Vol.2, No.6, 551-556 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.26069
Effect of prolonged intake of iron enriched diet on
testicular functions of experimental rats
Mohamed M. El-Seweidy*, Mervat E. Asker, Sousou I. Ali, Hebatallah H. Atteia
Biochemistry Department, Faculty of Pharmacy, Zagazig University; *Corresponding Author:
Received 28 December 2009; revised 8 January 2010; accepted 23 March 2010.
ABSTRACT
Iron deficiency anemia represents a common
nutritional problem which affects many socie-
ties allover the world and iron fortified diet has
been suggested as one of possible tools to
combat and solve such problem. Present study
was designed to illustrate the effect of dietary
iron intake on certain biochemical markers
dealing with oxidative stress, inflammatory re-
sponse and cellular alterations of testicular
tissues. Adult male rats which were fed on bis-
cuits fortified with iron (0.3% ferrous sulfate)
daily for 10 weeks (iron group) showed increa-
sed serum iron, ferritin, tumor necrosis factor-
alpha (TNF-α), nitric oxide (NO) and decreased
Testosterone level (p < 0.05). Testicular tissues
content of Malondialdehyde (MDA), hydroxypro-
line (Hyp), iron showed significant increase (p <
0.05) and decreased glutathione (GSH) as com-
pared to control group. Testicular tissues dem-
onstrated massive iron distribution in sertoli
interstial tissues and degeneration of germinal
epithelial cells. Apparent reduction in number of
sperms and spermatogenic cells were also obs-
erved. These symptoms may demonstrate that
prolonged intake of Biscuit fortified with iron
causes certain testicular damage through cert-
ain mechanism.
Keywords: Iron Overload; Oxidative Stress;
Inflammatory Response of Rat Testis
1. INTRODUCTION
Iron is an essential element for many cellular activities
like oxygen transport, electron transfer [1] and gene reg-
ulation. However, an increase in cellular iron content
may be toxic due to generation of reactive oxygen spe-
cies (ROS) that damage proteins, lipids and DNA [2,3].
Iron deficiency anemia represents a common nutri-
tional problem affecting large sector of individuals all-
over the world. Accordingly iron fortified diet was con-
sidered by some nutritionists as an effective tool and
long term strategy to combat such kind of anemia.
This program was adopted and applied during last
decades in different countries using different bakeries
and breads fortified with Iron. Objectors to these idea
stated that the possibility of toxicity from excess iron
absorption of fortified diet was too great and thus such a
measure should not be sponsored.Ten years ago Egyp-
tian ministry of education, through its distributed insti-
tutes had adopted iron fortification program for different
sectors of students especially young ages. This was
achieved through daily supplementation to students of
iron fortified biscuits (0.3% w/w). Till now this program
was not evaluated properly, taken in consideration that
additive iron here is greatly high. Healthy individuals
may however face iron storage state even with moderate
fortification of diet with iron, inducing inturn many va-
rieties of chronic diseases [4]. Continuous iron intake
may contribute to generation of free radicals and oxida-
tion of cell components, an example is hepatocellular
damage [5].
Testicular functions like liver cells are particularly
vulnerable to such kind of injury and mostly mediated
by reactive oxygen species (ROS) in consequence to iron
overload. Oxidative damage here can either affect sperm
cells or influence spermatogenic process which could
change sperm functions. ROS can also activate transc-
ription factors as nuclear factor-kappa B (NF-kB) which
upregulates the transcription of adhesion molecules, cy-
tokines and Enzymes as collective factors, mostly cont-
ributing to inflammatory response [6]. Chronic iron
overload (CIO) can enhance proliferation and increase
TNF-α. The latter can also induce nitric oxide (NO)
production from the activated polmorphonuclear leuko-
cytes in response to tissues inflammation [7,8]. NO is an
inorganic reactive nitrogen species synthesized in liver
by iNos located in hepatocytes, kupffer, endothelial cells
[9]. It represents a hepatoprotective mechanism against

M. M. El-Seweidy et al. / Natural Science 2 (2010) 551-556
552
CIO toxicity [10]. Decreased level of Testosterone in
male suffering from idiopathic hemochromatosis was
reported before [11]. Androgenic deficiency as mention-
ned there not only produced by pituitary failure but also
by testicular dysfunction due to hemosiderin deposition
in testes. Oxidative stress may suppress also steroido-
genesis. This is through substantial decrease in mRNA
of steroid Acute regulatory protein (Star) as well as ac-
tivities of testicular ∆5-3ß and 17-ß hydroxysteroid de-
hydrogenases [12]. Therefore present study aimed
mainly to illustrate the effect of prolonged intake of iron
enriched biscuits on oxidative stress, inflammation, tes-
ticular injuries and its degree in experimental rats. This
may add great focus on such program having higher for-
tification degree with iron.
