity
The SOD activity in the aorta was determined by the pro-
cedure of Madesh and Balasubramanian [49]. Briefly, the
reaction mixture contained 0.65 ml of PBS (pH 7.4), 30 µl
of 3-(4–5 dimethyl thiazol 2-yl) 2,5-diphenyl tetrazolium
bromide (1.25 mM), 75 µl of pyrogallol (100 µM) and 10 µl
supernatant of aorta homogenate (10%). The mixture was
incubated at room temperature for 5  min. and the reac-
tion was stopped by adding 0.75 ml of dimethyl sulfoxide.
The absorbance was read at 570 nm, and the activity was
expressed as unit/mg protein.
Determination of Catalase Activity
The CAT activity in the aorta was assayed by the spec-
trophotometric method of Aebi [50]. In brief, 1.99 ml of
phosphate buffer (50 mM, pH 7.0) and 10 µl supernatant of
homogenate (10%) were taken in a cuvette. The reaction was
started by adding 1 ml of ­H2O2 (10 mM), and the absorb-
ance was recorded at every 30 s for 3 min at 240 nm against
a water blank. The activity was expressed as mmol ­H2O2
utilized/min/mg protein.
Determination of Glutathione Peroxidase (GPx) Activity
The GPx activity was determined by the method of Paglia
and Valentine [51]. The reaction mixture contained
2.48 ml of phosphate buffer (50 mM, pH 7.0, containing
13

5 mM EDTA), 0.1 ml of NADPH (8.4 mM), 0.1 ml of GSH
(150 mM), 0.1 ml of sodium azide (112.5 mM), 4.6 U
glutathione reductase (Type III; Sigma Chemicals, USA)
and 10 µl supernatant of aorta homogenate. The reaction
was initiated by adding 0.1 ml of H­ 2O2 (2.2 mM) to the
mixture. The change in absorbance was read at 340 nm
for 3 min. The enzyme activity was expressed as µmol of
NADPH oxidized to NADP/min/mg protein.
Determination of Glutathione Reductase (GR) Activity
The GR activity in the aorta was measured following the
method of Goldberg and Spooner [52]. The 3 ml of reac-
tion mixture contained 2.6 ml of PBS (0.12 M, pH 7.2),
0.1 ml of EDTA (15 mM) and 0.1 ml of oxidized glu-
tathione (GSSG; 65.3 mM). To this, 10 µl supernatant of
aorta homogenate (10%) was added, and the volume was
made up to 2.95 ml with distilled water. After incuba-
tion at room temperature for 5 min, 0.05 ml of NADPH
(9.6 mM) was added. A decrease in absorbance/min was
recorded immediately at 340 nm for 3 min. Control was
run without GSSG. The activity of GR was expressed as
µmol NADPH oxidized to NADP/min/mg protein.
Determination of Reduced Glutathione (GSH) Content
GSH content of the aorta was measured by the method
of Sedlak and Lindsay [53]. Briefly, 1 ml supernatant of
homogenate, 0.8 ml of water and 0.2 ml of 50% trichlo-
roacetic acid solution were added and incubated at room
temperature for 15 min. This mixture was centrifuged at
3000 rpm for 15 min, and 0.4 ml of the supernatant was
taken. To it, 0.8 ml of 1 M Tris buffer (pH 8.0) was added
followed by 0.2 ml of 5,5’-dithiobis-2-nitrobenzoic acid
(DTNB) reagent (0.01 M). The absorbance was read at
412 nm within 5 min. Reagent blank had no sample, and
sample blank was without DTNB. The level of GSH was
expressed as mmol of GSH/g of aorta tissue homogenate.
Enzyme Linked Immunosorbent Assay (ELISA)
The level of interleukin-1β (IL-1β), interleukin-6 (IL-6),
tumor necrosis factor-α (TNF-α) (RayBiotech Inc., USA),
cyclooxygenase-2 ­(COX2) (Bioassay Technology Labora-
tory, Shanghai, China), prostaglandin ­E2 ­(PGE2) in aorta
tissue homogenate and angiotensin-II (Ang-II) (Elabsci-
ence® Biotechnology Inc, Wuhan, China) in plasma were
measured using ELISA kits as per the standard protocol
described by manufacturers.
13
B. R. Gowda et al.
Protein Estimation
The protein content in the supernatant of aorta homogen-
ates was determined by following the method described by
Lowry et al. [54] using bovine serum albumin as a standard.
Real‑Time PCR Analysis of ­Nox2 genes
A part of thoracic aorta collected into RNA later® solution
was homogenized in a microcentrifuge tube containing 1 ml
ice-cold Trizol™ reagent with mortar and pestle. Chloro-
form (200 µl) was mixed with the homogenate. The mixture
was vortexed for 30 s, kept for 10 min at room temperature
and centrifuged at 12,000 × g for 15 min at 4 °C to separate
the homogenate into 3 layers. The top aqueous layer was
collected, from which the RNA was precipitated with the
addition of an equal volume of ice-cold isopropanol. It was
mixed properly and kept for 10 min at room temperature.
Then centrifugation was done at 12,000 × g for 10 min at
4 °C. The supernatant was discarded, and the RNA pellet
was collected. The RNA pellet was washed with 75 percent
ethanol at 7,500 × g for 5 min. The pellet was air-dried, and
≈ 40 µl nuclease-free water was added to the pellet. The
purity of the RNA was quantified by NanoDrop® spectro-
photometer, and only RNA samples with an A260/A280
ratio of 1.8–2.0 were used for reverse transcription.
The synthesis of cDNA was carried out by using com-
mercially available kit (TaKaRa Bio Inc., Japan). cDNA syn-
thesis kit was synthesized from the mRNA present in the
total RNA using Moloney murine leukemia virus (MMLV)
reverse transcriptase.
The published primers were used for mRNA expression
of the Nox2 and GAPDH genes. Quantitative real-time PCR
reactions were run on QuantStudio™ 3 (Applied biosys-
tems®, by Thermo Fisher Scientific, USA). Real-time PCR
was carried out by using TB Green premix Ex Taq-II [(2X)
(TaKaRa®, Japan)]. The optimum annealing temperature
as determined by PCR for Nox2 using the specific primers
was 63 °C.
Each sample was run in triplicate in a 20 μl reaction.
The 20 μl reaction mixture contained 10 μl TB Green pre-
mix, 1 μl from 10 pmol working solutions of each of gene-
specific forward and reverse primers, 0.5 μl of cDNA, and
the volume was made up to 20 μl with nuclease-free water.
The real-time PCR reaction started with initial incubation
at 95 °C for 30 s followed by 40 cycles of amplification with
denaturation at 95 °C for 5 s, annealing at 63 °C for Nox2 for
5 s and extension at 72 °C for 5 s for each cycle.
The result obtained was expressed as threshold cycle
(CT). This value corresponds to the cycle number when the
fluorescence of the reporter dye appreciably higher than the
background fluorescence. The CT values adjusted automati-
cally by the instrument was used for the generation of CT
Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations in Redox Homeostasis,…

