Scholarly article on topic 'Acetylcholinesterase, glutathione and hepatosomatic index as potential biomarkers of sewage pollution and depuration in fish'

Acetylcholinesterase, glutathione and hepatosomatic index as potential biomarkers of sewage pollution and depuration in fish Academic research paper on "Veterinary science"

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Marine Pollution Bulletin
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{Biomarkers / Fish / "Sewage pollution" / Depuration / Acetylcholinesterase / Glutathione}

Abstract of research paper on Veterinary science, author of scientific article — Saif M. Al-Ghais

Abstract The current study was designed to validate the biomarkers of sewage pollution in Mozambique Tilapia (Tilapia mossambica, Peters) reared in sewage treatment plant (STP) effluent in Ras Al Khaimah, United Arab Emerates, before and following depuration/detoxification. Cellular biomarkers, cholinesterase activity using acetylcholine as a substrate (acetylcholinesterase AChE) and reduced glutathione (GSH) and hepatosomatic index (HSI) were investigated in fresh water fish, Tilapia, raised in a fish farm (Group I/Clean, as Control), treated sewage water/TSW (Group II/Sewage) and thereafter exposed to fresh water in an aquarium for 6weeks (Group III/Depurated) for depuration. The results showed significantly lower levels of AChE activities in liver (26% p <0.01) and muscle (30% p <0.01) of the fish reared in the STP water (Group II/Sewage) as compared to those recorded in the fish from fish farm (Group I/Clean). The depressed AChE level was fully restored in the muscle but partially in the liver after depuration (Group III/Depurated). In contrast, GSH levels were significantly raised in both liver (1.3-fold p <0.01) and muscle (4-fold) of Group II fish as compared to Group I (control) fish raised in fish farm and following depuration in fresh water (Group III/Depurated) elevated GSH level in liver restored to control values, while remained unchanged in muscle. The average hepatosomatic index (HSI=weight of liver×100/total fish weight), an indicator of hepatomegaly, in the Group II fish reared in TSW was also significantly higher than that in the reference Group I fish, but decreased to control level in Group III fish following depuration. This study suggests the importance of cellular biomarkers, AChE, GSH and hepatosomatic index in monitoring the impact of sewage water pollution on fish caused by a complex mixture of chemico-biological contaminants and its mitigation following depuration, an effective mean of fish detoxification.

Academic research paper on topic "Acetylcholinesterase, glutathione and hepatosomatic index as potential biomarkers of sewage pollution and depuration in fish"

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Acetylcholinesterase, glutathione and hepatosomatic index as potential biomarkers of sewage pollution and depuration in fish q

Saif M. Al-Ghais *

Department of Biology, Faculty of Science, UAE University, P.O. Box 17551, Al Ain, United Arab Emirates Environment Protection and Development Authority, P.O. Box 11377, Ras Al Khaimah, United Arab Emirates

ARTICLE INFO ABSTRACT

The current study was designed to validate the biomarkers of sewage pollution in Mozambique Tilapia (Tilapia mossambica, Peters) reared in sewage treatment plant (STP) effluent in Ras Al Khaimah, United Arab Emerates, before and following depuration/detoxification. Cellular biomarkers, cholinesterase activity using acetylcholine as a substrate (acetylcholinesterase AChE) and reduced glutathione (GSH) and hepatosomatic index (HSI) were investigated in fresh water fish, Tilapia, raised in a fish farm (Group I/ Clean, as Control), treated sewage water/TSW (Group Il/Sewage) and thereafter exposed to fresh water in an aquarium for 6 weeks (Group III/Depurated) for depuration. The results showed significantly lower levels of AChE activities in liver (26% p < 0.01) and muscle (30% p < 0.01) of the fish reared in the STP water (Group II/Sewage) as compared to those recorded in the fish from fish farm (Group I/Clean). The depressed AChE level was fully restored in the muscle but partially in the liver after depuration (Group III/Depurated). In contrast, GSH levels were significantly raised in both liver (1.3-fold p < 0.01) and muscle (4-fold) of Group II fish as compared to Group I (control) fish raised in fish farm and following depuration in fresh water (Group III/Depurated) elevated GSH level in liver restored to control values, while remained unchanged in muscle. The average hepatosomatic index (HSI = weight of liver x 100/total fish weight), an indicator of hepatomegaly, in the Group II fish reared in TSW was also significantly higher than that in the reference Group I fish, but decreased to control level in Group III fish following depuration. This study suggests the importance of cellular biomarkers, AChE, GSH and hepatosomatic index in monitoring the impact of sewage water pollution on fish caused by a complex mixture of chemico-bio-logical contaminants and its mitigation following depuration, an effective mean of fish detoxification.

© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Keywords:

Biomarkers

Sewage pollution Depuration Acetylcholinesterase Glutathione

1. Introduction

Fish farming using domestic sewage water i.e. grey water culture, has been practiced for centuries by many cultures across the world (WHO, 1989; FAO-ALCOM, 1994; