Invest Clin 66(4): 408 - 425, 2025 https://doi.org/10.54817/IC.v66n4a05

Corresponding author: Dandan Qiu. Department of Urology, The First Affiliated Hospital of Zhejiang Chinese

Medical University, Zhejiang Hospital of Traditional Chinese Medicine, Hangzhou Zhejiang, 310006, China. Phone:

+8613588454495. Email: qiudandan2000@outlook.com

Rutin as a potential therapeutic agent

for arsenic-induced testicular damage:

Antioxidant, anti-inflammatory,

and anti-apoptotic effects.

Yunyun Wang1,#, Zengrong Hua2,#, Jin Qing Hui3 and Dandan Qiu4

1Department of Pharmacy, The First Aﬃliated Hospital of Zhejiang Chinese Medical

University, Zhejiang Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang, China.

2Department of Urology, Huai'an Clinical Medical College of Jiangsu University

(Huai'an Hospital of Huai'an City), Huai'an, Jiangsu, China.

3Department of Surgical, Shaanxi Kangfu Hospital, Xian, Shaanxi, China.

4Department of Urology, The First Affiliated Hospital of Zhejiang Chinese Medical

University, Zhejiang Hospital of Traditional Chinese Medicine, Hangzhou Zhejiang, China.

#Yunyun Wang and Zengrong Hua are co-first authors; they contributed equally

to this work.

Keywords: Apoptosis; Arsenic; Inflammation; Rutin, Spermatogenesis; Testicular

Toxicity.

Abstract. Arsenic exposure is associated with male reproductive toxicity, in-

cluding impaired spermatogenesis, reduced sperm quality, and disrupted hormone

levels. Rutin, a natural flavonoid, has demonstrated protective effects in various or-

gans owing to its antioxidant, anti-inflammatory, and antiapoptotic properties. This

study aimed to assess the efficacy of rutin in ameliorating testicular toxicity caused

by sodium arsenite in laboratory rats. Sprague-Dawley rats were randomly assigned

to normal, arsenic control, rutin (25, 50, and 100 mg/kg), and Co-enzyme Q10 (10

mg/kg) treatment groups. Testicular damage was induced by oral administration of

sodium arsenite (10 mg/kg, for two days). Rutin and Co-enzyme Q10 were adminis-

tered orally for 15 and seven days, respectively. The serum hormone levels, sperm pa-

rameters, testicular mitochondrial enzymes, oxidative stress markers, inflammatory

cytokines, apoptotic proteins, and histopathology were assessed. Arsenic exposure

significantly decreased (p<0.001) sperm parameters (count, motility, and viabil-

ity), serum hormone levels (follicle-stimulating hormone, luteinizing hormone, and

testosterone), and mitochondrial enzyme activity. Rutin (50 and 100 mg/kg) sig-

nificantly (p<0.01 and p<0.001) attenuated arsenic-induced alterations in a dose-

dependent manner, improving organ weight, sperm parameters, and hormone lev-

els. Rutin also improved mitochondrial complex activity and testicular architecture.

In contrast, elevated oxidative stress (reduced glutathione, superoxide dismutase,

nitric oxide, and lipid peroxidation), inflammation (tumor necrosis factor-alpha,

Protective effects of rutin against arsenic-induced testicular damage 409

Vol. 66(4): 408 - 425, 2025

Rutina, un agente terapéutico potencial para el daño testicular

inducido por arsénico: Efecto antioxidante, antinflamatorio,

y anti-apoptótico.

Invest Clin 2025; 66 (4): 408 – 425

Palabras clave: Apoptosis; Arsénico; Inflamación; Rutina; Espermatogénesis; Toxicidad

Testicular.

Resumen. La exposición al arsénico se asocia con toxicidad reproductiva mas-

culina que incluye alteración de la espermatogénesis, reducción de la calidad del

esperma y alteración de los niveles hormonales. La rutina, un flavonoide natural, ha

demostrado efectos protectores en varios órganos debido a sus propiedades antio-

xidantes, antiinflamatorias y antiapoptóticas. El propósito del trabajo fue evaluar la

eficacia de la rutina para reducir la toxicidad testicular causada por el arsenito de

sodio en ratas de laboratorio. Ratas (Sprague-Dawley) fueron asignadas aleatoria-

mente a grupos de tratamiento normal, control de arsénico, rutina (25, 50 y 100

mg/kg) y coenzima Q10 (10 mg / kg). El daño testicular se indujo mediante la ad-

ministración oral de arsenito de sodio (10 mg/kg, 2 días). La rutina y la coenzima

Q10 se administraron por vía oral durante 15 y 7 días, respectivamente. Se evalua-

ron los niveles séricos de hormonas, los parámetros de los espermatozoides, las enzi-

mas mitocondriales testiculares, los marcadores de estrés oxidativo, las citocinas in-

flamatorias, las proteínas apoptóticas y la histopatología. La exposición al arsénico

disminuyó significativamente (p<0,001) los parámetros espermáticos (recuento,

motilidad y viabilidad), y los niveles séricos de hormonas (hormona estimulante del

folículo, hormona luteinizante y testosterona) y la actividad de las enzimas mitocon-

driales. La rutina (50 y 100 mg/kg) atenuó significativamente (p<0,01 y p<0,001)

las alteraciones inducidas por el arsénico de manera dosis-dependiente, mejorando

el peso de los órganos, los parámetros espermáticos y los niveles hormonales. La

rutina también mejoró la actividad del complejo mitocondrial y la arquitectura tes-

ticular, mientras que el estrés oxidativo elevado (glutatión reducido y superóxido

dismutasa, óxido nítrico y peroxidación lipídica), la inflamación (factor de necrosis

tumoral alfa, interleucina-6 e interleucina-1β) y la apoptosis (expresión de proteínas

caspasa-3 y caspasa-9) mejoraron con la rutina. En conclusión, la rutina demostró

efectos protectores significativos contra la toxicidad testicular inducida por el ar-

senito de sodio en ratas al reducir el estrés oxidativo, la inflamación y la apoptosis.