values. Relative quantification (RQ) value was determined mg/protein) (group-II) as compared to control group
(RQ = ­2−∆∆CT) and compared between different experimen- (768.20 ± 126.77 pmol of NBT reduced.min−1.mg−1 pro-
tal groups of rats. tein) (group-I). Telmisartan administration to ‘As’ exposed
rats (815.34 ± 43.41 pmol of NBTreduced/min/mg/protein)
Histopathology of the Aorta (group-III) significantly (p < 0.05) reduced the superoxide
radical generation (Fig. 1a).
Standard procedure was adopted to process the tissue for
histopathological examination. The tissues were processed LPO  The oxidative injury among various experimental
by the routine paraffin embedding technique and sections of groups was estimated as nmol of MDA/’g’ of tissue. Sub-
5-µm thickness were stained with haematoxylin and eosin chronic ‘As’ (36.94 ± 2.48  nmol MDA/g tissue) (group-II)
(H&E) for light microscopy [55]. Similarly, the standard exposure significantly (p < 0.05) elevated the LPO levels
procedure of Masson’s trichrome staining was employed to when compared to control group (17.40 ± 2.85 nmol MDA/g
stain histopathological sections of the aorta [56]. tissue) (group-I). Telmisartan administered to ‘As’ exposed
rats (24.03 ± 1.18 nmol MDA/g tissue) (group-III) showed
Statistical Analysis a significant (p < 0.05) reduction in LPO level when com-
pared to ‘As’ exposed rats (Fig. 1b). Further, the LPO levels
The values obtained from the various experiments were
expressed as mean ± S.E with ‘n’ equal to number of animals or
samples. Data obtained were statistically subjected to one-way
analysis of variance (ANOVA) followed by Duncan’s post hoc
multiple comparison test using SPSS statistics software (IBM®
SPSS® statistics software, Version 21.0.0, 2012, Armonk, NY,
USA). The difference was considered significant at p < 0.05
or lower. Graphical presentation of the data was carried out
by using GraphPad Prism software programme (GraphPad®
software Inc., Version 8.4.3; San Diego, CA, USA).

