Human Sperm Quality and Metal Toxicants: Protective Effects of some Flavonoids on Male Reproductive Function

Abstract

Background
Metals can cause male infertility through affection of spermatogenesis and sperm quality. Strong evidences confirm that male infertility in metal-exposed humans is mediated via various mechanisms such as production of reactive oxygen species (ROS). Flavonoids have antioxidant and metal chelating properties which make them suitable candidates for neutralizing adverse effects of metals on semen quality. In the current study, we have evaluated the effects of five types of flavonoids (rutin, naringin, kaempferol, quercetin, and catechin) on recovery of sperm motility and prevention of membrane oxidative damage from aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride (PbCl4).
Materials and Methods
In this experimental study, motility and lipid peroxidation of metalexposed sperm was investigated in the presence of different concentrations of five kinds of flavonoids. Malondialdehyde (MDA) production was assessed as a lipid peroxidation marker.
Results
Aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride (PbCl4) diminished sperm motility. Treatment of metal-exposed sperm with rutin, naringin, and kaempferol attenuated the negative effects of the metals on sperm motility. Quercetin and catechin decreased the motility of metal-exposed sperm.
Conclusion
Based on the MDA production results, only AlCl3 significantly induced lipid peroxidation. Treatment with rutin, naringin, and kaempferol significantly decreased MDA production.

Full text

215
Original Article
Human Sperm Quality and Metal Toxicants: Protective
Effects of some Flavonoids on Male Reproductive Function
Mostafa Jamalan, Ph.D.1, Mohammad Ali Ghaffari, Ph.D.2, 3*, Pooneh Hoseinzadeh, M.Sc.2, Mahmoud
Hashemitabar, Ph.D.2, 4, Majid Zeinali, Ph.D.5
1. Abadan School of Medical Sciences, Abadan, Iran
2. Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3. Department of Biochemistry, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
4. Department of Anatomical Sciences, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
5. Biotechnology Research Center, Research Institute of Petroleum Industry (RIPI), Tehran, Iran
Abstract
Background: Metals can cause male infertility through affection of spermatogenesis and
-
ated via various mechanisms such as production of reactive oxygen species (ROS). Flavonoids
have antioxidant and metal chelating properties which make them suitable candidates for neu-
tralizing adverse effects of metals on semen quality. In the current study, we have evaluated

recovery of sperm motility and prevention of membrane oxidative damage from aluminum
chloride (AlCl), cadmium chloride (CdCl), and lead chloride (PbCl).
Materials and Methods: In this experimental study, motility and lipid peroxidation of metal-
   

Results: Aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride
(PbCl4) diminished sperm motility. Treatment of metal-exposed sperm with rutin, nar-
ingin, and kaempferol attenuated the negative effects of the metals on sperm motility.
Quercetin and catechin decreased the motility of metal-exposed sperm.
Conclusion: Based on the MDA production results, only AlCl3-
 
MDA production.
Keywords: Metal Toxicity, Sperm Motility, Lipid Peroxidation, Flavonoids, Semen Quality
Citation: Jamalan M, Ghaffari MA, Hoseinzadeh P, Hashemitabar M, Zeinali M. Human sperm quality and metal toxi-
cants: protective effects of some avonoids on male reproductive function. Int J Fertil Steril. 2015; 9(3): 215-222.
Received: 10 Nov 2014, Accepted: 1 Sep 2015
*Corresponding Address: P.O.Box: 159, Cellular and Molecular
Research Center, Ahvaz Jundishapur University of Medical
Sciences, Ahvaz, Iran
Email: ghaffari@ajums.ac.ir Royan Institute
International Journal of Fertility and Sterility
Vol 10, No 2, Jul-Sep 2016, Pages: 215-222
Introduction
Metals are one of the main constituents of an
industrialized lifestyle that have a wide range of
applications. Metals such as lead (Pb), aluminum
(Al) and cadmium (Cd) induce toxicity in humans
and other living organisms by impacting enzyme
activity and generation of free radical production.
However, in terms of their unique characteristics,
their applications are expansive, even in medical
and drug industries (1, 2).
Metals can affect male and female fertility by in-
duction of reactive oxygen species (ROS) produc-
tion. Therefore, antioxidant therapy that inhibits
metal-induced toxicity is under active investiga-
tion (3). Flavonoids are a broad group of natural
      
    
low molecular weight polyphenols ubiquitously
synthesized by green plants that may show vari-
ous pharmacological attributes according to their
chemical structures (4). Direct antioxidant effects
     
have been previously researched (5-7). Researchers
report the existence of a cardioprotective role (8, 9)




Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016 216
of different plants (11). Protection against ultravio-
-
tion of pollinating insects are major proposed roles

can occur both in the free form and as glycosides.
Their structure is composed of a basic C6-C3-C6
phenyl-benzopyran backbone (Fig.1). The position
of the phenyl ring relative to the benzopyran moi-

and degree of polymerization determine chemical

Fig.1: Chemical structure of avonoids. A. Basic structure of a
avonoid with two benzene rings and a heterocyclic pyran ring
as the linker. Chemical structures of: B. Run, C. Naringin, D.
Kaempferol, E. Quercen, and F. Catechin.
ROS induce cellular membrane instability (16),
destruction of DNA structures, and promotion of
transformation, (17) ultimately resulting in cellular
aging (18), mutagenesis (17), carcinogenesis (19),
induction of coronary heart disease (CHD) (4), and
infertility (20). In addition to ROS, nitrogen reactive
species (NOS) can cause cardiovascular diseases

and increased release of matrix metalloproteinase-2
       

      
NOS-induced myocardial injuries (4).
   -
      
activities of these polyphenolic compounds. Func-
   
antioxidant effects by scavenging free radicals and/
or by chelating metal ions (4, 11). The chelating of
metals can be crucial in prevention of radical gen-
eration which damage target biomolecules (11). In
the current study, we have evaluated the effects of
-
ol, quercetin, and catechin) on recovery of sperm
motility and prevention of membrane oxidative
damage from aluminum chloride (AlCl3), cadmium
chloride (CdCl2), and lead chloride (PbCl4).
Materials and Methods
Materials
For this experimental study, AlCl3, CdCl2, PbCl4,
naringin, kaempferol, and quercetin were obtained
from Merck (Darmstadt, Germany). Rutin, cat-
echin and the remainder of chemicals and reagents
used in this research were purchased from Sigma-
Aldrich (St. Louis, MO, USA).
Sample collection and preparation of sperm
suspension
Sperm samples considered compatible to the world
health organization (WHO) reference value for hu-
     
  6     
    
40 healthy, non-smoking volunteers, that resided
in Ahvaz, Khuzestan Province, Iran. We compared
    -
oxidation of metal-exposed sperms using labora-
tory studies. The Institutional Ethics Committee of
Ahvaz University of Medical Sciences reviewed
and approved the protocol. All participants in the
current study signed informed consents. Collected
sperm samples were separated from semen plasma
for assessment of clinical attributes by washing three
times with an equal volume of M6 solution and sub-
sequent centrifugation for 10 minutes at 1600 g (25).
M6 solution contained (per liter, pH=7.4): 0.55%
NaCl, 0.03% KCl, 0.019% CaCl2, 0.016% K3PO4,
0.029% MgSO4, 0.031% NaHCO3, 0.496% HEPES,
0.26% sodium lactate, 36×10-4% sodium pyru-
vate, 0.11% glucose, 0.4% bovine serum albumin,
60×10-4% penicillin, and 50×10-4% streptomycin.
Separated pellets were suspended in M6 solution at
a density of 100 million sperm/ml and freshly were
used. Sperm counts were performed by a MMC-SK
Sperm Counting Chamber (Saint Petersburg, Russia).
Incubation of sperm samples with aluminum
chloride, cadmium chloride, and lead chloride
We evaluated the effects of AlCl3, CdCl2, and
PbCl4 on sperm motility and lipid peroxidation of
sperm cells at different concentrations (125 µM,
Flavonoids Effects on Metal-Exposed Human Sperm

Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016
217
250 µM, 500 µM, 1 mM, and 5 mM) of the metal
salts. The metal salt solutions were prepared in
M6 solution. Sperm samples were incubated in the

-

impacted sperm motility for additional experi-

Effects of avonoids on the motility of metal-
exposed sperm
   
with AlCl3 (1.0 mM), CdCl2 (500 µM) or PbCl4 (250
µM) in the presence of various concentrations (25,
50, 100, 200, 500, and 1000 µM) of rutin, naringin,
kaempferol, quercetin, and catechin. Subsequently,
we assessed sperm mobility by MMC Sperm. In or-
-
ed in a 1:1 (v/v) of Dimethyl sulfoxide (DMSO): M6
solution prior to their treatment of the sperm cells.
Effects of avonoids on lipid peroxidation of
metal-exposed sperm
Induction of lipid peroxidation was evaluated
in sperm samples in the presence of various con-
centrations of AlCl3, CdCl2, and PbCl4. Between
treated groups, sperm samples treated with 20 mM
of AlCl3 were simultaneously incubated with 25
µM, 50 µM, 100 µM, 200 µM, 500 µM, and 1 mM
each of rutin, naringin, kaempferol, quercetin, and

assessed for lipid peroxidation of the sperm cells
according to the indicated approach.
Analytical methods
Assessment of sperm motility
Evaluation of sperm motility was performed by
MMC Sperm (MultiMedia Catalog Sperm). MMC
Sperm is an automated image analysis software
package for sperm quality analysis according to
parameters recommended by the WHO laboratory
manual (26).
Measurement of lipid peroxidation
Lipid peroxidation was measured using malon-
dialdehyde (MDA) and thiobarbituric acid-reac-
-
droxytoluene (dissolved in ethanol) and 1.0 ml of
15% aqueous trichloroacetic acid were successive-
ly added to 2.0×107 sperm. The mixture was then
        

was added to 1.0 ml thiobarbituric acid (0.375% in

for 20 minutes. After cooling, the solution was an-
alyzed by a spectrophotometer at 532 nm.
Statistical analysis
All treatments were performed in triplicate. Each
experiment was run at least three times. Results
-
ference between treatment groups was determined
by the student’s t test. P<0.05 was considered sta-

Results
Effects of aluminum chloride, cadmium chlo-
ride, and lead chloride on sperm motility
AlCl3 is an abundant metal in the earth which has
toxic effects. High concentrations of AlCl3 induce
free radical-mediated cytotoxicity and can be toxic
for the male reproductive system (29, 30). In previ-
ous studies, it has been shown that treatment with
AlCl3 could decrease ejaculate volume, sperm con-
centration, and sperm motility (31). CdCl2 is a well-
known nephrotoxin and carcinogen (32, 33) that can
induce ROS production. Exposure to CdCl2 may re-
sult in decreased sperm concentration, diminished
sperm motility, creation of abnormal forms of sperm
following long-term exposure to CdCl2 (3, 34), and
infertility in treated male mice (35). PbCl4 poisoning
can result in decreased sperm motility. A number of
reports discuss DNA fragmentation in sperm cells ex-
posed to this metal in vitro (36). Our in vitro studies

different concentrations of AlCl3, CdCl2 and PbCl4
    
Fig.2). Mean sperm motility after a 2-hour incuba-
tion period in the presence of 5.0 mM AlCl3, CdCl2,
and PbCl4 were 93% (AlCl3), 75% (CdCl2), and 41%
(PbCl4) less than the control groups. As seen in Fig-
ure 2, the effect of Pb on sperm motility was higher
at the same concentrations of the three tested met-
als AlCl3
      
concentration of CdCl2 
   4  -
-
tion (Fig.2). The adverse effects of all three metals
on sperm motility were completely dose-dependent.
Jamalan et al.

Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016 218
Fig.2: Eects of aluminum chloride (AlCl3), cadmium chloride
(CdCl2), and lead chloride (PbCl4) on sperm molity. We evalu-
ated the eects of these compounds on sperm molity at dier-
ent concentraons (125 μM, 250 μM, 500 μM, 1 mM, and 5 mM)
of metal salts. Sperm samples were incubated in the presence
of the dened concentraons of metals for 2 hours at 37˚C. *;
P<0.05 and **; P<0.01 compared to the untreated control.
Effects of avonoids on motility of aluminum
chloride-exposed sperm
Previous studies reported an in vitro protective ef-
fect of ascorbic acid (vitamin C) and tocopherol (vi-
tamin E) on AlCl3-treated sperm (31, 37). As seen in
Figure 2, 1000 µM of AlCl3
   
used this concentration for additional studies with
-
tin, naringin, kaempferol, quercetin, and catechin
for motility recovery of AlCl3-exposed sperm. Com-
pared to the untreated control group, rutin increased
sperm motility by 9% at the 50 µM concentration
and 18% at the 200 µM concentration. Naringin,
       
      
was a gradual increase in recovery of sperm motil-
ity when the concentration of naringin increased to
500 µM (Fig.3). Kaempferol showed the most pro-

10% recovery of sperm motility at the kaempferol
concentration of 25 µM. On the other hand, effects
of quercetin and catechin on the sperm mobility

