Document Type : Original Article
Authors
1 Department of Zoology, Indira Gandhi National Tribal University, Amarkantak, Madhya Pradesh, India
2 Department of Zoology, Bundelkhand University, Jhansi, Uttar Pradesh, India
3 Department of Biomedical Sciences, Bundelkhand University, Jhansi, Uttar Pradesh, India
Abstract
Keywords
Synthetic pyrethroids insecticides are widely used because
of their high effectiveness against a large number
of insects, rapid biodegradation, low mammalian toxicity
and target-oriented mechanism of action (
Dietary antioxidants, chiefly plant phenolics, flavonoids
and carotenoids that have ROS scavenging activity, are
considered important for a healthy life. Curcumin, a polyphenolic
compound obtained from turmeric is an excellent
antioxidant and possesses a number of pharmacological
activities (
The present study was planned to investigate the role of curcumin and quercetin in Del and Cyp-induced reproductive toxicity in male Wistar rats. Apart from evaluating their antioxidant potential, we explored the effect of these phytochemicals on sperm parameters, hormones of the pituitary-gonadal axis and enzymes involved in testosterone biosynthesis.
Male Wistar rats, weighing about 200-250 g, were used in this controlled experimental study. Animals were kept in the animal house at 22 ± 3°C, with relative humidity of 45-55%, and 12 hours/12 hours dark/light cycles. The animals were fed with pelleted diet and water ad-libitum. All animal experiments were performed as per approval of the Institutional Animal Ethics Committee (BU/Pharma/ IAEC/12/032).
Technical grade Cyp (99.2%) and Del (98.5%) were obtained from Gharda chemicals (Mumbai, India). Curcumin (95%) was purchased from Sigma-Aldrich (St. Louis, MO. USA) and quercetin dihydrate (98%) from Himedia (Mumbai, India). All other chemicals used in this study were of high purity and purchased from standard firms.
Forty two male Wistar rats were randomly divided
into 7 groups of 6 animals. Cyp, Del, curcumin and
quercetin were dissolved in polyethylene glycol (
At the end of the experiment, rats were sacrificed by cervical dislocation, under ketamine-induced anesthesia. The testes and epididymis were removed and weighted. The epididymis was used for sperm motility and sperm morphology studies. One testis was used for sperm head counts and the other was used for estimation of lipid peroxidation, enzymatic and non-enzymatic antioxidants and steroidogenic enzymes. A part of testis was kept in 10% formaldehyde for histological studies. Blood was taken from the heart and used for estimation of various reproductive hormones.
Sperm head counts was performed using a hemocytometer
as described by Choi et al. (
A segment of distal cauda epididymis was removed and
kept in 2 ml of Dulbecco’s phosphate-buffered saline
(PBS), maintained at 36-38°C on a water bath. Cauda was
minced sufficiently to disperse the sperm for 1-5 minutes
and gently mixed using pasture pipette. The test sample
(5-10 µl) was loaded into the hemocytometer chamber
and the motile sperms were counted in white blood cells
(WBC) counting area. Sperms were counted as motile if
they exhibited any type of movement/motion. Hemocytometer
was placed on ice for 10-20 seconds to render all
the sperms immotile for counting the total sperms (
Cauda epididymis was minced with the help of a razor, in 1 ml of 0.9% saline and 1 ml of 10% neutral buffered formaldehyde was added. The suspension was diluted with water to a volume suitable for performing the assay. Next, 1-2 ml of 1% Eosin was added to 20 ml of the above-mentioned mixture and incubated at room temperature for 45 to 60 minutes. One drop of this suspension was taken on slide and a smear was prepared for studying sperm morphology. The head and tail abnormalities were expressed as percentage.
At the end of the experiment, blood was taken from the heart and centrifuged and serum was separated for the estimation of reproductive hormones. Testosterone (T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were estimated using rat specific ELISA kits (Qayee-Bio Life Science, China).
Testis tissue (100 mg) was rinsed and homogenized in 1 ml of 1X PBS and stored at -20°C, overnight. After two freeze-tha wcycles to break the cell membranes, the homogenate was centrifuged at 5000 g for five minutes in a refrigerated centrifuge. The supernatant was removed immediately and 3-ß hydroxyl steroid dehydrogenase (3ß-HSD) and 17ß-HSD were assayed using rat specific ELISA kits (Cusabio, USA)
A part of the testis was homogenized using homogenizing
buffer (10 times, w/v, 0.1 M phosphate buffer
(pH=7.4)+150 mM KCl) to prepare 10% homogenate.