2. MATERIALS AND METHODS
A total of 30 wistar male albino rats (180 ± 20 g, 3 mon-
ths old) were supplied by the Egyptian Organization for
Biological Products and Vaccines (Helwan farm, Cairo).
Rats were housed in stainless steel rodent cages and kept
under environmentally controlled conditions and allow-
ed one week for acclimatization at room temperature
with a 12 h dark/light cycle before starting the experim-
ental work. During such time animals received normal
rat chow (El-Nasr Pharmaceuticals Chemicals, Egypt),
and allowed free access of drinking water.
3. WORK DESIGN
The rats were then divided into 2 groups, first one re-
ceived in addition to normal chow, iron enriched biscuits
(0.3% ferrous sulphate w/w) [13] daily for ten weeks (Fe
group). Second group received iron free biscuits and
served as control. This protocol was approved by the
animal care and use committee of the Biochemistry De-
partment, Faculty of Pharmacy, Zagazig University.
Both groups, approximately consumed equal quantities
of biscuits, its main components were: wheat flour, eggs,
Butter, cane sugar and vanillin as flavor. Ten weeks after
feeding, rats were fasted overnight and blood was col-
lected via retro-orbital bleeding. Serum was prepared,
and aliquots were stored at –20℃ for later determina-
tions of: iron, ferritin, TNF-α, testosterone and nitric
oxide (NO).
Rats were killed by dissection. Testicular tissues were
removed, rinsed with cold normal saline, divided into
parts and dried with filter paper. First part was quickly
frozen in liquid nitrogen (–170℃) then stored at –20℃
for determination of biochemical parameters: malondial-
dehyde (MDA), Glutathione (GSH) Hydroxyproline
(Hyp) and iron contents.
The other part was kept in 10% formalin-saline at 4ºC
for 1 week, subsequently dehydrated with a series of
ethanol solution from 75 to 100% before embedding in
paraffin. Cross sections (5 µm thick) were stained with
hematoxylin and eosin (H & E) for routine light micros-
copy assessments, Perl’s Prussian blue stain to localize
deposited iron and Mallory trichrome stain to illustrate
collagen fibers.
4. ANALYTICAL PROCEDURES
I: Serum iron was determined Colorimetrically by using
The kits supplied by spinreact, (S.A., Spain) [14], NO as
nitrite [15]. TNF-α was measured by using ELISA kits
supplied by Biosource Int. (California, USA) [16]. Se-
rum testosterone and ferritin were measured by electro-
chemiluminscence immunoassay “ECLIA” using com-
mercial kits (Roche Diagnostics, USA) and Roche Elec-
sys 2010 immunoassay analyzers [17,18].
II: Testicular tissues: MDA was determined spectro-
photometrically by using thiobarbituric acid, TBA [19].
0.5 G tissue was homogenized in 5 ml phosphate buffer
(PH = 7.2), centrifuged at 2000 g for 10 min, supernatant
fraction was used for MDA determination. GSH content
was determined spectrophotometrically using Ellman’s
reagent according to modified method [20]. 0.1 G of
tissues was homogenized in 1 ml phosphate buffer (PH =
8) at 4℃. 0.5 ml of homogenate was mixed with 0.5 ml
of 10 percent TCA in 5 mM EDTA sodium, mixed well
and centrifuged at 2000 g for 5 min. Supernatant was
used for determination of reduced GSH. Iron content
was determined by flame atomic absorption Spectro-
photometer [21]. About 0.1 G tissues was digested in
2ml conc Nitric acid and 2 ml perchloric acid at room
temperature for 24 hours, the samples were filtered, di-
luted and absorption was measured at 248 nm. Hyp was
determined spectrophotometrically by Ehrlich reagent
[22]. 0.1 G tissues was pulverized ground, 500 ul of 6 N
Hcl were added and incubated overnight at 120 c. 5 ul of
acid hydrolysate were mixed with 5 ul of citrate acetate
buffer and 100 ul chloramines T in ELISA plate and
incubated for 20 minutes at room temperature before
addition of Ehrlich solution.
5. STATISTICAL ANALYSIS
Analysis was carried out by using SPSS program for
windows version 10 (SPSS, Chicago, USA). All results
are presented as “Mean ± SD”, student “t” test was used
for the comparison between groups. Statistical signifi-
cance was defined at P < 0.05.

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553
6. RESULTS
Rats received biscuits enriched with iron (BEI) demon-
strated significant increase in serum iron, ferritin, TNF-α,
NO and decreased testosterone level (p < 0.05) (Table
1). Testicular contents of MDA, Hyp, and total iron
demonstrated significant increase while GSH showed
significant decrease (p < 0.05) (Table 2).
Serum iron showed positive correlation with ferritin
r = 0.8, TNF-α r = 0.57 and negative one with testoster-
one r = –0.72 (p < 0.05). Testicular iron content was
positively correlated with MDA r = 0.948, Hyp r = 0.978
and negatively with GSH r = –0.861 (p < 0.05).
The histopathological changes associated with Fe are
illustrated in (Figures 1-6).
Table 1. Serum parameters of rats received iron fortified biscuits.
Item Control BEI Group
Iron (mg/dl) 264.6 ± 44.5 518.45 ± 90.2#
Ferritin (ng/ml) 4.83 ± 0.76 10.58 ± 90.2#
Testosterone (ng/dl) 123.63 ± 24.91 43.92 ± 9.3 #
NO (µmol/l) 34.23 ± 4.69 66.37 ± 6.38 #
TNF-α (pg/ml) 130.98 ± 7 213.6 ± 4.33#
# Significantly different from control at P < 0.05.
Table 2. Testicular parameters of rats received iron fortified
Biscuits.
Item Control BEI
MDA (nmol/g tissue) 669.17 ± 49.64 1439 ± 70.58#
GSH (nmol/g protein) 38.09 ± 1.93 18.63 ± 2.51#
Hyp (µg/g tissue) 130.98 ± 7 213.6 ± 4.33#
Iron (µg/gtissue) 122.67 ± 7.84 265.17 ± 11#
# Significantly different from control at P < 0.05.
Figure 1. A photomicrograph of adult male albino rat
testis (control group) showing seminiferous tubules
(arrow) separated by interstitial tissue (double arrow)
(H & E × 100).
Figure 2. A photomicrograph of adult male albino
rat testis of iron overload group showing adhesion of
seminiferous tubules. Some tubules revealed disor-
ganized germinal epithelium (arrow). Extensive area
of exudates can be seen (double arrow) (H & E ×
100).
Figure 3. A photomicrograph of adult male albino
rat testis (control group) showing distinctive bound-
ary formed of collagen fibers (Mallory trichrome ×
100).
Figure 4. A photomicrograph of adult male albino
rat testis (iron overload group) showing multiple
collagen fibers (Mallory trichrome × 100).
Figure 5. A photomicrograph of adult male albino
rat testis (control group) showing negative Perl’s
Prussian blue stain (Perl’s Prussian blue × 400).

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554
Figure 6. A photomicrograph of adult male albino rat
testis (iron overload group) showing positive Perl’s
prussian blue stain (arrow) in interstitial tissue (Perl’s
Prussian blue × 400).
7. DISCUSSION
As mentioned above prolonged intake of iron enriched
diet induced significant increase in serum iron and fer-
ritin levels, in agreement with reported studies [23,24].
Previous report suggested that iron overload in rats can
specifically activate target genes in the liver (i.e., L fer-
ritin and procollagen) [25]. Similar study supported this
suggestion, illustrated an increased expression of the
heavy-chain isoform of ferritin mRNA in liver of iron
overloaded rats [26]. Others suggested that the elevated
non-transferrin bound iron (NTB iron); observed in iron-
overloaded diseases may reflect two factors. First one,by
increasing saturation degrees of transferrin it will reduce
the number of plasma binding sites for NTB iron .This
may increase the total plasma iron fraction in the NTB
iron pool. The second factor may be liver damage [27].