Results

The current study was carried out with an aim to character-

ize the effect of inorganic arsenic (‘As’) on vascular per-
turbations in Wistar rats following sub-chronic exposure to
examine the potential role of telmisartan, an angiotensin-II
antagonist under such circumstances.
Sub-chronic exposure to ‘As’ did not show any acute
signs of illness or mortality in any of the experimental
groups. However, upon close clinical examination, there
was dehydration and a minor degree of weakness in rats
which received ‘As’ was observed. At term, the ‘As’ exposed
rats (group-II) showed a significant decrease in body weight
(b.wt.) in comparison to other experimental groups. The
b.wt. of telmisartan alone administered rats (group-IV) were
statistically similar to that of control group of rats.

Oxidative Stress Biomarkers

Superoxide Radical ( O2 ⋅)
The O2 ⋅ generation measured as pmol of NBT reduced/
min/mg protein indicated a significant (p < 0.05)
increase in levels of superoxide radical in ‘As’ exposed
rats (1232.75 ± 97.85  pmol of NBT reduced/min/
Fig. 1  Mean (±  SE) superoxide radical activity (pmol of NBT
reduced/min/mg/ protein) (a) and levels of MDA formed (nmol
MDA/g tissue) (b) in aorta tissue homogenate in different groups
of experimental rats (n = 6 each). Note: Bars bearing dissimilar
alphabets vary significantly between experimental groups; p < 0.05;
[C = control, As = arsenic, As + Tel = arsenic + telmisartan, Tel = tel-
misartan]

13
 B. R. Gowda et al.

in rats that received telmisartan alone did not influence the

basal level as recorded in the control group (group-I).

Enzymatic Antioxidants

SOD

Mean (± SE) SOD activity (U/mg protein) in the aorta

of rats belonging to control (group-I) and ‘As’ exposed
group (group-II) were 0.68 ± 0.07 and 0.41 ± 0.03 U/mg
protein, respectively; and these values were differed sig-
nificantly (p < 0.05). Telmisartan administration to ‘As’
exposed rats (group-III) significantly (p < 0.05) improved
the SOD activity (0.58 ± 0.04 U/mg protein). However, tel-
misartan alone administered rats (group-IV) (0.60 ± 0.02
U/mg protein) and control (group-I) rats were statistically
similar (p > 0.05) to each other (Fig. 2a).

CAT​

The CAT activity was significantly (p < 0.05) reduced in the

aorta of ‘As’ exposed rats (group-II) (186.15 ± 20.49 mmol
of ­H2O2 utilised/min/mg protein) when compared to control
group (group-1) (401.14 ± 10.33 mmol of ­H2O2 utilised/
min/mg protein). Administration of telmisartan to ‘As’
exposed rats (group-III) showed a significant (p > 0.05)
increase in CAT activity (285.68 ± 22.96 mmol of H ­ 2O 2
utilised/min/mg protein) (Fig. 2b).
GPx
The GPx activity in the aorta of ‘As’ exposed rats (group-II)
(5.22 ± 0.41 μmol of NADPH oxidized/min/mg protein) was
significantly (p < 0.05) reduced when compared to control
group (group-I) (14.20 ± 0.64 μmol of NADPH oxidized/
min/mg protein). However, administration of telmisartan in
‘As’ exposed rats (group-III) showed a significant recovery
in GPx activity (7.55 ± 0.83 μmol of NADPH oxidized/min/
mg protein) in the aorta (Fig. 3a).
GR  The GR activity in the aorta of control group (group-
I) and ‘As’ exposed rats (group-II) were 9223.37 ± 429.57
and 5214.59 ± 135.94  μmol of NADPH oxidized/min/
mg protein, respectively. Statistical comparison between
these two groups indicated that ‘As’ induced a signifi-
cant (p < 0.05) decrease in glutathione reductase activity.
However, telmisartan administration in ‘As’ exposed rats
(group-III) showed significant (p < 0.05) recovery in the
glutathione reductase activity (7511.18 ± 230.21 μmol of
NADPH oxidized/min/mg protein) (Fig. 3b).
Fig. 2  Mean (±  SE) superoxide dismutase (SOD) activity (Unit/
mg protein) (a) and catalase (CAT) activity (mmol of ­H2O2 utilized/
min/mg protein) (b) in aorta tissue homogenate in different groups
of experimental rats (n = 6 each). Note: Bars bearing dissimilar
alphabets vary significantly between experimental groups; p < 0.05;
[C = control, As = arsenic, As + Tel = arsenic + telmisartan, Tel = tel-
misartan]
Non‑enzymatic Antioxidant
GSH
The GSH content in the aorta of control group (group-
I) and ‘As’ exposed rats (group-II) were 0.21 ± 0.01 and
0.15 ± 0.002 mmol/ ‘g’ tissue, respectively. These values
differ significantly (p < 0.05). The ‘As’ induced depletion
in the reduced glutathione levels in the aorta was signifi-
cantly (p < 0.05) elevated (0.20 ± 0.01 mmol/ ‘g’ tissue)
in the telmisartan administered group (group-III) (Fig. 4).
Pro‑inflammatory Cytokines
IL‑1β
The IL-1β level in the control group (group-I) of rats was
2550.76 ± 322.01 pg/mg protein. Arsenic exposure (group-II)