A part of the homogenate was used for lipid peroxidation
(LPO) and glutathione (GSH) estimations. The
remaining part was centrifuged at 8500 g for 20 minutes
in a refrigerated centrifuge to get supernatant (S)
fraction. The ‘S’ fraction was used for measurement
of superoxide dismutase (SOD), catalase (CAT), glutathione
peroxidase (GPx), glutathione-S-transferase
(GST) and glutathione reductase (GR) activities (
Briefly, LPO and GSH were estimated by the methods
of Ohkawa et al. (
The testicular tissues, previously kept in 10% formaldehyde
were used for histological studies. The tissues were
washed overnight in running water to remove remaining
fixative. Dehydration was carried out to remove water
using a series of gradually increasing concentrations of
alcohol. These tissues were then cleared in xylol, embedded
in wax and cut in sections of 5-µm thickness. The
sections were recovered from wax blocks, stained with
haematoxylin and eosin and analyzed by trinocular microscope
with camera (
The results were expressed as mean ± SEM. Intergroup variations were evaluated by one way analysis of variance (ANOVA) followed by Dunnett’s test. Statistical significance was considered at P<0.05. The statistical analysis was performed using Graph Pad In Stat Software Inc., V. 3.06, San Diego, USA.
Non-significant decreases in weight of testis and
epididymis were observed between exposure (B, C, and
D) and treatment groups (E, F, and G) and control group A.
The treatment groups E, F and G showed non-significant
(P>0.05) increases in the weight of testes and epididymis
as compared to exposure group D (
Effect of curcumin and quercetin in Cyp and Del-induced changes in sex organs weight. Each bar represents mean ± SEM of 6 rats.
Cyp; Cypermethrin, Del; Deltamethrin, .; P>0.05, compared to group A, p.; Compared with group A and D both, A; Control, B; Cyp, C; Del, D; Cyp+Del, E; Cyp+Del+curcumin, F; Cyp+Del+quercetin, and G; Cyp+Del+curcumin+quercetin.
Sperm head counts were significantly decreased in
groups B, C, D, E, and F (29.38, 15.99, 40.46, 15.53
and 17.38%, respectively, P<0.01) and group G (5.15%,
P>0.05) as compared to group A. Sperm motility was
decreased significantly in groups B and (28.09 and
46.20%, respectively, P<0.01) and groups E and F (17.70
and 14.86%, respectively, P<0.05) whereas it decreased
non-significantly (P>0.05) in groups C (14.69%) and G
(2.09%) as compared to group A. On the other hand, in
group E, F and G, we observed significant (P<0.01) increase
in sperm head counts (41, 38.75 and 59.30%, respectively)
and sperm motility (52.98, 58.26 and 82%, respectively)
as compared to group D. Significant (P<0.01)
increases in sperm abnormality were observed in groups
B (61.37%), C (52.85%) and D (102.28%) as compared
to control group A. Groups E, F and G showed significant
increases in sperm abnormality (27.27% for group
E (P<0.05), 22.73% for group F (P>0.05) and 8.43% for
group G (P>0.05) as compared to group A. On the contrary,
a significant (P<0.01) reduction in sperm abnormality
was found in group E (37.07%), F (39.32%), and G
(46.39%) as compared to group D (
Testosterone levels decreased significantly in groups
B, C, and D (73.37, 65.47, and 90.70%, respectively,
P<0.01 for all groups) and groups E and F (54.99 and
56.47%, respectively, P<0.05 for all groups) and non-
significantly (P>0.05) in group G (18.84%) as compared
to group A. Significant (P<0.05) increases in testosterone was observed in groups E, F and G (p<0.01) when
compared to group D. A decrease in FSH level was observed
in groups B, C, E, F, and G (57.55, 47.08, 27.95,
29.06, and 16.77%, respectively, P>0.05 for all groups)
and D (64.55%) as compared to group A. Groups E, F
(p<0.05) and G (p<0.01) showed significant increases
in FSH level as compared to group D. A significant decrease
in LH level was observed in groups B, C, D, E,
and F (56.76, 52.52,72.97,49.69 and 57.92%, respectively,
p<0.01 or all groups) and G (37.37%, p<0.05)
as compared to group A. Groups E, F (p<0.05) and G
(p<0.01) showed significant increases in LH level as
compared to group D (
Non-significant (P>0.05) decreases 3ß-HSD was observed
in groups B (67.39%), C (49.34%), D (76.84%), E (17.28%),
F (31.95%) and G (3.26%) as compared to group A, whereas
significant (p<0.05) increases were found in groups E, F and
G (p<0.01) as compared to group D. 17ß-HSD activity was
significantly (p<0.01) decreased in groups B (58.65%), C
(52.88%), D (80.28%) and F (52.88%) and also in groups
E (44.23%, p<0.05) and G (5.76%, P>0.05) as compared to
group A. Significant (P>0.05) increases in 17ß-HSD activity
were observed in groups E, F and G (p<0.01) as compared
to group D (
Effect of curcumin and quercetin on Cyp and Del-induced changes in testicular sperm counts. A. Control, B. Cyp, C. Del, D. Cyp+Del, E. Cyp+Del+curcumin, F. Cyp+Del+quercetin, and G. Cyp+Del+curcumin+quercetin. Cyp; Cypermethrin, Del; Deltamethrin.