- rutin, naringin and kaempferol. The antioxidants,
quercetin and catechin did not protect sperm cells
from heavy metal-mediated damages; rather, they
showed inhibitory effects on sperm motility. When
we increased the concentrations of quercetin and
catechin from 0 to 1000 µM, there was a gradual
decrease in sperm motility compared to the untreat-
ed control group. Mean motility of AlCl3-exposed
sperm after a 2 hours incubation period in the pres-
ence of 1000 µM quercetin was 22% and for cat-
echin, it was 28%.
Fig.3: Eects of run, naringin, kaempferol, quercen, and cat-
echin on aluminum chloride (AlCl3)-exposed sperm. Sperm sam-
ples were treated for 2 hours at 37˚C with AlCl3 (1.0 mM) in the
presence of various concentraons (25, 50, 100, 200, 500, and
1000 μM) of run, naringin, kaempferol, quercen, and catechin.
Sperm mobility was assessed by MMC Sperm. *; P<0.05 and **;
P<0.01 compared to the avonoid untreated control.
Effects of avonoids on motility of cadmium
chloride-exposed sperm
Previous studies by El-Demerdash et al. (3) in

  2 on
the male reproductive system. In the current study,
we observed that treatment with rutin, naringin and
kaempferol resulted in recovery of motility in CdCl2-
exposed sperm cells. Our results showed that rutin,

2-exposed sperm
cells in a dose-dependent manner (Fig.4). In contrast,
quercetin and catechin did not induce any protective
effect against CdCl2 toxicity; they reduced the motil-
ity of CdCl2-exposed sperm compared to the untreat-
ed control samples (Fig.4). These results disagreed
with an in vivo study by Farombi et al. (38) about
the antioxidative nature of quercetin. They showed
   
quercetin prevented Cd-mediated decreased sperm
motility in adult male rats. Other researchers report-
ed the positive effects of quercetin on sperm capacity
under both in vitro and in vivo conditions (39). Sup-
plementation of quercetin restored the decrease in
glutathione (GSH) level, and superoxide dismutase
(SOD) and GSH peroxidase activities in Cd-exposed
mice. This discrepancy between in vitro and in vivo
results might be attributed to the difference in querce-
tin exposure time or to in situ metabolic alteration of
quercetin (40).
Flavonoids Effects on Metal-Exposed Human Sperm

Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016
219
Fig.4: Effects of rutin, naringin, kaempferol, quercetin, and
catechin on cadmium chloride (CdCl2)-exposed sperm. Sperm
samples were treated for 2 hours at 37˚C with CdCl2 (500 μM)
in the presence of various concentrations (25, 50, 100, 200,
500, and 1000 μM) of rutin, naringin, kaempferol, quercetin,
and catechin. Sperm mobility was assessed by MMC Sperm. *;
P<0.05 and **; P<0.01 compared to the flavonoid untreated
control.
Effects of avonoids on motility of lead chloride-
exposed sperm
Toxic effects of PbCl4 on sperm quality, motility,
DNA fragmentation, and acrosome reaction have been
investigated extensively in mice and humans (36, 41-
44). According to our results (Fig.2), PbCl4 compared
to AlCl3 and CdCl2 had more adverse effects on sperm
motility at the 0.125 to 5.0 mM concentrations. We
used the 250 µM concentration of PbCl4 for additional
  
decreased motility of PbCl4-exposed sperm cells in a
dose-dependent manner. However, as seen in Figure
5, the 500 µM concentration of rutin, naringin, and
     
65% (rutin), 60% (naringin) and 63% (kaempferol).
       
against PbCl4-induced harmful attacks.
Fig.5: Eects of run, naringin, kaempferol, quercen, and cat-
echin on lead chloride (PbCl4)-exposed sperm. Sperm samples
were treated for 2 hours at 37˚C with PbCl4 (250 μM) in the
presence of various concentraons (25, 50, 100, 200, 500, and
1000 μM) of run, naringin, kaempferol, quercen, and catechin.
Sperm mobility was assessed by MMC Sperm. *; P<0.05 and **;
P<0.01 compared to avonoid untreated control.
Sperm lipid peroxidation in the presence of alu-
minum chloride, cadmium chloride and lead
chloride
Sperm membranes are rich in polyunsaturated
fatty acids (PUFAs) (45). Previous in vivo studies
have demonstrated that Al could increase peroxi-
dation of PUFAs in sperm samples (31, 46). The
presence of a high level of PUFA in the sperm plas-
ma membrane is required for membrane fusion
-
ity as a result of lipid peroxidation can diminish
the rates of sperm-oocyte fusion (47). Our in vitro
studies have shown that AlCl3 at concentrations
  

Fig.6). MDA is an end-product of enzymatic and
oxygen radical-initiated oxidative decomposition
of PUFAs and most frequently used as an indica-
tor of lipid peroxidation. We have shown that the
effect of AlCl3 on sperm lipid peroxidation was
dose- and time-dependent (Fig.6). There were

observed following incubation with 0.5-30 mM
of CdCl2 or PbCl4 (data not shown). Therefore,

MDA formation in AlCl3-exposed sperm cells.
Fig.6: Sperm lipid peroxidaon in the presence of aluminum
chloride (AlCl3). Sperm samples were treated with AlCl3 (20
mM) for 2 hours at 37˚C. Aer incubaon, we assessed the
amount of lipid peroxidaon of the sperm cells with MDA. **;
P<0.01 compared to the untreated control group and MDA;
Malondialdehyde.
Effects of avonoids on lipid peroxidation of
aluminum chloride-exposed sperm
Researchers previously reported the protective
effect of ascorbic acid as an antioxidant against
induction of lipid peroxidation by AlCl3 in sperm
cells (46). However, to the best of our knowledge
there was no report about the protective effect
Jamalan et al.

Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016 220
      
exposed sperm cells. Moretti et al. showed that
quercetin, rutin and, to a lesser extent, naringenin,
   
induced lipid peroxidation in human sperm (48).
Their studies indicated that epicatechin was not ef-
    
against oxidants. Our investigations showed that
kaempferol was the most effective amongst the
tested products in protection of sperm cells against
AlCl3-induced lipid peroxidation (Fig.7). Kaemp-
ferol, at a concentration of 100 µM, reduced MDA
production from 250 nmol/ml (in untreated cells)
to approximately 80 nmol/ml. Naringin and rutin
were less effective in protection of AlCl3-exposed
sperm cells against lipid peroxidation compared to
kaempferol. We observed that quercetin and cat-
echin did not protect sperm. Quercetin, as an an-
tioxidant, did not protect sperm cells against lipid
peroxidation; rather, it had inhibitory effects on
sperm motility. Khanduja et al. (49) have reported
2+-ATPase activ-
ity following quercetin treatment. Ca2+-ATPase is
the responsible enzyme that provides energy for
progressive movement of sperm cells. Inhibition
of Ca2+-ATPase activity has been shown to result
in Ca2+ accumulation in the cells and blockage of
the sperm motility apparatus (50).
Fig.7: Eects of run, naringin, kaempferol, quercen, and cat-
echin on lipid peroxidaon of aluminum chloride (AlCl3)-exposed
sperm. Sperm samples were treated with AlCl3 (20 mM) and
simultaneously incubated with dierent concentraons of ru-
n, naringin, kaempferol, quercen, and catechin for 2 hours
at 37˚C. Aer incubaon, we assessed the lipid peroxidaon of
sperm cells with MDA. *; P<0.05, **; P<0.01 compared to the
avonoid untreated control group and MDA; Malondialdehyde.
Discussion
The impact of heavy metal toxicity, even at low
concentrations, on the male reproductive system
     
(51-54). Sperm motility depends on the synchro-
nized actions of proteins, sugars, ions, and small
organic molecules. It is one of the main factors that
facilitates the journey of sperm toward the egg and
the subsequent fertilization process (55). Defects
in sperm motility are a common reason for infertil-
ity in humans (56). In the current study we have
shown that AlCl3, CdCl2 and PbCl4 
affected sperm motility. PbCl4 had the most toxic
effect.
Infertility due to metal toxicity usually occurs as
a result of ROS induction (57). Therefore, antioxi-
dant therapy is a promising strategy for treatment
of individuals with heavy metal poisoning (58).
   
likely to exert protective activities against metal
toxicity compared to carotenoids and vitamin E
       
rutin, naringin, and kaempferol have been shown
to restore motility of AlCl3-, CdCl2-, and PbCl4-
      
catechin and quercetin, had no positive effects on
motility of metal-exposed sperm; rather, they de-
creased sperm motility compared to untreated con-
trol samples.
We conducted additional research on the pro-

against heavy metal-induced lipid peroxidation.
MDA formation was assessed in AlCl3-exposed


free radical scavenging and metal chelating abili-
ties has been extensively investigated (60). How-
ever, according to the obtained results, quercetin
and catechin did not protect sperm cells from
ROS-mediated damages. They adversely affected
sperm motility. Inhibition of sperm motility with-
out considerable effects on peroxidation of PUFAs
would indicate involvement of other inhibitory
mechanisms. In contrast, increased motility of Al-
exposed sperm cells treated with rutin, naringin
and kaempferol was accompanied by decreased
levels of MDA formation. We have concluded that
antioxidant or chelating properties were not suf-
   
damages of heavy metals. Flavonoids, as naturally
occurring compounds may have some inhibitory
effects on enzyme activities (49) or exert their
growth inhibitory activities through binding to
human receptors (61). Therefore, it is essential to
Flavonoids Effects on Metal-Exposed Human Sperm

Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016
221
know the exact mechanisms of metal-induced tox-
    -
scribing medications to combat the adverse effects
of heavy metals on infertility.
Acknowledgments

from the Cellular and Molecular Research Center
of Ahvaz Jundishapur University of Medical Sci-
ences (Ahvaz, Iran), project number CMRC-003.
        
portion of this manuscript.
References
1. Nordberg GF, Fowler BA, Nordberg M, Friberg L. Hand-
book on the toxicology of metals. 3rd ed. Oxford: Academ-
ic; 2007; 117-145.
2. Cadmium in the human environment: toxicity and carcino-
genicity. Symposium proceedings. IARC Sci Publ. 1992;
(118): 1-464.
3. El-Demerdash FM, Yousef MI, Kedwany FS, Baghdadi
HH. Cadmium-induced changes in lipid peroxidation,
blood hematology, biochemical parameters and semen
quality of male rats: protective role of vitamin E and beta-
carotene. Food Chem Toxicol. 2004; 42(10): 1563-1571.
4. Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxi-
dants: chemistry, metabolism and structure-activity rela-
tionships. J Nutr Biochem. 2002; 13(10): 572-584.
5. Plaza M, Pozzo T, Liu J, Gulshan Ara KZ, Turner C, Nord-
berg Karlsson E. Substituent effects on in vitro antioxidiz-
ing properties, stability and solubility in avonoids. J Agric
Food Chem. 2014; 62(15): 3321-3333.
6. Rice-Evans C. Flavonoid antioxidants. Curr Med Chem.
2001; 8(7): 797-807.
7. Flora SJ. Structural, chemical and biological aspects of
antioxidants for strategies against metal and metalloid ex-
posure. Oxid Med Cell Longev. 2009; 2(4): 191-206.
8. Mazur A, Bayle D, Lab C, Rock E, Rayssiguier Y. Inhibi-
tory effect of procyanidin-rich extracts on LDL oxidation in
vitro. Atherosclerosis. 1999; 145(2): 421-422.
9. Kondo K, Hirano R, Matsumoto A, Igarashi O, Itakura
H. Inhibition of LDL oxidation by cocoa. Lancet. 1996;
348(9040): 1514.
10. Korkina LG, Afanas'ev IB. Antioxidant and chelating prop-
erties of avonoids. Adv Pharmacol. 1997; 38: 151-163.
11. Harborne JB, Williams CA. Advances in avonoid re-
search since 1992. Phytochemistry. 2000; 55(6): 481-504.
12. Hammerstone JF, Lazarus SA, Schmitz HH. Procyani-
din content and variation in some commonly consumed
foods. J Nutr. 2000; 130(8S Suppl): 2086S-2092S.
13. Carando S, Teissedre PL, Pascual-Martinez L, Cabanis
JC. Levels of avan-3-ols in French wines. J Agric Food
Chem. 1999; 47(10): 4161-4166.
14. Prior RL, Cao G. Antioxidant capacity and polyphenolic
components of teas: implications for altering in vivo anti-
oxidant status. Proc Soc Exp Biol Med. 1999; 220(4): 255-
261.
15. Kumar S, Pandey AK. Chemistry and biological activities
of avonoids: an overview. ScienticWorldJournal. 2013;
2013: 162750.
16. Mora A, Payá M, Ríos JL, Alcaraz MJ. Structure-activity
relationships of polymethoxyavones and other avonoids
as inhibitors of non-enzymic lipid peroxidation. Biochem
Pharmacol. 1990; 40(4): 793-797.
17. Takabe W, Niki E, Uchida K, Yamada S, Satoh K, Noguchi
N. Oxidative stress promotes the development of transfor-
mation: involvement of a potent mutagenic lipid peroxidation
product, acrolein. Carcinogenesis. 2001; 22(6): 935-941.
18. Sastre J, Pallardó FV, Viña J. Mitochondrial oxidative
stress plays a key role in aging and apoptosis. IUBMB
Life. 2000; 49(5): 427-435.
19. Kawanishi S, Hiraku Y, Oikawa S. Mechanism of guanine-
specic DNA damage by oxidative stress and its role in
carcinogenesis and aging. Mutat Res. 2001; 488(1): 65-
76.
20. Sheweita SA, Tilmisany AM, Al-Sawaf H. Mechanisms
of male infertility: role of antioxidants. Curr Drug Metab.
2005; 6(5): 495-501.
21. Thomas SR, Davies MJ, Stocker R. Oxidation and anti-
oxidation of human low-density lipoprotein and plasma
exposed to 3-morpholinosydnonimine and reagent perox-
ynitrite. Chem Res Toxicol. 1998; 11(5): 484-494.
22. Moore KP, Darley-Usmar V, Morrow J, Roberts LJ 2nd.
Formation of F2-isoprostanes during oxidation of human
low-density lipoprotein and plasma by peroxynitrite. Circ
Res. 1995; 77(2): 335-341.
23. Wang W, Sawicki G, Schulz R. Peroxynitrite-induced myo-
cardial injury is mediated through matrix metalloprotein-
ase-2. Cardiovasc Res. 2002; 53(1): 165-174.
24. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker
HW, Behre HM, et al. World Health Organization refer-
ence values for human semen characteristics. Hum Re-
prod Update. 2010; 16(3): 231-245.
25. Farrell PB, Foote RH, Simkin ME, Clegg ED, Wall RJ. Re-
lationship of semen quality, number of sperm inseminated,
and fertility in rabbits. J Androl. 1993; 14(6): 464-471.
26. World Health Organization. [Laboratory manual of the
WHO for the examination of human semen and sperm-
cervical mucus interaction]. Ann Ist Super Sanita. 2001;
37(1): I-XII, 1-123.
27. Buege JA, Aust SD. Microsomal lipid peroxidation. Meth-
ods Enzymol. 1978; 52: 302-310.
28. Janero DR. Malondialdehyde and thiobarbituric acid-reac-
tivity as diagnostic indices of lipid peroxidation and per-
oxidative tissue injury. Free Radic Biol Med. 1990; 9(6):
515-540.
29. Dawson EB, Ritter S, Harris WA, Evans DR, Powell LC.
Comparison of sperm viability with seminal plasma metal
levels. Biol Trace Elem Res. 1998; 64(1-3): 215-219.
30. Yousef MI, Salama AF. Propolis protection from reproduc-
tive toxicity caused by aluminium chloride in male rats.
Food Chem Toxicol. 2009; 47(6): 1168-1175.
31. Yousef MI, El-Morsy AM, Hassan MS. Aluminium-induced
deterioration in reproductive performance and seminal
plasma biochemistry of male rabbits: protective role of
ascorbic acid. Toxicology. 2005; 215(1-2): 97-107.
32. Waalkes MP, Anver M, Diwan BA. Carcinogenic effects of
cadmium in the noble (NBL/Cr) rat: induction of pituitary,
testicular, and injection site tumors and intraepithelial pro-
liferative lesions of the dorsolateral prostate. Toxicol Sci.
1999; 52(2): 154-161.
33. Waalkes MP, Anver MR, Diwan BA. Chronic toxic and
carcinogenic effects of oral cadmium in the Noble (NBL/
Cr) rat: induction of neoplastic and proliferative lesions of
the adrenal, kidney, prostate, and testes. J Toxicol Environ
Health A. 1999; 58(4): 199-214.
34. Oliveira H, Spanò M, Santos C, Pereira Mde L. Adverse
effects of cadmium exposure on mouse sperm. Reprod
Toxicol. 2009; 28(4): 550-555.
35. Monse M, Alaee S, Moradshahi A, Rohani L. Cadmium-
Jamalan et al.