13
Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations in Redox Homeostasis,…
Fig. 3  Mean (± SE) glutathione peroxidase (GPx) activity (μmol of
NADPH oxidized to NADP/mg/min) (a) and glutathione reductase
(GR) activity (μmol of NADPH oxidized to NADP/mg/min) levels
(b) in aorta tissue homogenate in different groups of experimental
rats (n = 6 each). Note: Bars bearing dissimilar alphabets vary signifi-
cantly between experimental groups; p < 0.05; [C = control, As = arse-
nic, As + Tel = arsenic + telmisartan, Tel = telmisartan]
Fig. 4  Mean (±  SE) reduced glutathione (GSH) content (mmol
GSH/g tissue) in aorta tissue homogenate in different groups of
experimental rats (n = 6 each). Note: Bars bearing dissimilar alpha-
bets vary significantly between experimental groups; p < 0.05;
[C = control, As = arsenic, As + Tel = arsenic + telmisartan, Tel = tel-
misartan]
(4760.49 ± 129.94  pg/mg protein) caused significantly
increased production of IL-1β, while telmisartan adminis-
tration to ‘As’ exposed rats (group-III) significantly (p < 0.05)
(3416.10 ± 435.49 pg/mg protein) reduced its production back
to levels as observed in the control group of rats (Fig. 5a).
IL‑6  The IL-6 level in the control group (group-I) of rats
was 77.37 ± 5.90 pg/mg protein. Arsenic exposure (group-
II) (111.04 ± 7.88  pg/mg protein) caused a significant
(p < 0.05) increase in production of IL-6, while telmisartan
administered rats (group-III) significantly (p < 0.05) reduce
its production (82.21 ± 4.46 pg/mg protein) (Fig. 5b).
TNF‑α
The level of TNF-α production in control (group-I)
(46.71 ± 4.06 pg/mg protein) and telmisartan alone (group-
IV) administered (51.62 ± 2.94 pg/mg protein) rats were
significantly comparable (p > 0.05). Rats exposed with
‘As’ (group-II) exhibited a significant (p < 0.05) increase
in its production (79.31 ± 3.51 pg/mg protein). Telmisartan
administration (group-III) significantly (p < 0.05) inhibited
‘As’ mediated increase in TNF-α levels (48.06 ± 3.47 pg/
mg protein) (Fig. 5c).
Inflammatory Mediators
COX2
The ­COX2 activity in control group of rats (group-I) was
0.60 ± 0.00  ng/mg protein. Arsenic exposure (group-II)
(0.72 ± 0.01 ng/mg protein) caused significantly increased
production of C ­ OX2, while telmisartan administration to
group-III rats significantly (p < 0.05) reduced its activity as
of control group of rats (0.66 ± 0.01 ng/mg protein) (Fig. 6a).
PGE2
The ­PGE2 levels in the aorta of ‘As’ exposed rats (group-
II) was significantly (p < 0.05) increased (48.99 ± 4.50 pg/
mg protein) when compared to control group of rats
(group-I) (36.04 ± 1.04 pg/mg protein). While telmisar-
tan administration in ‘As’ exposed (group-III) rats showed
a significant (p < 0.05) decrease in the levels of ­P GE 2
(38.10 ± 1.57 pg/mg protein) in the aorta (Fig. 6b).
Ang‑II
The Ang-II level was significantly (p < 0.05) increased in
plasma of ‘As’ exposed rats (group-II) (17.05 ± 0.85 pg/
13