Testicular iron content demonstrated significant in-
crease (Table 2), in agreement with previous studies
[28,29] which considerd testicular tissues as secondary
target for accumulated iron. This observation also im-
plicates a regulation of iron transport which greatly dif-
fers from liver. It considers also that testis produce their
own transferrin, and participates in iron shuttle system
which fulfill the requirements of this metal for sper-
matogenesis [30].
Pro-oxidant effects of iron in testicular tissue are rea-
sonable factor which mediates MDA increase and GSH
decrease [31]. Other studies demonstrated GSH deple-
tion, reduced activities of antioxidant enzymes like glu-
tathione peroxidase, catalase, and superoxide dismutase
[32]. In vitro study reported also similar findings [33].
Testicular content of Hyp demonstrated also signific-
ant increase in consistent with. Previous study [34] wh-
ich may be due to increased activity of prolyl hydroxy-
lase [35]. It was demonstrated before that iron overload
induced moderate fibrosis in testes interstitium, intersti-
tial macrophages, fibroblast and Leydig cells [36] mean-
while present study configurated similar findings (Fig-
ure 4).
Last years many studies has been oriented to illustrate
iron-induced oxidative stress, its effect on activation of
the transcription factor nuclear factor-kappa B (NF-kB)
[37]. NF-kB exists in an inactive state in the cytosol,
usually as two subunits, p50and p65, complexed with
inhibitory proteins (IkB). Degradation of inhibitory pro-
teins can translocate NF-kB to the nucleus where it alters
transcription of a number of genes involved in cell
growth, differentiation, inflammation, and immune func-
tion. Such study suggested that NF-kB activation can be
modulated by the redox status of the cell [38]. ROS can
increase NF-kB activation while, antioxidant compounds
including iron chelators can inhibit it [39].
NF-kB activation also plays a key role in the inflam-
matory process and upregulates the transcription of ad-
hesion molecules, cytokines and enzymes involved in
the inflammatory responses [40]. Present study demon-
strated significant increase in TNF-α of iron group and,
in agreement with recent study [13].
TNF-α may also activate NO production from the ac-
tivated polymorphnuclear leukocytes in response to tis-
sue inflammation [6]. The presence of nitrogen reactive
species can explain the iron sequestration pattern which
characterizes macrophages under inflammatory condi-
tions.It may refer also to a complex relationship between
iron and NO so that iron overload can also increase iron
uptake by the liver Kupffer cells while No increase is
mostly attributed to activated nitric oxide synthase [41].
NO expression is controlled also by the redox-sensit-
ive transcription factor NF-KB [10], acting in iron over-
load state as hepatoprotective agent against iron toxicity.
This may be associated with increased NF-kB DNA
binding to the iNOS gene promoter and higher expres-
sion and activity of iNOS mRNA [42].
Decreased serum testosterone in Fe group may be at-
tributed to haemosiderin deposition in the pituitary gland
[43] or cytotoxic effects of iron on the gonadotrophs
[44].
Reported histopathological study indicated that exces-
sive iron deposition caused degranulation of the adeno-
hypophysiocytes and decreased hormone storage within
such cells [45], additionally iron deposition in the ante-
rior lobe of the pituitary gland can decrease its response
to gonadotropin-releasing hormone [12]. Involvement of
oxidative stress in the suppression of steroidogenesis
was also reported before. This may be due to substantial
reduction in the mRNA of steroid acute regulatory pro-
tein as well as activities of testicular Δ5-3β and 17-β hy-
droxysteroid dehydrogenases (HSD) through strong af-
finity of divalent heavy metal to the thiol groups of these
proteins and enzymes [46].
Other reported studies indicated that TNF-α induced

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an early and transient increase in serum luteinizing hor-
mone (LH) , followed by a transient decrease in serum
testosterone levels, while FSH demonstrated no change
[47].
8. CONCLUSIONS
Taken together, the present study indicates that iron ove-
rload causes impairment in testicular activity and affects
the androgenicity of male rats .This may be a reflection
of iron deposition in testicular tissues, a matter which
deserve great focus and attentions regarding such dietary
program which have higher fortification degree with
iron.
9. ACKNOWLEDGEMENTS
We acknowledge the financial support (Research grant) for the study
presented from Biochemistry Department, Faculty of Pharmacy-Zagazig
University.
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