1. Role of Se+Zn in Regeneration (Ki67)
Following Pb Toxicity in Wistar rat.
B.A Falana, O.M Ogundele, F.I Duru,
A.A.Osinubi, D.T Falode

2. Introduction
• Lead poisoning is a serious health [reblem in
the developing countries, (WHO 2002)
• Causes reproductive toxicity via the
suppression of spermatogenesis and
Androgenesis in males Apostoli and Catalini 2011), (Benoff et
al., 2000; Akinloye et al 2006 ), Fukushima et al., (2005), Yi Lu ( 2009),
Salgado et al (2010).

3. Introduction
• This study addresses the effect of Pb toxicity
on the germinal epithelium and the
proliferative effect of selenium and zinc.

4. Overall aim
• The aim of this study is to determine
the effect of selenium and zinc on
lead-induced testicular damage in
Sprague-Dawley Rats.

5. The Specific objectives
• The specific objective of this study are to
determine the effect of
(a) Selenium and zinc supplementation on the
activities of Ki 67 (a proliferation marker)
following treatment with lead
(b)Selenium and zinc on the activities of P53
and CaD in lead induce-testicular damage

6. Statement of Problem
• Lead poisoning in Nigeria (Doctors without borders 2010)
• Occupational and Environmental Health
• Developed and Developing Countries
• Acute and Chronic exposure

7. Background
• The Germinal epithelium
• Blood-testis barrier
• Lead, Selenium and zinc ( Antioxidants)

8. Methods
• 60F1 generation of Wistar rats were divided
into four groups 1-4
• Group 1: Normal saline control
• Group 2: Pb only
• Group 3: Pb+Se+Zn
• Group 4: Se+Zn

9. Histology
• Embedded tissues were sectioned to
obtainn 7 microns thickness for
routine H&E (Kareema et al., 2012)

10. Immunohistochemistry
• Paraffin wax embedded sections were mounted
on a glass slide in preparation for antigen
retrieval.
• Immersed in Urea overnight and placed in
microwave for 45 mins
• Primary and secondary antibodies application
( Biotinylated goat serum for 1hr. And 1%
Bovine serum albumin)

11. Results

12. Fig. 1(a-d): General morphology of the germinal
epithelium of wild type adult wistar rats (stained with
Hematoxylin and Eosin) (a) Control, (b) 100 mg kg-1 Pb-
Acetate, (c) Pb-Acetate+Se+Zn and (D) Se+Zn only,
Degeneration was most prominent in the lead treatment
group, cell proliferation is most prominent in the Se+Zn
treatment group. (‡) represents regions of cell
proliferation and (†) represents regions of cellular
degeneration, (n) represents normal cells of the
epithelium, (BM) basement membrane, arrow head
indicates the lumen of the seminiferous tubule (Scale bar

14. Fig. 2(a-d): p53 Immunohistochemistry (for cell cycle
dysregulation) in the germinal epithelium of wild type
adult Wistar rats, (a) Control, (b) 100 mg kg 1 Pb-Acetate,
(c) Pb-Acetate+Se+Zn and (d) Se+Zn only, The Pb
treatment groups showed reduced p53 activity (2B)
compared with the control (2A). The basement membrane
(rich in spermatogonia and stem cell-lines) showed
increased p53 activity in the Pb+Se+Zn (2C) and the Se+Zn
treatment groups (2D), (†) represents regions of Ki-67
immunopositivity and (†) represents regions of cellular
degeneration, (n) represents normal cells of the
epithelium, (BM) basement membrane, arrow head
indicates the lumen of the seminiferous tubule (Scale bar
is 30 μm)

15. Fig. 3(a-d): Cathepsin D (CAD) immunohistochemistry
in the germinal epithelium of wild type adult Wistar
rats, (a) Control, (b) 100 mg kg 1 Pb-Acetate, (c) Pb-
Acetate+Se+Zn and (D) Se+Zn only. CAD activity is
high in the BM of the control (3a), in the BM and
degenerating cells of the lead treated group (3b), in
the BM of Pb+Se+Zn and it is widely diffused in the
epithelial cells of the Se+Zn treated group (3d). (‡)
represents regions of CAD immunopositivity and (†)
represents regions of cellular degeneration, (n)
represents normal cells of the epithelium, (BM)
basement membrane, arrow head indicates the
lumen of the seminiferous tubule (Scale bar is 30
μm)