Fig. 5  Interleukin-1β (IL-1β) (a), interleukin-6 (IL-6) (b) and tumor
necrosis factor-α (TNF-α) (c) levels (pg/mg protein) in aorta tis-
sue homogenate in different groups of experimental rats (n  = 6
each). Note: IL-1β, IL-6 and TNF-α were measured with ELISA
and recorded at absorbance 450 nm, and data are expressed as mean
(± SE) pg/mg protein; bars bearing dissimilar alphabets vary signifi-
cantly; p < 0.05;[C = control, As = arsenic, As + Tel = arsenic + tel-
misartan, Tel = telmisartan]
13
B. R. Gowda et al.
Fig. 6  Cyclooxygenase-2 (COX2) (a) (ng/mg protein) and Prosta-
glandin ­E2 ­(PGE2) (pg/mg protein) (b) in aorta tissue homogenate in
different groups of experimental rats (n = 6 each). Note: ­COX2 (ng/
mg protein) and ­PGE2 (pg/mg protein)was measured with ELISA
and recorded at absorbance 450 nm and data are expressed as mean
(± SE) per unit protein; bars bearing dissimilar alphabets vary sig-
nificantly; p < 0.05; [C = control, As = arsenic, As + Tel = arsenic + tel-
misartan, Tel = telmisartan]
ml plasma) when compared to control group (group-I)
(10.38 ± 0.56  pg/ml plasma). Telmisartan administra-
tion to ‘As’ exposed rats (group-III) showed a significant
(p > 0.05) decrease in (13.03 ± 0.66 pg/ml plasma) Ang-II
levels in plasma (Fig. 7).
Quantitative RT‑PCR: ­Nox2 mRNA Expression
The Nox2 mRNA expression in aorta was observed in all
the samples belonging to various experimental groups of
rats. There was a significant (p < 0.05) (2.8 fold) upregula-
tion of Nox2 mRNA expression in rats exposed to ‘As’ (RQ:
2.915 ± 0.16) (group-II) when compared to the control
group of rats (group-I) (RQ: 0.188 ± 0.04). Administration
Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations in Redox Homeostasis,…
Fig. 7  Angiotensin-II (Ang-II) (pg/ml plasma) levels in plasma in
different groups of experimental rats (n = 6 each). Note: Ang-II (pg/
ml plasma) was measured with ELISA and recorded at absorbance
450  nm, and data are expressed as mean (± SE) pg/ml plasma; bars
bearing dissimilar alphabets vary significantly; p < 0.05; [C = control,
As = arsenic, As + Tel = arsenic + telmisartan, Tel = telmisartan]
of telmisartan in ‘As’ exposed rats (group-III) showed a
significant (p > 0.05) (1.2 fold) downregulation in Nox2
gene expression (RQ: 2.234 ± 0.04) when compared to rats
exposed to ‘As’ (Fig. 8).
Histological Evaluation
Figures 9A, and D shows that the control rats (group-I) and
the telmisartan-administered rats (group-IV) did not show
any appreciable alterations in the aortic histology. Arse-
nic exposure caused disruption of tunica adventitia and
Fig. 8  Relative expression levels of Nox2 gene ofmRNA isolates of
rats belonging to various experimental groups. Note: Bars bearing
dissimilar alphabets vary significantly between experimental groups;
p < 0.05; [C = control, As = arsenic, As + Tel = arsenic + telmisartan,
Tel = telmisartan]
thickening of aorta and tunica media (group-II) (Fig. 9B).
Telmisartan appreciably attenuated these vascular histologi-
cal lesions in the arsenic-exposed rats (group-III) (Fig. 9C).
Discussion
In the present study sub-chronic exposure to sodium (meta)
arsenite (‘As’) (group-II) resulted in a significant (p < 0.05)
decrease in body weight (b.wt.) compared to other groups
when assessed as per cent change over the initial weight of
rats. The present result is consistent with the findings of
Shila et al. [33] and Garcia et al. [57]. The probable reason
for decrease in body weight of ‘As’ exposed rats (group-II)
may be due to a decrease in feed and water consumption due
to the inherent toxic nature of ‘As’. The per cent change over
the initial weight of rats that are treated with telmisartan
alone (group-IV) showed only a marginal decrease in body
weight when compared to control group of rats (group-I).
The present findings are in tune with the results of previous
studies in mice [58] and rats [59]. The decrease in body
weight may be attributed to partial peroxisome proliferator-
activated receptor-γ (PPAR-γ) agonistic activity of telmisar-
tan which may have the capacity to improve glucose and
lipid metabolism [58].
Superoxide radical production can sequentially lead to
­H2O2 formation and then generate hydroxyl radicals ­(OH⋅),
the ultimate toxicant. In the present study, sub-chronic ‘As’
exposure (group-II) increased the production of O2 ⋅ in rats,
suggesting the development of oxidative stress and conse-
quent vascular endothelial dysfunction [27, 60]. The rise
in O2 ⋅ levels might be due to increased activity of NADPH
oxidase enzyme and surge in the Ang-II level [36]. Admin-
istration of telmisartan to ‘As’ exposed group (group-III)
significantly decreased ‘As’ induced O2 ⋅ levels. A similar
observation in the aorta was also made previously [46], and
the authors opined that telmisartan decreased the production
of O2 ⋅ via reducing NADPH-dependent oxidase activity.
Sub-chronic exposure of ‘As’ resulted in a significant
(p < 0.05) increase in the levels of TBARS called MDA,
and our results are in conformity with the results of Waghe
et al. [25]. Maintaining redox balance in the vascular system
plays a vital role, failing which redox signaling leading to
oxidative stress and contributes to endothelial dysfunction
or vascular disease. An increase in MDA levels is regarded
as one of the basic mechanisms of tissue damage caused by
free radicals and extensively used as a biomarker of oxida-
­ H⋅ primarily responsible for
tive stress. The production of O
the peroxidation of lipid membranes by MDA [61], and it
could be related to deleterious effects of ROS and the fail-
ure of antioxidants to re-establish the redox homeostasis in
vascular tissue [25]. The antioxidant property of telmisar-
tan can be considered as the main factor responsible for the
13