Int J Fertil Steril, Vol 10, No 2, Jul-Sep 2016 222
induced infertility in male mice. Environ Toxicol. 2010;
25(1): 94-102.
36. Gomes M, Gonçalves A, Rocha E, Sá R, Alves A, Silva
J, et al. Effect of in vitro exposure to lead chloride on se-
men quality and sperm DNA fragmentation. Zygote. 2015;
23(3): 384-393.
37. Yousef MI, Kamel KI, El-Guendi MI, El-Demerdash FM. An
in vitro study on reproductive toxicity of aluminium chlo-
ride on rabbit sperm: the protective role of some antioxi-
dants. Toxicology. 2007; 239(3): 213-223.
38. Farombi EO, Adedara IA, Akinrinde SA, Ojo OO, Eboh AS.
Protective effects of kolaviron and quercetin on cadmium-
induced testicular damage and endocrine pathology in
rats. Andrologia. 2012; 44(4): 273-284.
39. Gibb Z, Butler TJ, Morris LH, Maxwell WM, Grupen CG.
Quercetin improves the postthaw characteristics of cryo-
preserved sex-sorted and nonsorted stallion sperm. The-
riogenology. 2013; 79(6): 1001-1009.
40. Metodiewa D, Jaiswal AK, Cenas N, Dickancaite E, Se-
gura-Aguilar J. Quercetin may act as a cytotoxic prooxi-
dant after its metabolic activation to semiquinone and
quinoidal product. Free Radic Biol Med. 1999; 26(1-2):
107-116.
41. Zribi N, Chakroun NF, Elleuch H, Abdallah FB, Ben Ham-
ida AS, Gargouri J, et al. Sperm DNA fragmentation and
oxidation are independent of malondialdheyde. Reprod
Biol Endocrinol. 2011; 9: 47.
42. Raque M, Khan N, Perveen K, Naqvi A. The effects of
lead and zinc on the quality of semen of albino rats. J Coll
Physicians Surg Pak. 2009; 19(8): 510-513.
43. Graça A, Ramalho-Santos J, de Lourdes Pereira M. Effect
of lead chloride on spermatogenesis and sperm param-
eters in mice. Asian J Androl. 2004; 6(3): 237-241.
44. Mushina EV. Study of the combined effects of lead and
cadmium on experimental animals. Gig Sanit. 1989; (9):
89-90.
45. Sikka SC. Relative impact of oxidative stress on male re-
productive function. Curr Med Chem. 2001; 8(7): 851-862.
46. Ige SF, Akhigbe RE. The role of Allium cepa on aluminum-
induced reproductive dysfunction in experimental male rat
models. J Hum Reprod Sci. 2012; 5(2): 200-205.
47. Aitken RJ. Free radicals, lipid peroxidation and sperm
function. Reprod Fertil Dev. 1995; 7(4): 659-668.
48. Moretti E, Mazzi L, Terzuoli G, Bonechi C, Iacoponi F,
Martini S, et al. Effect of quercetin, rutin, naringenin and
epicatechin on lipid peroxidation induced in human sperm.
Reprod Toxicol. 2012; 34(4): 651-657.
49. Khanduja KL, Verma A, Bhardwaj A. Impairment of human
sperm motility and viability by quercetin is independent of
lipid peroxidation. Andrologia. 2001; 33(5): 277-281.
50. Breitbart H, Rubinstein S, Nass-Arden L. The role of cal-
cium and Ca2+-ATPase in maintaining motility in ram
spermatozoa. J Biol Chem. 1985; 260(21): 11548-11553.
51. Iavicoli I, Fontana L, Bergamaschi A. The effects of metals
as endocrine disruptors. J Toxicol Environ Health B Crit
Rev. 2009; 12(3): 206-223.
52. Pizent A, Tariba B, Živković T. Reproductive toxicity of
metals in men. Arh Hig Rada Toksikol. 2012; 63 Suppl 1:
35-46.
53. Ghaffari MA, Motlagh B. In vitro effect of lead, silver, tin,
mercury, indium and bismuth on human sperm creatine
kinase activity: a presumable mechanism for men infertil-
ity. Iran Biomed J. 2011; 15(1-2): 38-43.
54. Järup L. Hazards of heavy metal contamination. Br Med
Bull. 2003; 68: 167-182.
55. Yoshida M, Kawano N, Yoshida K. Control of sperm motil-
ity and fertility: diverse factors and common mechanisms.
Cell Mol Life Sci. 2008; 65(21): 3446-3457.
56. McLaren JF. Infertility evaluation. Obstet Gynecol Clin
North Am. 2012; 39(4): 453-463.
57. Lavranos G, Balla M, Tzortzopoulou A, Syriou V, Angelo-
poulou R. Investigating ROS sources in male infertility:
a common end for numerous pathways. Reprod Toxicol.
2012; 34(3): 298-307.
58. Niederberger C. Re: the role of sperm oxidative stress
in male infertility and the signicance of oral antioxidant
therapy. J Urol. 2012; 187(4): 1377.
59. Mansuri ML, Parihar P, Solanki I, Parihar MS. Flavonoids
in modulation of cell survival signalling pathways. Genes
Nutr. 2014; 9(3): 400.
60. Hu JP, Calomme M, Lasure A, De Bruyne T, Pieters L, Vli-
etinck A, et al. Structure-activity relationship of avonoids
with superoxide scavenging activity. Biol Trace Elem Res.
1995; 47(1-3): 327-331.
61. Garrett SD, Lee HA, Morgan MR. A nonisotopic estrogen
receptor-based assay to detect estrogenic compounds.
Nat Biotechnol. 1999; 17(12): 1219-1222.
Flavonoids Effects on Metal-Exposed Human Sperm