17. Fig. 4(a-d): Ki-67 Immunohistochemistry (for cell
proliferation) in the germinal epithelium of wild type
adult Wistar rats, (a) Control, (b) 100 mg kg-1 Pb-
Acetate, (c) Pb-Acetate+Se+Zn and (d) Se+Zn only.
Cell proliferation marked by Ki-67 immunopositivity
is higher in the Se+Zn treatment group (Fig. 4d),
followed by the control (4a). (‡) represents regions
of Ki-67 immunopositivity and (†) represents regions
of cellular degeneration, (n) represents normal cells
of the epithelium, (BM) basement membrane, arrow
head indicates the lumen of the seminiferous tubule
(Scale bar is 30 μm)

18. Literature review
• Se+Zn supplementation attenuates lead-
induced reproductive toxicity in rats (Falana and
Oyeyipo 2012)
• Primary mechanism of Pb Toxicity is majorly
induction of oxidative stress via inhibition of
allosteric sites in metalloenzyme ALP, CcOx:
Kumari et al 2013, Musatoy and Robinson 2012)

19. Literature review
• Molecular oxygen reacts with electrons from
the reduction of food substances to generate
ROS
• The primary role of ROS is peroxidation of
lipids (Humphrey et al.,2012)
• The first response involes production of
mtNOS to counter the no that is formed from
the reaction of ROS wth nitrogen containing
groups (Ekici et al., 2012)

20. Literature review
• This study evaluates the role of Pb as
an agent capable of inducing
degeneration via cell cycle proteins
as well as role of selenium and zinc
2.25mg/kg BW as agents capable of
reducing such toxicity by evaluation
of proliferation

21. Literature review
• Degeneration and toxicity can be accessed
through mitochondria and cytoplasmic pathways
by measuring immunohistochemically the level of
expression of P53 (a 53 Kda Tumor supressor
protein that is usually activated in the Gap phases
of the cell cycle to proof read the genome for
errors such as cleavage and deletions which are
induced by Pb toxicity
( National Toxicology programme 2007)

22. Literature review
• P53 is a nucleolase that will digest the genetic
materialsin case of such errors ( Matteo et al
1995
• The Mitochondrial pathway can be tracked via
the caspase 3 and 9 syytems through the P21
shunt to Cathepsin D(CAD) ( Zeng et al)

23. Discussion
• The cytoplasmic pathway was observed to be
the most predominant in lead toxicity,
although oxidative stress is primary, it is
important to distinguish the resultant forms of
cell death in the germinal epithelium
• Apoptotic pathway was predominant as seen in
the lead treated group 2( Fig 1b,2b,3b, and 4b),
although necrosis was observed in the sertoli
cells closer to the BM

24. Discussion
• The expression of P53 was found to
be prominent in the BM region
• This suggests alteration in membrane
integrity and in essence structural
conformation of the BM and the
junctional complexes that forms the
basis of the barrier.

25. Discussion
• Although toxicity was observed in group treated with
Pb the Se and Zn(Se+Zn),it was observed that greatly
reduced.
• The observed cellular changes were limited to slight
enlargement in cell size with no obvious
cytorchitectural alteration thus conforming that at
moderate doses Se+Zn can reduce the toxicity of Pb
either by functioning as co-factors to activate radical
scavengers or by functioning as as a competitor to
reduce the ability of Pb to alter the allosteric sites in
oxidative enzymes (Fig 1c,2c,3c and 4c)

26. Discussion
• A second control was set upto confirm the
proliferative effect of se+zn trace on normal
germinal epithelium without toxicity.
• It was observed that the se+zn induced cell
proliferation (fig 1d), and ki67
immunohistochemistry figure 4d is similar to
the findings of Chen et al., 2013

27. Discussion
• Cathepsin D and P53 expression were also
greatly reduced showing that neither the
cytoplasmic or mitochondria pathway was
activated nor oxidative stress induced and if at
all induced was not significant enough to
cause ant obvious structural damage

28. Discussion
• The proliferative effect of (Se+Zn)
inki67 studies where theSe+Zn (fig
4d) was more immuno positive for
the protein than the control (4a)

29. Summary and Conclusion
• Pb toxicity can follow a mitochondria pathway
(CAD) or a cytoplasmic pathway involving
P53, the most predominant form of cell death
is apoptosis which can result from both
pathways.
• Se+Zn treatment improves proliferation and
counters Pb toxicity by substitution, activation
of enzymes and Growth factors, Endothelial
factors, and radical scavengers.