Fig. 9  Light microscopy of A B
aorta showing normal archi-
tecture in control (A) and
telmisartan administered rats
(D). Sections of arsenic (‘As’)
exposed rats (B) showed disrup-
tion of tunica adventitia (➞)
and loss of connective tissue
and elastic fibres (➘), whereas
sections of telmisartan followed
by arsenic (‘As’) administered
rats showed near-normal
architecture of aorta (Masson’s
trichrome stain × 20)
C D
protective role and reduced lipid peroxidation as in the case
of cisplatin-induced nephropathy in rats [62] or by virtue of
its ability to augment the expression of PPAR-γ [37].
Sub-chronic ‘As’ exposure (group-II) not only signifi-
cantly reduced the enzymatic antioxidant parameters assayed
(viz., SOD, CAT, GPx, GR) but also depleted the GSH con-
tent of the aorta. Previous studies have demonstrated that
exposure to ‘As’ can reduce the activity of SOD, GR [25],
CAT, GPx and tissue GSH content [28] in rat aorta. This
reduction in antioxidant enzymes level could be attributable
to increased production of O2 ⋅ by NADPH oxidase activ-
ity or elevated Ang-II levels [63], removal of accumulated
­H2O2 through a GSH dependent mechanism in the aorta of
experimental rats.
Telmisartan co-administration to ‘As’ exposed rats
(sub-chronic) significantly improved the ‘As’ induced inhi-
bition in the levels of antioxidant enzymes (viz., SOD,
CAT, GPx, GR) and in the GSH content of the aorta. The
reduced antioxidant enzyme levels could be attributable
to (i) increased utilization of enzymes for maintenance
of redox homeostasis, (ii) direct binding of arsenicals to
dithiol targets [64] or (iii) might be because telmisartan
can exert partial PPAR-γ agonistic activity at therapeutic
doses which could be responsible for its antioxidant, anti-
proliferative and anti-inflammatory effect [62].
13
B. R. Gowda et al.
Sub-chronic exposure of ‘As’ (group-II) showed a sig-
nificant increase in levels of pro-inflammatory cytokines,
viz., IL-1β, IL-6 and TNF-α. Kesavan et al. [26] and Zhao
et al. [29] in the aorta and cardiac tissue, respectively,
have also observed similar trend of increase in the levels
of cytokines. The probable mechanism involved might be
the activation of signaling pathways like ERK, JNK, p38-
MAPK and NF-κB, which in turn induces the expression
of variety of pro-inflammatory genes [65].
Fouad et al. [66] reported that concomitant adminis-
tration of telmisartan with sodium arsenite significantly
inhibited the TNF-α production in hepatic tissue of mice,
so as in testicular tissue of rats [67]. Further, apart from
decline in levels of IL-1β and IL-6, a significant decline
in the levels of TNF-α (in the aorta) in ‘As’ exposed rats
that received telmisartan (group-III) could be attributed to
either anti-inflammatory mechanism via PPAR-γ [68] path-
way or through suppression of NF-κB gene activation [69].
Arsenic exposure (sub-chronic) significantly increased
the ­PGE2 levels on account of increased activity of cycloox-
ygenase activity (­ COX2). Telmisartan administration to ‘As’
exposed rats decreased the levels of C ­ OX2 significantly
might be due to exposure of cells to arsenite causes a sig-
nificant ­COX2 expression in an IKKB/NF-κB-dependent
manner due to oxidative stress [70]. Sukumaran et al. [45]
also reported that telmisartan can decrease the TNF-α and
Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations in Redox Homeostasis,…