Effect of curcumin and quercetin on Cyp and Del-induced changes in sperm parameters
Parameter | Group A | Group B | Group C | Group D | Group E | Group F | Group G |
---|---|---|---|---|---|---|---|
Sperm count×106/g tissue | 72.22 ± 1.176 | 51.00 ± 0.730q** | 60.666 ± 2.028r** | 43.00 ± 1.751** | 61 ± 2.129r** | 59.666 ± 2.275r** | 68.5 ± 2.487r• |
Sperm motility (%) | 70.475 ± 3.367 | 50.675 ± 2.54q** | 60.12 ± 3.245r• | 37.911 ± 2.909** | 60.00 ± 2.745r* | 58.00 ± 2.160r* | 69.00 ± 1.506r• |
Sperm abnormality (%) | 43.998 ± 3.246 | 71.00 ± 1.807r** | 67.251 ± 2.627r** | 89.00 + 3.183** | 56.00 ± 3.724r* | 54.00 ± 2.769r• | 47.71 ± 2.911r• |
Data are presented as mean ± SEM of 6 rats in each group. Cyp; Cypermethrin, Del; Deltamethrin, •; P>0.05, *; P<0.05, **; P<0.01 compared with group A, p; P>0.05, q; P<0.05, r; P<0.01 compared with group D, Group A; Control, B; Cyp, C; Del, D; Cyp+Del, E; Cyp+Del+curcumin, F; Cyp+Del+quercetin, and G; Cyp+Del+curcumin+quercetin.
Effect of curcumin and quercetin on Cyp and Del-induced changes in sex hormone level
Parameter | Group A | Group B | Group C | Group D | Group E | Group F | Group G |
---|---|---|---|---|---|---|---|
Testosterone (ng/ml) | 3.635 ± 0.745 | 0.968 ± 0.302p** | 1.255 ± 0.170p** | 0.338 ± 0.137** | 1.636 ± 0.492q* | 1.585 ± 0.495q* | 2.95 ± 0.556r• |
FSH (mIU/ml) | 2.415 ± 0.446 | 1.025 ± 0.112p• | 1.278 ± 0.169p• | 0.856 ± 0.213* | 1.74 ± 0.494q• | 1.713 ± 0.492q• | 2.01 ± 0.454r• |
LH (mIU/ml) | 1.98 ± 0.325 | 0.856 ± 0.213p** | 0.94 ± 0.220p** | 0.535 ± 0.126** | 0.996 ± 0.118q** | 0.833 ± 0.113q** | 1.24 ± 0.112r* |
Data are presented as mean ± SEM of 6 rats in each group.
Cyp; Cypermethrin, Del; Deltamethrin, •; P>0.05, *; P<0.05, **; P<0.01 compared with group A, p; P>0.05, q; P<0.05, r; P<0.01 compared with group D, Group A; Control, B; Cyp, C; Del, D; Cyp+Del, E; Cyp+Del+curcumin, F; Cyp+Del+quercetin, and G; Cyp+Del+curcumin+quercetin.