30. Thanks
• Thanks for listening.

31. References
• Apostoli P and Catalini, S (2011). Metal ions Affecting Reproduction and
Development. Met ions life sci.8: 263-303
• Adamopoulos DA., Pappa A, Nicopoulou, S. et al., (1996). Seminal
volume and total sperm number trends in men attending sub-fertility clinics
in the greater Athens area during the period 1977-1993. Reprod.,9, 1936-
1941
• Atar D, Backx,PH, Appel MM. et al .,(1995). Excitation-Transcription
coupling mediated by zinc influx through voltage-dependent calcium
chanels.J.Biol.Chem, 270,2473-2477
• Becker, S. and Berhane, K (1997) A met-analysis of 61 sperm count studies
revisited. Fertil.steril., 67,1103-1108
• Bennof, S, Jabbob, A and Hurley, R (2000).Male fertility and
environmental exposure to lead and cadmium. Human reproduction update
(6) 2:107-121
• Jenny.P. glusker, Amy K. Kats and Charles W. Bock (1999).Metal ions in
biological system .The Rigaku Journal 16(2):1-10

32. References
• Benin AL, Sargent JD., DaltonM, et al., (1999).High Concentrations of
Heavy metals in Neighborhoods near Ore smelters in Northern
Mexico.Environ.Health Perspect.,107,279-284
• Buchancova, J.,Knizkova, M.,Hyllova, D.et al (1994) Content of selected
trace elements (Al, As,Cd,Cu,Fe,Hg,Mn,Ni,Pb,Zn) in blood urine, hair of
blood donors without occupational exposure to theses metals.Cent
Eur.J.Public health,2,82-87
• Carlsen, E.,Giwercman, A.,Keiding,Net al (1992). Evidence for decreasing
quality of semen during the past 50 years.Br.Med.J.305, 609-613
• Falana, B.A and Oyeyipo, I.P (2012). Selenium and Zinc Attenuate Lead-
induced reroductive toxicity in male Sprague-Dawley Rats. Research
Journal of Medical Sciences 6(2):66-70
• Fisch, H and Goluboff E.T (1996).Geographic Variations in Sperm Counts:
A Potential Cause o Bias in Studies of Semen Quality. Fertil.Steril.65,1044-
1046

33. References
• Fukushima et al., (2005). Effects of Male Reproductive
Toxicant on Gene Expression in Rat Testes.J.Toxicol.Sci.
30(3):195-206
• Hidiroglou M, and Knipfel J.E (1984). Zinc in Mammalian
Sperm: A Review.J.Diary. Sc.i 67:1147-1156
• Glusker, JP, Kats AK, and Bock C.W (1999).Metal ions in
Biological Systems. The Rigaku Journal 16(2):1-10
• Matzui MM, and Lamb DJ. Genetic Dissection of
Mammalian Fertility Pathways.Nat.med 8 suppl:533-540
• Markku Saaranen (1990). Glutathione Peroxidase and Some
Metal ions in Male Reproductive System 69,(5): 453-454
• Merker HJ and Gunther T.(1997). Testis Damage Induced
by Zinc deficiency in Rat. J.Trace element 11:19-22

34. References
• Miura T, Ando A, Miura C, Yamauchi K .(2002). Comparative Studies
Between Invivo and Invitro Spermatogenesis of Japanesse Eel(Anguina
japonica). Proc Natl Acad Sci USA 88:5774-5778
• Miura T, Higuchi M, Ozaki Y, Ohta ,T and Miura C.(2006) .Progestin is an
essential factor for the initiation of meiosis in spermatogenic cell of the
eel.Proc. Natl .Acad.Sci .USA 103:7333-7338
• Miura T, Yamauchi K, Takahashi H, and Nagahama, Y (1991) Hormonal
Induction of all the Stages of Spermatogenesis invitro in the Male Japanese
Eel (Anguilla japonica) Proc. Natl .Acad .Sci. USA ,88:5774-5778
• Morisawa M, and Mohri H. (1972).Heavy Metals and Spermatozoon
Motility. I. distribution of iron, zinc, and copper in sea urchin spermatozoa.
Exp.Cell.Res.70:311-316
• Salgado, E.N et al.,(2010) Metal Templated design of protein interfaces.
Proc.Natl. Acad .Sci .USA ,107:1827-1832

35. References
Ogunlesi Modupe(2009) Determination of the
concentration of zinc and vitamin c in oyster and some
medicinal plants used to correct male factor fertility.
Journal of Natural Product 2:89-97.
Yamamoto et al.,(2005). Protein expression analysis of
rat testes induced testicular toxicity with several
reproductive toxicants. The Journal of Toxicological
Sciences 30(2):111-126
Yi Lu (2009). Metal ions as matchmakers for protein.
Current Issue 107(5):234-239.
Xiao X, Mruk DD, Cheng FL, Cheng CY (2012). C-Src and
C-Yes are two unlikely partners of spermatogenesis and
their roles in blood-testes barrier dynamics. Adv Exp
Med Biol. 763: 295-317.