Fig. 10  Light microscopy of
aorta showing normal archi-
tecture in control (A) and tel-
misartan administered rats (D).
Disruption of tunica adventitia
was evident (➞) following ‘As’
exposure (60 days), whereas
sections of telmisartan followed
by arsenic (‘As’) administra-
tion showed marked recovery
in the architecture of the aorta
(H&E × 20)

­ OX2 activity in cardiac tissue subjected to porcine cardiac

C rats exposed to sodium arsenite which received candesar-
myosin-induced autoimmune myocarditis. As opined by
Fouad et al. [66], this could be probably due to the ability of
telmisartan to prevent the activation of the NF-κB signaling
pathway which promotes the transcription of the ­COX2 gene.
Sub-chronic exposure to ‘As’ significantly increased
the plasma Ang-II levels and our results are in tune with
the findings of Waghe et  al. [27] in rats which can be
associated with concomitant modulation of extracellular
signal-regulated kinase vascular endothelial growth fac-
tor (ERK-VEGF) signaling, as well as due to increased
generation of O2 ⋅ radicals [71]. Telmisartan alone treated
group showed a marginal decrease in the levels of Ang-
II in plasma which could be primarily due to selective
antagonistic activity of telmisartan at the ­AT1 receptors.
Concurrent administration of telmisartan to ‘As’ exposed
rats showed a significant decrease in Ang-II levels which
can be attributed to its vasoprotective effect. A similar
observation was recorded by Khuman et al. [72] in Wistar
tan, another ­AT1 receptor blocker.
Arsenic exposure (sub-chronic) showed a significant
(p < 0.05) upregulation of Nox2 mRNA expression, similar
to a study conducted by Waghe et al. [27]. Increased levels
of Ang-II and its binding to ­AT1 receptor leads to the acti-
vation of phospholipase C, releasing DAG inturn activates
protein kinase C-mediated Nox2 activation to generate O2 ⋅
by phosphorylating p47phox [73]. Telmisartan being an ­AT1
receptor blocker can inhibit Nox2 activation, thus decreasing
the upregulation of Nox2 in the aorta [42].
Experimental exposure (sub-chronic) of ‘As’ led to the
disruption of tunica adventitia of the aorta along with loss
of connective tissues and elastic fibres unlike control group
which has shown classical architecture of the aorta charac-
terized by intact layers of tunica intima, tunica media and
tunica adventitia. However, telmisartan co-administration
restored the normal histology of the aorta (Fig. 10), which
may be invariably due to restoration of redox balance and