Estos hallazgos sugieren que la rutina tiene potencial terapéutico para mitigar la

toxicidad reproductiva asociada al arsénico.

Received: 12-08-2025 Accepted: 22-09-2025

interleukin-6, and interleukin-1β), and apoptosis (caspase-3 and caspase-9 protein

expression) were ameliorated by rutin. In conclusion, rutin demonstrated signifi-

cant protective effects against sodium arsenite-induced testicular toxicity in rats by

reducing oxidative stress, inflammation, and apoptosis. These findings suggest that

rutin has therapeutic potential in mitigating arsenic-related reproductive toxicity.

410 Wang et al.

Investigación Clínica 66(4): 2025

INTRODUCTION

Arsenic is a metalloid commonly found

in the environment, known for its toxicity

and important public health effects 1,2. It

mainly impacts populations through con-

taminated drinking water, often from natural

sources and human activities such as indus-

trial discharge and the use of arsenic-based

pesticides 3,4. According to the World Health

Organization (WHO), more than 140 million

people worldwide are exposed to arsenic-

contaminated water exceeding the safe limit

of 10 µg/L 5. Furthermore, long-term expo-

sure to arsenic has been associated with nu-

merous health problems, including cancer

and various non-cancerous conditions like

cardiovascular diseases, diabetes, and repro-

ductive issues 6,7. Sodium arsenite is recog-

nized for causing testicular toxicity through

mechanisms such as oxidative stress, apop-

tosis, and inflammation. Researchers believe

that inflammation, oxidative/nitrosative

stress, and apoptosis are key factors in arse-

nic-induced testicular damage 6,8.

The impact of arsenic toxicity on re-

productive health has become an increas-

ing concern, particularly regarding the male

reproductive system 9. Arsenic exposure is

linked to various reproductive problems,

including male infertility, decreased sperm

quality, and disruptions in testosterone pro-

duction. This results in reduced weights of

reproductive organs, more sperm abnormali-

ties, and apoptosis in testicular cells 10. The

harmful effects of arsenic on male fertility

involve several mechanisms, such as inter-

ference with steroidogenesis and mitochon-

drial dysfunction in reproductive tissues 9.

These disruptions lead to lower testoster-

one levels, decreased sperm count, and ab-

normal sperm morphology 11. Additionally,

changes in spermatogenesis and reduced

gonadotropin levels have been observed, fur-

ther emphasizing the reproductive risks of

arsenic 3. Moreover, arsenic exposure causes

increased production of reactive oxygen spe-

cies (ROS), indicating the role of oxidative

stress in its genotoxic effects 12. Research-

ers have documented that testicular toxicity

caused by sodium arsenite is partly due to its

ability to induce apoptosis through the p53

pathway, as shown in zebrafish studies where

sodium arsenite exposure elevated apoptosis

markers and decreased global DNA methyla-

tion 13. Furthermore, experiments with Cae-

norhabditis elegans demonstrated that sodi-

um arsenite can cause cell cycle arrest and

germline apoptosis, both of which depend on

concentration and exposure time 12.

Several protective agents have been stud-

ied to reduce testicular toxicity caused by so-

dium arsenite. Melatonin has been shown to

decrease arsenic-induced testicular cell death,

oxidative stress, and tissue damage by boost-

ing antioxidant enzyme activity and lowering

lipid peroxidation 14. Similarly, Salvia hispani-

ca (chia seeds) proved effective in decreasing

testicular toxicity by improving sperm quality,

serum sex hormone levels, and antioxidant en-

zyme activity, thanks to its flavonoid content

and antioxidant properties 15. Hesperidin and

lipoic acid were also tested for their protec-

tive effects against liver, kidney, and testicular

toxicity when administered with sodium arse-

nite16. Rutin is a natural flavonoid with a wide

range of pharmacological benefits, including

antioxidant, antimicrobial, antifungal, anti-

allergic, anti-cancer, anti-inflammatory, and

antiapoptotic effects 4,17-22. Additionally, rutin

has been shown to help manage neurodegen-

erative diseases and metabolic conditions like

diabetes due to its cell-protective effects 19. Its

protective properties have been documented

in the liver, kidneys, and heart due to its anti-

inflammatory, antioxidant, and antiapoptotic

actions, making it a candidate for protecting

these organs from toxic agents 4. However, the

impact of rutin on testicular toxicity caused

by sodium arsenite has not yet been studied.

Given its protective effects on other organs, ru-

tin might provide similar benefits in reducing

testicular damage from sodium arsenite. This

study examined the effectiveness of rutin in al-

leviating testicular toxicity caused by sodium

arsenite in male rats.

Protective effects of rutin against arsenic-induced testicular damage 411

Vol. 66(4): 408 - 425, 2025

MATERIALS AND METHODS

Experimental design

Rats (male, Sprague-Dawley, weighing

200-220 g, sourced from the animal house

of Shaanxi Kangfu Hospital, China) were

randomly divided into the following groups

(n=6 per group): normal, arsenic control

(As), rutin (Sigma Chemical Co., St Louis,

Missouri, United States; 25, 50, and 100

mg/kg), and Co-enzyme Q10 (Medicines Pvt.