Effect of quercetin and curcumin on Cyp and Del-induced changes in steroidogenic enzymes
Parameter | Group A | Group B | Group C | Group D | Group E | Group F | Group G |
---|---|---|---|---|---|---|---|
3-β HSD (pg/ml) | 0.92 ± 0.283 | 0.30 ± 0.125p• | 0.466 ± 0.105p• | 0.213 ± 0.106• | 0.761 ± 0.213q• | 0.626 ± 0.175q• | 0.89 ± 0.274r• |
17-β HSD (ng/ml) | 1.04 ± 0.112 | 0.43 ± 0.104p** | 0.49 ± 0.105p** | 0.205 ± 0.095** | 0.58 ± 0.124q* | 0.49 ± 0.100q** | 0.98 ± 0.113r• |
Data are presented as mean ± SEM of 6 rats in each group. Cyp; Cypermethrin, Del; Deltamethrin, •; P>0.05, *; P<0.05, **; P<0.01 compared with group A, p; P>0.05, q; P<0.05, r; P<0.01 compared with group D, Group A; Control, B; Cyp, C; Del, D; Cyp+Del, E; Cyp+Del+curcumin, F; Cyp+Del+quercetin, and G; Cyp+Del+curcumin+quercetin.
Significant (p<0.01) increases in LPO level were observed
in groups B (142.65%), C (80%), D (188.94%),
E (144.70%), F (127.48%) and G (47.70%) as compared
to group A. Significant (p<0.05) decreases in LPO level
were observed in groups E (15.52), F (86.87%) and
also in group G (48.87%, p<0.01) as compared to group
D. GSH level was significantly (p<0.01) decreased in
group B (40%), C (31.98%), D (75.82%), E (54.69%)
and F (46.36%) and non-significantly (P>0.05) in group
G (6.67%) as compared to group A. Groups E (87.39%,
p<0.05), F(121.81%, p<0.01) and G (285.97%, p<0.01)
showed significant increases in GSH level compared to
group D (
However, significant (p<0.05) increases were observed in groups E (89.18%), F (90.27%), and G (97%) when compared to group D. Significant (p<0.05) decreases in GPx activity were observed in groups B (36.32%), D (55.59%, p<0.01) and non-significant (P>0.05) decreases were found in groups C (21.23%), F (22.21%), E (27.54%) and G (3.84%) as compared to group A. Significant (p<0.05) increases in GPx activity were observed in groups E (63.17%), F (75.18%) and G (116.55%, p<0.01) as compared to group D. GR activity was significantly (p<0.05) reduced in groups B (31.94%), E (27.77%), F (25.83%) and D (61.55%, p<0.01) and non-significantly (P>0.05) in groups C (20.96%) and G (6.38%) as compared to group A.
On the other hand, significant (p<0.01) increases were observed
in groups E (87.84%), F (92.89%) and G (143.48%)
as compared to group D. The GST activity was significantly
(p<0.01) decreased in groups B (7.19%), D (17.62%), E
(14.13%), F (14.40%), G (8.76%) and C (4.60%, p<0.05)
as compared to group A. Significant increases in GST were
recorded in groups E and F (4.24, 3.91%, respectively,
p<0.05 for both groups) and group G 10.75%, p<0.01, as
compared to group D (
Effect of curcumin and quercetin on Cyp and Del-induced changes in lipid peroxidation, non- enzymatic and enzymatic antioxidants
Parameter | Group A | Group B | Group C | Group D | Group E | Group F | Group G |
---|---|---|---|---|---|---|---|
LPO (nmoles MDA/hours/gtissue) | 3.165 ± 0.2749 | 7.68 ± 0.166q** | 5.726 ± 0.148r** | 9.145 ± 0.489** | 7.725 ± 0.319q* * | 7.2 ± 0.402r** | 4.675 ± 0.309r* |
GSH(µmole/g tissue) | 2.920 ± 0.288 | 1.735 ± 1.145r** | 1.986 ± 0.224r** | 0.706 ± 0.071** | 1.566 ± 0.03827r** | 1.323 ± 0.0388q** | 2.725 ± 0.1385r• |
SOD (nmole/minutes/mg protein) | 31.911 ± 1.262 | 17.648 ± 1.631r** | 22.858 ± 1.474r** | 10.631 ± 0.5518** | 21.726 ± 0.9145r** | 19.453 ± 1.170r** | 28.874 ± 0.7624r• |
CAT(µ mole/minutes/mg protein) | 74.14 ± 1.193 | 47.62 ± 1.335r** | 60.596 ± 1.295r** | 36.538 ± 1.331** | 69.521 ± 0.659r* | 69.123 ± 0.620r* | 72.00 ± 0.7857r• |
GPx (nmole/minutes/mg protein) | 8.396 ± 1.005 | 5.346 ± 0.715q* | 6.613 ± 0.774q• | 3.728 ± 0.760** | 6.531 ± 0.771q• | 6.083 ± 0.736q• | 8.073 ± 0.719r• |
GR (nmole/minutes/mg | 3.553 ± 0.339 | 2.418 ± 0.250q* | 2.808 ± 0.2778r• | 1.366 ± 0.1686** | 2.635 ± 0.185r* | 2.566 ± 0.2008r* | 3.326 ± 0.1578r• |
GST (µmole/minutes/mg protein) | 91.015 ± 0.681 | 84.471 ± 1.141r** | 86.823 ± 0.775r* | 74.973 ± 1.284** | 78.153 ± 0.673p** | 77.906 ± 0.741p** | 83.038 ± 0.791r** |
Data are presented as mean ± SEM of 6 rats in each group.