13

downregulation of pro-inflammatory cytokines. Our results
are in conformity with the previous studies in rats [26, 28].
To conclude the present study revealed that sub-chronic
exposure to sodium (meta) arsenite in Wistar rats through
drinking water induces lipid peroxidation leading to redox
signaling, enhanced COX activity and trigger pro-inflamma-
tory cascade in vascular tissue (the aorta). However, concomi-
tant administration of telmisartan, an A ­ T1-receptor blocker
can restore the arsenic-induced perturbations in redox homeo-
stasis, suppress the pro-inflammatory cytokines and mediator
­(PGE2) and prevent damage to aortic architecture. The obser-
vations made in this study have clinical relavence in situa-
tions where animals are likely to expose to arsenic-epidemic
geopgraphical locations. Therefore, the results of the present
study explicitly confirm the pharmacological potential of tel-
misartan to exploit for its antioxidant, anti-inflammatory and
antiapoptotic activities to overcome aortic dysfunction.
Acknowledgements  The authors are also thankful to the Dean, Vet-
erinary College, Bengaluru, Karnataka (India) for providing necessary
facilities to conduct the study.
Author Contribution  Authors whose names appear on the submis-
sion have contributed sufficiently to the scientific work and therefore
share collective responsibility and accountability for the results. RGB
and PN conceived and designed the study. SCR, PBH, RR and MLS
made substantial contributions in reviewing the design of the study and
acquiring the data. SGR and MY coordinated sample collection, while
PKW oversaw various laboratory assays. AKR assisted in conduct of
RT-PCR and interpreted the data obtained. SR contributed substantially
in histopathological analysis. All authors contributed to final approval
of the version of the manuscript submitted for publication.
Funding  The authors are grateful to the Karnataka Veterinary, Animal
and Fisheries Sciences University (KVAFSU), Nandinagar, Bidar, Kar-
nataka State, India, Pin code: 585 226 (India), for providing financial
support in conducting the study.
Data Availability  No data have been fabricated or manipulated (includ-
ing images) to support your conclusions. The data that support the
findings of this study are available from the corresponding author, upon
reasonable request.
Declarations 
Ethics Approval and Consent to Participate  Prior approval of the Insti-
tutional Animal Ethics Committee (IAEC) was obtained (No. VCH/
IAEC/2019/125 dated 03/12/2019) to carry out the current investigation
as per the guidelines of the Committee for Purpose of Control and Super-
vision of Experiments on Animals (CPCSEA), New Delhi. Experimental
rats were housed in Small Animal House facility, Veterinary College,
Hebbal, Bengaluru (Registration No. 493/GO/ReBiBt-S/Re-L/01/CPC-
SEA, New Delhi) in polypropylene cages and maintained under standard
management practices including light: dark cycle of 12 h each.
Consent for Publication  Consent to submit has been received from all
co-authors and responsible authorities at the institute where the work
has been carried out before the work is submitted. The corresponding
author was given consent on behalf of all author for the publication.
13
B. R. Gowda et al.
Competing Interests  The authors declare no competing interests.
Disclaimer  Further, the article is the author’s original work, has not
received prior publication and is not under consideration for publica-
tion elsewhere.
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Authors and Affiliations
B. Rudresh Gowda1   · N. Prakash2   · C. R. Santhosh1   · B. H. Pavithra1   · Rashmi Rajashekaraiah1   ·
M. L. Sathyanarayana3   · Suguna Rao3   · Prashantkumar Waghe4   · K. R. Anjan Kumar3   · G. R. Shivaprasad1   ·
Y. Muralidhar1 
B. Rudresh Gowda
rudreshbrudri414@gmail.com
C. R. Santhosh
crsanthoshvet@gmail.com
B. H. Pavithra
pavithra.bh@gmail.com
Rashmi Rajashekaraiah
rashmirvet7@gmail.com
M. L. Sathyanarayana
mlspathology@yahoo.com
Suguna Rao
sugunabg@gmail.com
13
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Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Prashantkumar Waghe
drprashant1985@gmail.com
K. R. Anjan Kumar
drvet_anjan@hotmail.com
G. R. Shivaprasad
shivaprasad_gr@rediffmail.com
Y. Muralidhar
murali4357@gmail.com
1
Department of Veterinary Pharmacology and Toxicology,
Veterinary College, Karnataka Veterinary, Animal
and Fisheries Sciences University, Hebbal, Bengaluru,
Karnataka 560 024, India
Effect of Telmisartan on Arsenic‑Induced (Sub‑chronic) Perturbations in Redox Homeostasis,…

2 4
Veterinary College, Karnataka Veterinary, Animal Department of Veterinary Pharmacology and Toxicology,
and Fisheries Sciences University, Vinobanagar, Veterinary College, Karnataka Veterinary, Animal
Shivamogga, Karnataka 577 204, India and Fisheries Sciences University, Nandinagar, Bidar,
3 Karnataka 585 226, India
Department of Veterinary Pathology, Veterinary College,
Karnataka Veterinary, Animal and Fisheries Sciences
University, Hebbal, Bengaluru, Karnataka 560 024, India

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