Ltd. Mumbai, India; 10 mg/kg). Testicular

damage was induced in rats (except the nor-

mal group) by oral administration of sodium

arsenite (Otto Chemicals, India; 10 mg/kg,

for two days) 23,24. Rats in the arsenic control

and normal groups were treated with double-

distilled water (10 mL/kg). Three different

dosages of rutin (25, 50, and 100 mg/kg)

were selected based on previous studies 21,22

and were administered orally for 15 days. Co-

enzyme Q10 was administered for seven con-

secutive days before arsenite administration

and continued for up to 15 days. On the 16th

day, the rats were put under anesthesia us-

ing ether, and blood was drawn through ret-

ro-orbital puncture. Each blood sample was

placed in a separate vial for analysis of se-

rum parameters. Serum follicle-stimulating

hormone, luteinizing hormone, and testos-

terone levels were quantified according to

the manufacturer’s instructions for the rat-

specific ELISA kit (Thermo Scientific, Rock-

ford, IL, USA). The rats were euthanized by

carbon dioxide asphyxiation, and the testes

were rapidly removed and stored at -80°C for

biochemical parameters. Other organs, such

as the epididymis and prostate, were isolated

and weighed. The epididymal sperm count

and motility were determined according to a

previously reported method 25.

Biochemical estimation of testis

homogenate

Supernatant of the tissue homogenate

was employed to estimate lipid peroxidation

[malondialdehyde (MDA) content], nitric

oxide (NO content), reduced glutathione

(GSH), and superoxide dismutase (SOD)]

as described previously 26-29. Testicular mito-

chondrial enzyme activities, including Com-

plex I (nicotinamide-adenine dinucleotide

(NADH) dehydrogenase activity), Complex

II (succinate dehydrogenase (SDH) activity),

Complex III (mitochondrial redox activity),

and Complex IV (cytochrome oxidase assay)

were estimated according to previously re-

ported methods 30,31.

Testicular interleukins (interleukins

(ILs); IL-6 (ERA31RB) and IL-1β (BMS630))

and tumor necrosis factor-alpha (TNF-α;

ERA56RB) were quantified in the testis ho-

mogenate using Enzyme-linked immunosor-

bent assay (ELISA) kits (Thermo Scientific,

Rockford, IL, USA). Briefly, 500 mg of testis

tissue samples were homogenized with a me-

chanical homogenizer in 5 ml of phosphate-

buffered saline at 3000 rpm. The homoge-

nate was centrifuged for 30 min at 20,000

rpm (4°C) in a cryo centrifuge (Eppendorf),

and the supernatant was used to determine

ILs and TNF-α. Briefly, the quantification of

ILs and TNF-α was performed using the Ther-

mo Scientific Rat ILs and TNF-α immunoas-

say kit, following the instructions provided.

The Rat ILs and TNF-α immunoassay was a

4.5 h solid phase designed to measure rat

ILs and TNF-α levels. The assay employed a

sandwich enzyme immunoassay principle. A

monoclonal antibody specific for rat ILs and

TNF-α was pre-coated on the microplates.

Standards, control, and samples were pipet-

ted into the wells, and the immobilized an-

tibody thus bound any rat ILs or TNF-α pres-

ent in the sample. After washing away the

unbound substance, an enzyme-linked poly-

clonal antibody specific for rat ILs or TNF-α

was pipetted into the microtitre wells. Any

unbound antibody was washed off, and then

a substrate solution was added to the wells.

The enzymatic reaction produced a blue

product that turned yellow upon addition of

the stop solution. The intensity of the gener-

ated color was measured and was proportion-

al to the amount of rat ILs or TNF-α bound

in the initial steps. A standard curve was run

412 Wang et al.

Investigación Clínica 66(4): 2025

on each assay plate using recombinant ILs or

TNF-α in serial dilutions. The sample values

were then read and calculations made ac-

cording to the standard curve. Values were

expressed as means ± S.E.M. The levels of

ILs or TNF-α were expressed as units per mg

of gastric tissue.

The protein expression of caspase-3 and

caspase-9 was assessed by western blot in the

testis 32-34. Briefly, the testis was sonicated in

Tissue Protein Extraction Reagent (Thermo

Fisher Scientific, Inc., Mumbai, Maharash-

tra, India). The lysates were centrifuged at

10,000 × g for 10 min at 4°C. Protein con-

centration was determined using a Bicin-

choninic Acid (BCA) assay kit (Beyotime

Shanghai, China) on ice for 30 min. Equal

amounts of extracted protein samples (50

µg) were separated by 10% SDS-PAGE (sodi-

um dodecyl sulfate-polyacrylamide gel elec-

trophoresis) and transferred onto polyvinyli-

dene difluoride membranes. The membranes

were blocked with 5% non-fat dry milk at

37°C for 1 hr and incubated overnight at 4°C

with the primary antibodies recognizing cas-

pase-3 (ab4051; 1/200 dilution; 31 kDa) and

caspase-9 (ab202068; 1/2000 dilution; 46

kDa; Abcam, Cambridge, MA, USA). In ad-

dition, an anti-rabbit horseradish-linked sec-

ondary antibody (goat anti-rabbit IgG H&L;

ab97051) was used, which was incubated at

37°C for 2 hr. Protein bands were visualized

using the Chemiluminescent kit (Bio-Rad

Laboratories, Inc., Mumbai, Maharashtra,

India), and glyceraldehyde 3-phosphate de-

hydrogenase (GAPDH; EPR6256, ab128915;

1/10000 dilution; 36 kDa) served as the

loading control.

Histopathological analysis of testis

Other testis samples were preserved in

10% formalin for 24 hours. These samples

underwent dehydration and were immersed

in xylene for one hour, repeated three times,

followed by treatment with ethyl alcohol at

concentrations of 70%, 90%, and 100% for

two hours. The infiltration and impregnation

process involved treating the samples with

paraffin wax twice, with each session lasting

1 hour. The tissue samples were then sliced

into sections with a thickness of 3-5µm and

stained using hematoxylin and eosin (H&E).