Cyp; Cypermethrin, Del; Deltamethrin, •; P>0.05, *; P<0.05, **; P<0.01 compared with group A, p; P>0.05, q; P<0.05, r; P<0.01 compared with group D, Group A; Control, B; Cyp, C; Del, D; Cyp+Del, E; Cyp+Del+curcumin, F; Cyp+Del+quercetin, and G; Cyp+Del+curcumin+quercetin.
Effect of curcumin and quercetin on Cyp and Del-induced changes in testicular sperm counts. A. Control, B. Cyp, C. Del, D. Cyp+Del, E. Cyp+Del+curcumin, F. Cyp+Del+quercetin, and G. Cyp+Del+curcumin+quercetin.
Cyp; Cypermethrin, Del; Deltamethrin, LC; Leydig cells, BM; Basement membrane, SG; Spermatogonia, PSC; Primary spermatocyte, SSC; Secondary spermatocyte, LST; Lumen filled with spermatids, SC; Sertoli cells, RBM; Ruptured basement membrane, DSV; Disorganized structure of seminal vesicles, NSC; Necrosis of sertoli cells, VLM; Vacuolation of lumen, NLC; Necrosis of Leydig cells, AS; Arrested stages of spermatogenesis, DSG; Disorganized spermatogonia, NST, Necrosis of spermatids, DSV; Disorganization of seminal vesicles, LLC; Loosed Leydig cells, DSC; Degeneration of sertoli cells, AS; Arrested spermatogenesis, LMDST; Lumen filled with dead spermatids, DDSV; Disorganization and disappearance of seminal vesicles, NSC; Necrosis of sertoli cell, FSG; Fading of spermatogonia, NMST non-motile spermatids, SLC; Scattering of Leydig cells, LDTST; Lumen filled with dead and tailless spermatids, TBM; Thick basement membrane, ISG; Increased spermatogonia, WLC; Well-developed Leydig cells, IS; Increased spermatogenesis, OCS; Organized sertoli cells, LMSZ; Lumen filled with spermatozoa, RBM; Recovery of basement membrane, RLC; Recovery of Leydig cell, RSG; Recovery of spermatgonia, US; Unaffected spermatogenesis, LST; Lumen filled with spermatids, SZT; Spermatozoa with tail, WBM; Well-shaped basement membrane, DSG; Dense structured spermatogonia, DPS; Densely packed primary spermatocyte, SSC; Secondary spermatocyte, WST; Well-developed spermatids with tails, LMD; Lumen filled with dense materials, OLC; Organized Leydig cells, and WCS; Well-shaped sertoli cells.
The histology of testes of control rats showed seminiferous
tubules separated by basement membrane containing
Leydig cells. The germinal epithelium consisted
of concentric layers of germs cells viz. spermatogonia,
primary and secondary spermatocytes, lumen filled with
spermatids and spermatozoa with tail and sertoli cells
(
Cyp and Del showed toxic effects in reproductive system
of male Wistar rats. The reproductive toxicity caused
by these insecticides was ameliorated by curcumin and
quercetin. The weight of the testes and epididymis decreased
following single as well as combined exposure to
Cyp and Del, as compared to the control. The male reproductive
toxicity of Cyp (
The endocrine disruptive action of Del (
StAR protein transports cholesterol from the cytoplasm
to the mitochondrial matrix. The transport of
cholesterol is a rate-limiting step in testosterone biosynthesis.
