Impact of sublethal concentrations of Lead Nitrate on histopathology and enzyme activities in the gills of the Indian stinging catfish, Heteropneustes fossilis (Bloch) Archisman Mahapatra1, Sumanta Kar, Arup Mistri, Rahul Kumar Singh, Swati Mittal* Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi -221005, U.P., India 1Presenter. E-mail:
[email protected], *Corresponding author. E-mail:
[email protected] Histology and Enzyme Histochemistry
➢ Heavy metals are essential to maintain the body metabolism, but they show toxicity at higher concentrations via metabolic interference and mutagenesis. They can bioaccumulate through the food chain ➢ Highest permissive value of consumption of Pb is 25 μg/kgBW (WHO). Heteropneustes fossilis is chosen for their easy handling, culture and maintenance in the laboratory. ➢ H. fossilis is considered as a very common freshwater fish used in ecotoxicological studies. 𝐿𝐶50 of 𝑃𝑏(𝑁𝑂3 )2 in H. fossilis is 925 mg/L ( Parashar and Banerjee, 2002). ➢ Gills are chosen for the test organ as it is the primary place for gaseous exchange, ion regulation, and excretion of metabolic waste products. Gills are the first organ targeted by several xenobiotics because they have a very large interface area. It is also reported that heavy metal accumulation in fish tissue is highest in gills (Oladimeji and Offem ,1989).
Fixed tissues were embedded in paraffin wax and serial sections of 6 µM thickness was cut and slides were then stained in Ehrlich’s Heamatoxylin and Eosin (H/E). For enzyme histochemical studies fixed tissues were mounted in 100% OCT and cryosections of 16 µM thickness was cut in cryostat. Enzyme histochemical studies for Alkaline and Acid phosphatases were performed following the azo dye coupling method (Bancroft and Gamble, 2008). Whereas, for enzyme Peroxidase DAB method (Graham and Karnovsky, 1996) was performed and for Catalase we followed the method described by Stoward and Pearse (1991).
Enzymes ALP
ACP
Enzyme assays
The total protein content in gills of three different experimental groups was estimated by the method of Lowry et al. (1951). The activity of Alkaline Phosphatase and Acid Phosphatases was performed followed by Walter and Schutt (1974). The activity of enzymes Catalase and Peroxidase were determined following Worthington Enzyme Manual (1988). All observations were made on a Leitz Wetzlar microscope and digital camera system of Leica.
CAT
PER
Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) (where p < 0.05) using Microsoft Excel.
Maintenance of fish
Healthy specimens of H. fossilis were collected from local fish market and were acclimatized for 15 days in tap water in large plastic aquaria (60 L capacity) under normal photoperiod and temperature (13L: 11D, 28 ± 2 ℃). The heavy metal salt, Lead Nitrate (Pb(NO3 ) 2 ) (99.5% purity; MW 331.23) was used in the experiment. Experiments in all groups were performed simultaneously for 15 days.
Fig 3. : Graphical representation of activity of different enzymes in gills of H. fossilis at different doses of Lead Nitrate
Erythrocyte
+ + + + + + -
++ +++ +++ + ++ +++ +++ ++ + +++ ++ +
➢ Acid Phosphatase helps in tissues reorganization and tissue repair. Elevated level indicates tissue alteration and cell damage (Karan et al.,1998). ➢ Alkaline Phosphatase involved in metabolic processes, such as molecule permeability, growth, cell differentiation and steroidogenesis (Ram and Sathayanesan 1985). Elevated level indicates cellular damage and metabolic disruptions (Karan et al.,1998). Lead act as an activator of both ALP and ACP (Demayo 1981). ➢ Catalase & Peroxidase both are antioxidant enzyme involved in scavenging 𝐻2 𝑂2 thereby inhibiting oxidative stress. Declined level indicating binding of Pb to – SH groups of the enzyme molecule (Gurer and Ercal 2000).
Fig 2. Histopathological changes in gills of H. fossilis exposed to Lead Nitrate
CONTROL (CONT)
H. fossilis
+ + + + -
Mucus cell
Being a vital organ gill reflect the physiological condition of the fish as well as severity of the contamination in the surrounding environment. Alteration in the activity of metabolic enzymes reflects the level of tissue damage and metabolic disruption. Whereas, the increased level of antioxidant enzymes states the condition of oxidative stress. Therefore, the study provides a potential use of H. fossilis as a biomarker for aquatic pollution.
Fig 1. : Experimental model : Indian Stinging catfish (Heteropneustes fossilis)
Experimental Design
CONTROL LOW DOSE HIGH DOSE CONTROL LOW DOSE HIGH DOSE CONTROL LOW DOSE HIGH DOSE CONTROL LOW DOSE HIGH DOSE
Epithelial cell
LOW DOSE (LD)
HIGH DOSE (HD)
Tap Water
0.4 mg/L 𝑷𝒃(𝑵𝑶𝟑 )𝟐 4 mg/L 𝑷𝒃(𝑵𝑶𝟑 )𝟐
Histopathological examination of gills revealed hypertrophy and swelling of epithelial cells, fusion of epithelium of gill filaments and secondary lamellae, and alteration of the structure of secondary lamellae. Biochemical assays and histochemical localization shows pronounced effect on the activity of enzyme Alkaline Phosphatase (ALP) and Acid Phosphatase (ACP) in the gills of both groups of treated fishes. While significant decrease was noted in the activity of enzyme Catalase (CAT) and Peroxidase (PER).
➢ ➢ ➢ ➢ ➢ ➢ ➢ ➢ ➢ ➢ ➢
Fig 4. : Photomicrographs of cross-sections of the gill of H. fossilis, showing localization of the (1) Alkaline phosphatase, (2) Acid Phosphatase, (3) Catalase and (4) Peroxidase.
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