The sections were mounted on slides with

Distrene Pthalate Xylene (DPX) serving as

the mounting medium. A light microscope

was used to examine the sections for histo-

pathological characteristics and cell infiltra-

tion. The observed changes in histological

features were categorized into grades rang-

ing from 0 to 4 according to a previously re-

ported method 35.

Statistical analysis

The data are presented as mean ±

standard error of the mean (SEM). Graph-

Pad Prism 5.0 software (GraphPad, San Di-

ego, CA, USA) was utilized for data analysis.

Biochemical parameter data were examined

using one-way analysis of variance, followed

by Tukey’s multiple range test for paramet-

ric results, while the Kruskal-Wallis test was

used for non-parametric outcomes. A p of

less than 0.05 was deemed statistically sig-

nificant. Correlation coefficients were calcu-

lated using a two-sided Fisher’s test.

RESULTS

Attenuation of arsenic-induced alteration

in body weight and organ weights by rutin

Table 1 presents the descriptive results

for various body and organ weight parame-

ters across the different treatment groups.

The arsenic (As) control group had a signifi-

cant reduction (p<0.001) in organ weights

(testes, epididymis, and prostate) and body

weight compared with the normal group.

CoQ treatment resulted in a significant im-

provement (p<0.001) in body weight and

organ weights compared to the arsenic con-

trol group. Rutin (50 and 100 mg/kg) ad-

ministration also effectively (p<0.01 and

p<0.001) and dose-dependently increased

the body, testes, epididymis, and prostate

weights relative to the arsenic-exposed

group.

Protective effects of rutin against arsenic-induced testicular damage 413

Vol. 66(4): 408 - 425, 2025

Table 1. Attenuation of arsenic-induced alteration in body weight, organ weights (testes, epididymis, and prostate),

and sperm parameters (count, motility, and viability) by rutin.

Parameters Normal As Control CoQ (10) R (25) R (50) R (100)

Body weight (gm) 281.8 ± 2.86 217.50 ± 3.73### 275.70 ± 2.69*** 214.70 ± 2.80 247.00 ± 4.02** 262.20 ± 2.77***

Testes weight (gm) 1.79 ± 0.04 1.03 ± 0.07### 1.59 ± 0.04*** 1.07 ± 0.05 1.51 ± 0.06** 1.68 ± 0.02***

Testes weight/Body weight (×10-3) 6.34 ± 0.15 4.75 ± 0.39### 5.76 ± 0.18*** 4.99 ± 0.21 6.13 ± 0.33** 6.41 ± 0.12***

Epididymis weight (gm) 0.51 ± 0.01 0.19 ± 0.02### 0.45 ± 0.02*** 0.23 ± 0.01 0.33 ± 0.01** 0.45 ± 0.01***

Epididymis weight/Body weight (×10-3) 1.80 ± 0.03 0.87 ± 0.07### 1.62 ± 0.07*** 1.09 ± 0.07 1.32 ± 0.04** 1.73 ± 0.04***

Prostate weight (mg) 612.30 ± 10.94 308.30 ± 9.78### 560.70 ± 9.02*** 298.70 ± 11.44 478.30 ± 11.78** 541.70 ± 11.45***

Prostate weight/Body weight (×10-3) 2.18 ± 0.04 1.42 ± 0.05### 2.03 ± 0.04*** 1.39 ± 0.04 1.94 ± 0.07** 2.07 ± 0.05***

Sperm count (millions/mL) 60.17 ± 1.25 34.00 ± 0.97### 56.83 ± 1.30*** 33.83 ± 0.75 45.00 ± 1.21** 51.83 ± 1.25***

Sperm motility (%) 71.33 ± 1.05 40.33 ± 1.02### 62.00 ± 1.00*** 44.33 ± 0.95 49.17 ± 1.49** 59.50 ± 0.67***

Dead sperm (%) 28.67 ± 1.05 59.67 ± 1.02### 38.00 ± 1.00*** 55.67 ± 0.95 50.83 ± 1.49** 40.50 ± 0.67***

Abnormal sperm (%) 12.17 ± 0.60 36.50 ± 0.43### 17.17 ± 0.54*** 34.17 ± 0.48 24.67 ± 0.42** 19.33 ± 0.76***

Data analysis: One-way ANOVA (post-hoc test: Tukey’s multiple range test). Data are reported as mean ± SEM (n=6). Statistically significant compared with

###normal rats, ** and ***as control rats. ###p < 0.001, **p < 0.01 and ***p < 0.001. Arsenic (As), Co-enzyme Q10 (CoQ (10)) and Rutin (R).

Table 2. Attenuation of arsenic-induced alteration in serum luteinizing hormone, follicle stimulating hormone,

testosterone levels and testicular mitochondrial complex activities by rutin.

Parameters Normal As Control CoQ (10) R (25) R (50) R (100)

Serum parameters

Luteinizing hormone (ng/mL) 1.88 ± 0.04 0.26 ± 0.05### 0.33 ± 0.03 0.36 ± 0.04 0.71 ± 0.05** 1.21 ± 0.04***

Follicle stimulating hormone (ng/mL) 1.37 ± 0.02 0.16 ± 0.02### 0.15 ± 0.02 0.26 ± 0.02 0.64 ± 0.02** 0.92 ± 0.02***

Testosterone (ng/mL) 3.69 ± 0.04 1.41 ± 0.05### 1.32 ± 0.06 1.5 ± 0.06 2.39 ± 0.04** 2.86 ± 0.05***

Testicular parameters

Complex I (nmole of NADH oxidized/

min/mg protein) 33.36 ± 1.66 7.41 ± 1.80### 30.21 ± 1.79*** 10.07 ± 1.35 16.91 ± 1.25** 28.31 ± 2.10***

