Histological Study of Gonads Tissues Adult Stage Artemia salina (Linnaeus 1758) and Immunohistochemistry by Caspase 3 and HSP70 to Detect Specific Apoptosis Markers on Gonads Tissue After Exposure to TBTCl
Najla Mohamed Abushaala(1*), Abdulfattah Mohamed Elfituri(2) and Syaizwan Zahmir Zulkifli(3)
1- Zoology Department, Faculty of Science, University of Tripoli, Tripoli, Libya
2- Marine Biology Research Centre, Tajoura, Tripoli, Libya
3- Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Background: Several types of research have been recently performed on the biological effects of TBTs, including investigations of genitals in invertebrates in response to exposure to TBTs in marine water.
Aim: The objective of this research to investigate acute effects of TBTCl on gonad in the adult stage of Artemia salina by use normal histology and immunohistochemistry (IHC) (Caspase 3 and HSP70) to see specific apoptosis markers.
Methods: After exposure of A. salina to different concentrations of TBTCl (25, 50, 100, 200, and 300 ng.L-1), 50 adult A. salina (25 male and 25 female) were selected randomly from each concentration to study histological gonad. The gonad tissue was sectioned (5µm) and some slides stained with hematoxylin and eosin and others stained with IHC Avidin-Biotin Complex (ABC-Kit), were examined under a light microscope.
Results: The results showed significant differences (P < 0.05) in histological lesions between different concentrations of TBTCl. The histological lesions in the testis and ovary section were undifferentiated cells, degenerating yolk globules and follicle cells enveloping the oocyte which was then compared with control tissue, and these effects were found to be increasing in females more than in males with the highest concentration of TBTCl. IHC showed that positive immunostaining was observed in testis and ovary as brownish deposits to Caspase 3 and HSP70 antibody after exposure to TBTCl. While testis and ovary section in control tissue had no immunoreactivity to Caspase 3 and HSP70 antibody; these effects were profoundly increasing with the highest concentration of TBTCl in females more than in males. Finally, the histological lesions and IHC (Caspase 3 and HSP70) revealed that the apoptosis and immune system stress of A. salina gonad tissue damage in females to be more sensitive to TBTCl toxicity as compared to white males.
Conclusion: In general, the present study is aimed to observe the effects TBTCl on A. salina gonad by using histological sections and IHC (Caspase 3 and HSP70), which were evaluated for the first time and have been proven to possess an important function in apoptosis marker and immune system stress in Artemia. Finally, the specific mechanisms through which TBTCl affects A. salina Caspase 3 and HSP70 expression need further investigation.
Keywords: Tributyltin chloride; A. salina; Apoptosis; Immune system stress; Caspase 3; Heat shock protein; Ecotoxicology.