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
Introduction• Lead poisoning is a serious health [reblem inthe developing countries, (WHO 2002)• Causes reproductive toxicity via thesuppression of spermatogenesis andAndrogenesis in males Apostoli and Catalini 2011), (Benoff etal., 2000; Akinloye et al 2006 ), Fukushima et al., (2005), Yi Lu ( 2009),Salgado et al (2010).
Introduction• This study addresses the effect of Pb toxicityon the germinal epithelium and theproliferative effect of selenium and zinc.
Overall aim• The aim of this study is to determinethe effect of selenium and zinc onlead-induced testicular damage inSprague-Dawley Rats.
The Specific objectives• The specific objective of this study are todetermine the effect of(a) Selenium and zinc supplementation on theactivities of Ki 67 (a proliferation marker)following treatment with lead(b)Selenium and zinc on the activities of P53and CaD in lead induce-testicular damage
Statement of Problem• Lead poisoning in Nigeria (Doctors without borders 2010)• Occupational and Environmental Health• Developed and Developing Countries• Acute and Chronic exposure
Background• The Germinal epithelium• Blood-testis barrier• Lead, Selenium and zinc ( Antioxidants)
Methods• 60F1 generation of Wistar rats were dividedinto four groups 1-4• Group 1: Normal saline control• Group 2: Pb only• Group 3: Pb+Se+Zn• Group 4: Se+Zn
Histology• Embedded tissues were sectioned toobtainn 7 microns thickness forroutine H&E (Kareema et al., 2012)
Immunohistochemistry• Paraffin wax embedded sections were mountedon a glass slide in preparation for antigenretrieval.• Immersed in Urea overnight and placed inmicrowave for 45 mins• Primary and secondary antibodies application( Biotinylated goat serum for 1hr. And 1%Bovine serum albumin)
Fig. 1(a-d): General morphology of the germinalepithelium of wild type adult wistar rats (stained withHematoxylin 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 treatmentgroup, cell proliferation is most prominent in the Se+Zntreatment group. (‡) represents regions of cellproliferation and (†) represents regions of cellulardegeneration, (n) represents normal cells of theepithelium, (BM) basement membrane, arrow headindicates the lumen of the seminiferous tubule (Scale bar
Fig. 2(a-d): p53 Immunohistochemistry (for cell cycledysregulation) in the germinal epithelium of wild typeadult Wistar rats, (a) Control, (b) 100 mg kg 1 Pb-Acetate,(c) Pb-Acetate+Se+Zn and (d) Se+Zn only, The Pbtreatment groups showed reduced p53 activity (2B)compared with the control (2A). The basement membrane(rich in spermatogonia and stem cell-lines) showedincreased p53 activity in the Pb+Se+Zn (2C) and the Se+Zntreatment groups (2D), (†) represents regions of Ki-67immunopositivity and (†) represents regions of cellulardegeneration, (n) represents normal cells of theepithelium, (BM) basement membrane, arrow headindicates the lumen of the seminiferous tubule (Scale baris 30 μm)
Fig. 3(a-d): Cathepsin D (CAD) immunohistochemistryin the germinal epithelium of wild type adult Wistarrats, (a) Control, (b) 100 mg kg 1 Pb-Acetate, (c) Pb-Acetate+Se+Zn and (D) Se+Zn only. CAD activity ishigh in the BM of the control (3a), in the BM anddegenerating cells of the lead treated group (3b), inthe BM of Pb+Se+Zn and it is widely diffused in theepithelial 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 thelumen of the seminiferous tubule (Scale bar is 30μm)
Fig. 4(a-d): Ki-67 Immunohistochemistry (for cellproliferation) in the germinal epithelium of wild typeadult 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 immunopositivityis higher in the Se+Zn treatment group (Fig. 4d),followed by the control (4a). (‡) represents regionsof Ki-67 immunopositivity and (†) represents regionsof cellular degeneration, (n) represents normal cellsof the epithelium, (BM) basement membrane, arrowhead indicates the lumen of the seminiferous tubule(Scale bar is 30 μm)
Literature review• Se+Zn supplementation attenuates lead-induced reproductive toxicity in rats (Falana andOyeyipo 2012)• Primary mechanism of Pb Toxicity is majorlyinduction of oxidative stress via inhibition ofallosteric sites in metalloenzyme ALP, CcOx:Kumari et al 2013, Musatoy and Robinson 2012)
Literature review• Molecular oxygen reacts with electrons fromthe reduction of food substances to generateROS• The primary role of ROS is peroxidation oflipids (Humphrey et al.,2012)• The first response involes production ofmtNOS to counter the no that is formed fromthe reaction of ROS wth nitrogen containinggroups (Ekici et al., 2012)
Literature review• This study evaluates the role of Pb asan agent capable of inducingdegeneration via cell cycle proteinsas well as role of selenium and zinc2.25mg/kg BW as agents capable ofreducing such toxicity by evaluationof proliferation
Literature review• Degeneration and toxicity can be accessedthrough mitochondria and cytoplasmic pathwaysby measuring immunohistochemically the level ofexpression of P53 (a 53 Kda Tumor supressorprotein that is usually activated in the Gap phasesof the cell cycle to proof read the genome forerrors such as cleavage and deletions which areinduced by Pb toxicity( National Toxicology programme 2007)
Literature review• P53 is a nucleolase that will digest the geneticmaterialsin case of such errors ( Matteo et al1995• The Mitochondrial pathway can be tracked viathe caspase 3 and 9 syytems through the P21shunt to Cathepsin D(CAD) ( Zeng et al)
Discussion• The cytoplasmic pathway was observed to bethe most predominant in lead toxicity,although oxidative stress is primary, it isimportant to distinguish the resultant forms ofcell death in the germinal epithelium• Apoptotic pathway was predominant as seen inthe lead treated group 2( Fig 1b,2b,3b, and 4b),although necrosis was observed in the sertolicells closer to the BM
Discussion• The expression of P53 was found tobe prominent in the BM region• This suggests alteration in membraneintegrity and in essence structuralconformation of the BM and thejunctional complexes that forms thebasis of the barrier.
Discussion• Although toxicity was observed in group treated withPb the Se and Zn(Se+Zn),it was observed that greatlyreduced.• The observed cellular changes were limited to slightenlargement in cell size with no obviouscytorchitectural alteration thus conforming that atmoderate doses Se+Zn can reduce the toxicity of Pbeither by functioning as co-factors to activate radicalscavengers or by functioning as as a competitor toreduce the ability of Pb to alter the allosteric sites inoxidative enzymes (Fig 1c,2c,3c and 4c)
Discussion• A second control was set upto confirm theproliferative effect of se+zn trace on normalgerminal epithelium without toxicity.• It was observed that the se+zn induced cellproliferation (fig 1d), and ki67immunohistochemistry figure 4d is similar tothe findings of Chen et al., 2013
Discussion• Cathepsin D and P53 expression were alsogreatly reduced showing that neither thecytoplasmic or mitochondria pathway wasactivated nor oxidative stress induced and if atall induced was not significant enough tocause ant obvious structural damage
Discussion• The proliferative effect of (Se+Zn)inki67 studies where theSe+Zn (fig4d) was more immuno positive forthe protein than the control (4a)
Summary and Conclusion• Pb toxicity can follow a mitochondria pathway(CAD) or a cytoplasmic pathway involvingP53, the most predominant form of cell deathis apoptosis which can result from bothpathways.• Se+Zn treatment improves proliferation andcounters Pb toxicity by substitution, activationof enzymes and Growth factors, Endothelialfactors, and radical scavengers.
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