Complex II (mmole/mg protein) 14.35 ± 0.82 4.38 ± 0.78### 11.86 ± 0.84*** 4.74 ± 0.62 9.42 ± 0.67** 11.24 ± 0.58***

MTT assay (OD at 540 nm) 0.45 ± 0.03 0.17 ± 0.03### 0.45 ± 0.03*** 0.21 ± 0.02 0.32 ± 0.01** 0.39 ± 0.02***

Complex-IV (nmol cyto-C oxidized/

min/mg protein) 6319.00 ± 280.30 1008.00 ± 212.70### 5293.00 ± 254.70*** 1586.00 ± 191.90 3224.00 ± 201.30** 4515.00 ± 342.00***

Data analysis: One-way ANOVA (post-hoc test: Tukey’s multiple range test). Data are reported as mean ± SEM (n=6). Statistically significant compared with

###normal rats, ** and ***as control rats. ###p < 0.001, **p < 0.01 and ***p < 0.001. Arsenic (As), Co-enzyme Q10 (CoQ (10)), 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) and Rutin (R).

414 Wang et al.

Investigación Clínica 66(4): 2025

Attenuation of arsenic-induced alteration

in sperm parameters by rutin

The arsenic control group showed a

significant reduction (p<0.001) in cell

count and motility compared to the con-

trol group. However, the percentage of dead

and abnormal sperm was markedly higher

(p<0.001) in the arsenic control group

than that in the normal group. Treatment

with CoQ resulted in significant improve-

ments (p<0.001) in sperm count and mo-

tility compared with the arsenic control

group. Furthermore, CoQ administration

resulted in a more effective (p<0.001) re-

covery of the percentage of dead and abnor-

mal sperm than arsenic control. Rutin (50

and 100 mg/kg) treatment demonstrated a

marked (p<0.01 and p<0.001) and dose-

dependent improvement in sperm param-

eters relative to the arsenic-exposed group

(Table 1).

Attenuation of arsenic-induced alteration

in serum follicle-stimulating hormone,

luteinizing hormone, and testosterone

levels by rutin

Exposure to sodium arsenite significant-

ly decreased (p<0.001) the levels of serum

follicle-stimulating hormone, luteinizing hor-

mone, and testosterone in the arsenic con-

trol group compared with the normal group.

Treatment with CoQ and rutin (25 mg/kg)

had a minimal impact on serum luteinizing

hormone, follicle-stimulating hormone, and

testosterone hormone levels. However, rutin

(50 and 100 mg/kg) treatments demonstrat-

ed effective, dose-dependent increases in all

three hormones (p<0.01 and p<0.001, re-

spectively) compared with the arsenic control

group (Table 2).

Attenuation of arsenic-induced alteration

in testicular mitochondrial complex

activities by rutin

Arsenic exposure led to a significant

reduction (p<0.001) in mitochondrial com-

plex activity compared to the normal group.

The results presented in Table 2 demonstrate

that both CoQ and rutin (50 and 100 mg/

kg) significantly improved mitochondrial

function in arsenic-exposed rats. Specifical-

ly, rutin (50 and 100 mg/kg) administration

led to notable (p<0.01 and p<0.001) and

dose-dependent increases in mitochondrial

complex activities compared with the arse-

nic control group. However, rutin (25 mg/

kg) did not show any significant potential to

improve mitochondrial function or mitigate

arsenic-induced damage.

Attenuation of arsenic-induced alteration

in testicular oxido-nitrosative stress

by rutin

The results presented in Table 3 show

the effects of rutin treatment on testicular

oxido-nitrosative stress. Arsenic exposure

caused a substantial (p<0.001) decrease in

GSH and SOD levels and a marked (p<0.001)

elevation in nitric oxide and MDA levels in

the arsenic control group compared to the

normal group. CoQ treatment significantly

(p<0.001) improved GSH and SOD levels

and effectively reduced (p<0.001) nitric ox-

ide and MDA levels compared with the arse-

nic control group. Rutin (25 mg/kg) treat-

ment showed minimal effects in attenuating

arsenic-induced elevated testicular oxido-

nitrosative stress compared with the arsenic

control group. However, rutin (50 and 100

mg/kg) demonstrated marked (p<0.01 and

p<0.001) and dose-dependent protective ef-

fects, reflected by restored antioxidant sta-

tus (GSH and SOD levels) and reduced oxi-

dative stress markers (nitric oxide and MDA)

to normal levels (Table 3).

Attenuation of arsenic-induced alteration

in testicular inflammatory markers

activities by rutin

The results in Table 3 show significant

differences in inflammatory cytokine lev-

els between the normal and arsenic control

groups. The arsenic control group exhibited

markedly elevated (p<0.001) levels of ILs

(IL-6 and IL-1β) and TNF-α compared with

Protective effects of rutin against arsenic-induced testicular damage 415

Vol. 66(4): 408 - 425, 2025

the normal group. CoQ treatment substan-

tially (p<0.001) lowered ILs (IL-6 and IL-

1β) and TNF-α levels compared to the arse-

nic control group. Rutin (50 and 100 mg/

kg) administration resulted in effective

(p<0.01 and p<0.001) and dose-dependent

reductions in cytokine levels compared with

the arsenic control group. However, a lower

dose of rutin (25 mg/kg) did not reduce cy-

tokine levels to levels comparable to those of

the arsenic control group, indicating failure

to ameliorate arsenic-induced inflammation

(Table 3).

Attenuation of arsenic-induced alteration

in testicular caspase-3 and caspase-9

protein expressions by rutin

Fig. 1 illustrates the impact of arsenic

exposure on markers of apoptosis in the tes-

tes, along with their correlation with sperm

count. The caspase-3 and caspase-9 pro-

tein expression was significantly increased

(p<0.001) in the arsenic control group

compared with that in the normal group.