There are reports that pyrethroids reduce StAR
protein expression (
Increased lipid peroxidation impairs membrane functions
by decreasing membrane fluidity and changing
the activity of membrane-bound enzymes and receptors.
Lipid peroxidation products (lipid radical and lipid peroxide)
are harmful to the cells and are associated with a
number of pathological conditions. In the present study,
significant increases in the level of LPO was observed following
single and combined exposure to Cyp and Del as
compared to control group A. Increased LPO has been reported
after Cyp exposure in rats brain and liver (
Superoxide dismutase is the first line of defense against
deleterious effects of oxygen radicals in the cell. It acts
by catalyzing the dis-mutation of superoxide radicals to
hydrogen peroxide and molecular oxygen. Significant decreases
in the SOD activity were observed in the present
study, which may be due to the decrease in the ability of
the tissues to handle extra free radical. These extra free
radicals may attack the thiol group of cysteine residues
of proteins and polysaturated fatty acids of biological
membranes. Catalase is present ubiquitously in nearly all
living organisms exposed to oxygen and catalyzes the decomposition
of hydrogen peroxide to water and oxygen
(
The present study showed significant decreases in GPx
activity which may be due to reduced level of GSH, a substrate
of GPx. GR is a member of the pyridine-nucleotide
disulfide oxidoreductase family of flavo enzymes which
catalyzes the reduction of glutathione disulfide (GSSG)
to its reduced form GSH, in the presence of NADPH.
The level of GR was decreased in this study, possibly
due to the damage caused by Cyp and Del to the tertiary
structure of the enzyme. GST catalyzes the conjugation
of GSH to electrophiles and protects cellular components
from oxidative damage (
Additionally, exposure to Cyp and Del resulted in marked histo-architectural disturbances in testis. These changes were possibly caused by ROS-induced cell damage. The damage in sperm mother cells as well as supporting sertoli cells resulted in gross changes in sperm parameters and decrease in testosterone.
Curcumin and quercetin are phytochemicals with proven antioxidant and cyto-protective activities. Treatment of Cyp and Del-exposed rats with curcumin and quercetin in the present study, increased sex organs weights, sperm count, sperm motility, sex hormones (testosterone, LH and FSH) levels, and steroidogenic enzymes (3ß-HSD and 17ß-HSD) and decreased sperm abnormalities. Treatment with curcumin and quercetin also restored Cyp and Del-induced histo-architectural disturbances by their antioxidant and cyto-protective activities. Since we observed direct cytoprotective effect of these antioxidants, particularly the restoration of testicular histo-architecture, it is possible that curcumin and quercetin have crossed the blood-testes-barrier. The increase in antioxidant defense of the testis, as reflected by increased GSH, CAT, SOD, GPx, GST and GR activities and decreased LPO levels, indicated curcumin and quercetin-mediated scavenging of the hydroxyl, peroxy, and superoxide radicals.
Moreover, increased antioxidant defense protected
sertoli and Leydigcells with concomitant increases in
the level of sex hormones viz. testosterone, FSH and
LH. There are reports of feedback regulation of testosterone
biosynthesis by FSH and LH. Hu et al. (
This study indicated that the combined exposure to Cyp and Del was more toxic than exposure to each of the insecticide alone. The 45-day exposure to Cyp and Del showed marked decreases in sperm motility and sperm head counts, increases in sperm abnormality and decreases in testosterone, FSH, LH, 3ß-HSD and 17ß-HSD in serum. Enhanced activities of steroidogenic enzymes (3ß-HSD and 17ß-HSD) and concomitant increased levels of testosterone were mainly responsible for ameliorating effect of curcumin and quercetin. We also observed decreases in enzymatic and non-enzymatic antioxidants and disturbance in testicular histo-architecture in the exposed rats. Treatment with curcumin and quercetin ameliorated Cyp and Del-induced toxicity by improving the reproductive system. Curcumin showed slightly better activity as compared to quercetin. Our study further showed that combined treatment of curcumin and quercetin possesses higher activity as compared to treatment with each one alone.