The substantial elevation in caspase-3 and

caspase-9 protein expression was markedly

downregulated (p<0.001) by CoQ treat-

ment compared to the arsenic control

group. Treatment with rutin (50 and 100

mg/kg) significantly (p<0.01 and p<0.001)

and dose-dependently reduced both cas-

pase-3 and caspase-9 relative densities com-

pared to the arsenic control (Figs. 1A and

1B). Furthermore, Figs. 1C and 1D reveal

a strong inverse correlation between sperm

count and the relative densities of caspase-3

(R² = 0.8168, p<0.001) and Caspase-9 (R²

= 0.7075, p<0.01), respectively, indicating

that lower sperm counts are associated with

increased apoptotic activity.

Attenuation of arsenic-induced alteration

in testicular histopathology by rutin

Fig. 2 illustrates the histopathologi-

cal changes in rat testicular tissue after

exposure to arsenic and their ameliora-

tion across different treatment groups. In

the normal group (Fig. 2A), testicular ar-

chitecture appeared normal, with intact

seminiferous tubules containing organized

spermatogenic cells and healthy Leydig

cells, and no evidence of necrosis. However,

it showed mild inflammation (indicated by

black arrows). In contrast, the arsenic con-

Table 3. Attenuation of arsenic-induced alteration in testicular oxido-nitrosative stress

and inflammatory markers by rutin.

Parameters Normal As Control CoQ (10) R (25) R (50) R (100)

SOD (U/mg

of protein) 13.20 ± 1.10 5.19 ± 0.93### 10.66 ± 0.87*** 5.45 ± 0.61 8.91 ± 0.93** 10.66 ± 0.96***

GSH (µg/mg

of protein) 13.76 ± 0.64 3.90 ± 0.62### 11.04 ± 0.59*** 5.36 ± 0.50 8.31 ± 0.76** 10.43 ± 0.56***

MDA (nM/mg

of protein) 0.46 ± 0.10 3.14 ± 0.18### 1.97 ± 0.18*** 3.00 ± 0.27 2.11 ± 0.04** 1.59 ± 0.19***

NO (µg/mg

of protein) 119.00 ± 9.08 284.00 ± 13.96### 143.90 ± 9.97*** 261.40 ± 13.64 222.10 ± 12.14** 167.90 ± 9.82***

TNF-α (pg/mL) 157.00 ± 8.15 409.70 ± 12.69### 222.80 ± 14.07*** 377.20 ± 14.06 330.30 ± 8.02** 253.70 ± 12.06***

IL-1β (pg/mL) 17.28 ± 3.86 72.12 ± 1.85### 30.57 ± 2.76*** 68.46 ± 1.62 48.95 ± 2.70** 42.78 ± 2.41***

IL-6 (pg/mL) 40.50 ± 5.53 152.60 ± 5.18### 59.41 ± 9.32*** 155.50 ± 6.21 130.40 ± 7.87** 71.78 ± 7.13***

Data analysis: One-way ANOVA (post-hoc test: Tukey’s multiple range test). Data are reported as mean ± SEM

(n=6). Statistically significant compared with ###normal rats, ** and ***As control rats. ###p < 0.001, **p < 0.01

and ***p < 0.001. Arsenic (As), Co-enzyme Q10 (CoQ (10)), Glutathione (GSH), Interleukin-1 beta (IL-1β), Inter-

leukin-6 (IL-6), Malondialdehyde (MDA), Nitric Oxide (NO), Rutin (R), Superoxide Dismutase (SOD), and Tumor

Necrosis Factor-alpha (TNF-α).

416 Wang et al.

Investigación Clínica 66(4): 2025

trol group (Fig. 2B) demonstrated histo-

logical aberrations characterized by severe

inflammatory infiltration (black arrows),

necrosis (red arrows), vacuolated spermato-

genic cells (blue arrows), and Leydig cell

damage, indicating arsenic-induced toxic-

ity. The quantitative histopathological score

in Fig. 2G supports these visual observa-

tions, as the arsenic control group exhibited

significantly (p<0.001) elevated scores for

inflammatory infiltration, necrosis, Leydig

cell damage, and vacuolated spermatogenic

cells than the normal group. Treatment with

CoQ significantly reduced (p<0.001) these

histopathological aberrations compared to

the arsenic control group (Fig. 2C), sug-

gesting a protective effect against arsenic-

induced testicular injury. The rutin (25 mg/

kg) group showed a modest reduction in

these scores, but this was not significantly

Fig. 1. Attenuation of arsenic-induced alterations in testicular caspase-3 (A) and caspase-9 (B) protein ex-

pression by rutin. Correlation of sperm count with caspase-3 (C) and caspase-9 (D) protein expres-

sion. Data analysis: One-way ANOVA (post-hoc test: Tukey’s multiple range test). Data are reported as

mean ± SEM (n=6). Statistically significant compared with ###normal rats, ** and ***As control rats.

### p < 0.001, **p<0.01 and ***p<0.001. Correlation coefficients were determined using a two-sided

Fisher’s test. Arsenic (As), Co-enzyme Q10 (CoQ (10)), Glyceraldehyde-3-Phosphate Dehydrogenase

(GAPDH), and Rutin (R). Inflammatory infiltration (black arrow), necrosis (red arrow), and Leydig

cells damage (blue arrow).

Protective effects of rutin against arsenic-induced testicular damage 417

Vol. 66(4): 408 - 425, 2025

Fig. 2. Attenuation of arsenic-induced alterations in testicular histopathology by rutin treatment. Repre-

sentative images of testes from each group (A-F). Quantitative analysis of arsenic-induced alterations

in testicular histopathology and its attenuation by rutin (G). Data analysis: One-way ANOVA (post-hoc

test: Kruskal–Wallis test). Data are reported as mean ± SEM (n=6). Statistically significant compared

with ###normal rats, ** and ***As control rats. ###p < 0.001, **p < 0.01 and ***p < 0.001. Arsenic

(As), Co-enzyme Q10 (CoQ (10)), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), and Rutin (R).

418 Wang et al.

Investigación Clínica 66(4): 2025

different from the higher rutin or CoQ doses

(Fig. 2D). Treatment with rutin (50 and 100

mg/kg) showed a significant (p<0.01 and

p<0.001) reduction in arsenic-induced his-

tological aberrations in the testes, reflected

in a decrease in inflammatory infiltration,

necrosis, Leydig cell damage, and vacuolat-

ed spermatogenic cells compared to the ar-

senic control group (Figs 2E, 2F, and 2G).

DISCUSSION

Arsenic exposure has extensive human

health effects, with systemic toxicity affect-

ing multiple organs. It is particularly notori-

ous for its carcinogenic properties, causing

skin, lung, and bladder cancers, among oth-

ers 6. However, it is crucial to evaluate its ad-

verse effects on male reproductive health as

it directly affects male fertility. Arsenic expo-

sure impairs spermatogenesis and reduces

sperm quality through oxidative stress and

interference with crucial hormonal signaling

pathways 11. Researchers have explored the

protective efficacy of phytonutrients, such

as lutein and α-lipoic acid, against arsenic-

induced oxidative damage in testicular tis-

sues 36. In the present study, rutin, an herbal

intervention, was evaluated for its effects on

arsenite-induced testicular damage, and the

findings suggested that rutin ameliorated

testicular toxicity through its anti-apoptot-

ic, anti-inflammatory, and mitochondrial-

protective effects in experimental rats.

Integrative proteomic and metabolo-

mic analyses have shown that arsenic ex-

posure significantly alters the proteome

and metabolome in rat testes, affecting

spermatogenesis and fertilization through

disrupted signaling pathways 37. Sodium ar-

senite causes significant testicular damage

primarily through oxidative stress, apop-

tosis, and inflammatory pathways. Studies

have demonstrated that chronic exposure

to sodium arsenite can lead to a significant

decrease in both absolute and relative tes-

ticular weight 38. This reduction in testicu-

lar weight is attributed to the toxic effects

of arsenic on testicular tissue and its inter-

ference with normal spermatogenesis and

enzyme activities, including decreased acid

phosphatase, sorbitol dehydrogenase, and

17beta-hydroxy-steroid dehydrogenase activ-

ities, while increasing lactate dehydrogenase

and gamma-glutamyl transpeptidase activi-

ties. Additionally, arsenite exposure leads to

a notable increase in abnormal sperm forms,

along with a decrease in sperm count and

motility 38. Furthermore, arsenic is known to

accumulate significantly in reproductive tis-

sues, underscoring its potential to cause pro-

longed toxic effects 38. This accumulation in

the testes, epididymis, and prostate is linked

to oxidative stress and histopathological al-

terations, indicating that arsenic’s effects

are more profound at the cellular level 8,14.

In the present study, administration of ru-

tin significantly attenuated arsenite-induced

decreases in testes, epididymis, and prostate

weights, suggesting its protective efficacy in

reproductive organs.

Sodium arsenite led to decreased levels

of serum testosterone, luteinizing hormone,

and follicle-stimulating hormone, and to

significant changes in sperm parameters,

including reduced sperm count and motil-

ity, and an increase in abnormal sperm15.

Serum hormone levels, such as testosterone,

FSH, and LH, play crucial roles in regulating

male reproductive function 39. Testosterone,

which is primarily produced in the testes,

is vital for normal male reproductive func-

tions and secondary sexual characteristics.

FSH and LH, secreted by the pituitary gland,

regulate testicular function, including ste-

roidogenesis and spermatogenesis 40. Chron-

ic exposure to arsenite is often associated

with reduced testosterone levels owing to

impaired testicular function. This can be at-

tributed to the direct cytotoxic effects of ar-

senite on Leydig cells, which are responsible

for testosterone production. Additionally, al-

terations in hormone levels can disrupt the

hypothalamic-pituitary-gonadal axis, leading

to compensatory changes in LH and FSH lev-

els. In this study, arsenite-induced testicular

Protective effects of rutin against arsenic-induced testicular damage 419

Vol. 66(4): 408 - 425, 2025

damage was reflected in changes in serum

testosterone, FSH, and LH levels. However,

administration of rutin restored the dimin-

ished levels of these hormones in the serum.

Oxidative stress plays a crucial role in

arsenic-induced toxicity, impacting various

biological systems and leading to significant

health issues. Arsenic, a toxic metalloid, can

cross cellular barriers and accumulate in tis-

sues, contributing to oxidative stress by gen-

erating ROS 41,42. ROS generation is central

to the pathogenesis of arsenic toxicity, as it

leads to mitochondrial dysfunction, a criti-

cal factor in neurotoxicity, and other health

issues 43,44. Arsenic-induced oxidative stress

leads to mitochondrial damage by disrupting

the normal balance between antioxidants

and pro-oxidants within cells. This disruption

impairs mitochondrial membrane potential,

promotes cytochrome c release, and triggers

apoptosis through caspase activation42. On

a systemic level, oxidative stress also causes

chromosomal instability and DNA damage,

leading to further complications, such as car-

cinogenesis and neurological disorders44-47.

Moreover, arsenic exposure has been shown

to deplete antioxidant capacities and elevate

oxidative damage biomarkers, such as malo-

ndialdehyde (MDA) and nitric oxide, owing

to the heightened oxidative environment48,49.

These changes underline the oxidative stress

mechanism as a cornerstone of arsenic tox-

icity, which affects various systems, includ-

ing the reproductive system 50. Therapeutic

strategies to combat arsenic-induced oxida-

tive stress focus on enhancing mitochondrial

function and increasing antioxidant defense.

Acetyl-L-carnitine (ALC), for instance, has

been shown to counteract arsenic-induced

oxidative stress by improving antioxidant

mechanisms and mitochondrial function,

thus offering a potential therapeutic pathway

42. Other antioxidants, such as curcumin and

apigenin, also exhibit protective effects by

reducing ROS and supporting cellular anti-

oxidant systems, highlighting their possible

roles in mitigating arsenic toxicity51,52. In the

present investigation, arsenic-induced toxic-

ity, associated with elevated oxidative stress,

contributed to various cellular and systemic

damage, as reflected by diminished testicu-

lar mitochondrial enzyme activity. However,

rutin treatment effectively increased tes-

ticular glutathione and superoxide levels

and reduced malondialdehyde and nitric ox-

ide levels, suggesting its protective effects

against arsenic-induced mitochondrial dys-

function, potentially by mitigating oxidative

stress and enhancing mitochondrial energy

production.

Inflammation is a significant contribu-

tor to arsenic-induced testicular toxicity.

Pro-inflammatory cytokines, such as TNF-α

and interleukins, mediate this process.

TNF-α induces apoptosis and promotes in-

flammatory responses 53. Inflammation

induced by TNF-α can activate various sig-

naling pathways, culminating in oxidative

stress and tissue damage 54-56. It has been ob-

served that arsenic exposure results in the

upregulation of inflammatory mediators,

such as TNF-α and interleukins, contribut-

ing to testicular damage 57. Furthermore,

Caspases 3 and 9 play crucial roles in the

apoptotic pathways underlying arsenic-in-

duced testicular toxicity. Caspase-9 is part

of the intrinsic or mitochondrial apoptosis

pathway, which is often activated by cellular

stressors, including toxins such as arsenic.

It is responsible for activating downstream

effector caspases, including caspase-3 58,59.

Caspase-3 then executes apoptosis by cleav-

ing cellular substrates, leading to system-

atic breakdown of cellular components and

cell death 60-62. In arsenic-induced testicu-

lar toxicity, activation of these caspases is

indicative of enhanced apoptotic activity,

contributing to the observed tissue damage

and dysfunction 57. In the current study,

arsenic-induced elevated inflammation and

apoptosis, mediated by cytokines such as

TNF-α and enzymes such as caspases 3 and

9, highlighted the development of arsenic-

induced testicular toxicity. However, rutin

treatment mitigated the toxic effects in-

duced by chronic exposure to arsenic via

420 Wang et al.

Investigación Clínica 66(4): 2025

the amelioration of these pathways, sug-

gesting its therapeutic potential against

arsenic-induced testicular toxicity.

This study has some limitations. First,

the study focused on acute arsenic exposure

(two consecutive days), which may not re-

flect the effects of chronic, long-term expo-

sure often observed in human populations.

Second, the study only used sodium arsenite,

whereas environmental arsenic exposure of-

ten involves multiple arsenic species. Third,

this study did not address the bioavailabil-

ity and metabolism of rutin in rats, which

could affect its efficacy in humans. Lastly,

the study focused on rutin alone and did not

explore its potential synergistic effects with

other protective agents.

As a conclusion, rutin demonstrated

significant protective effects against sodium

arsenite-induced testicular toxicity in rats.

Rutin ameliorated arsenic-induced damage

by reducing oxidative stress, inflammation,

and apoptosis in testicular tissue, while im-

proving sperm parameters and hormone lev-

els. These findings suggest that rutin may

have therapeutic potential in mitigating ar-

senic-related reproductive toxicity, although

further research is needed to elucidate its

mechanisms of action and clinical applica-

bility fully.

Acknowledgement

Medical writing support for the devel-

opment of this manuscript, under the direc-

tion of the authors, was provided by Yonnova

Scientific Consultancy Pvt. Ltd. in accor-

dance with the Good Publication Practice

guidelines.

Funding

This study was funded by the Zheji-

ang Province Medical and Health Science

and Technology Plan Project (2021RC096)

and the Zhejiang Traditional Chinese Medi-

cine Science and Technology Plan Project

(2023ZL370).

Conflicts of interest

The authors declare that they have no

conflict of interest regarding this study.

Availability of data

The datasets generated and/or analyzed

during the current study are not publicly

available due to confidentiality agreements;

supporting data can only be made available

to bona fide researcher’s subject to a non-

disclosure agreement.

Ethical approval

All experiments were performed be-

tween 09:00 and 17:00. The experimental

protocol was approved by the Institutional

Animal Ethics Committee of the Shaanxi

Kangfu Hospital (approval no. sxkf2025).

All procedures involving animals were con-

ducted in accordance with the National In-

stitute of Health Guide for the Care and Use

of Laboratory Animals.

ORCID number of authors

• Yunyun Wang (YW):

0009-0007-3462-5323

• Zengrong Hua (ZH):

0009-0008-8248-8148

• Jin Qing Hui (JQH):

0009-0008-5583-9213

• Dandan Qiu (DQ):

0009-0009-1376-7853

Author contributions

Each author has made significant con-

tributions to the development of this manu-

script. YW: conceived and designed the eval-

uation and drafted the manuscript; ZH and

DQ: performed data acquisition; YW: par-

ticipated in developing the assessment, per-

formed parts of the statistical analysis, and

helped to draft the manuscript; JQH: revised

the manuscript and performed the statisti-

cal analysis. All the authors have read and

approved the final version of the manuscript.

Protective effects of rutin against arsenic-induced testicular damage 421

Vol. 66(4): 408 - 425, 2025

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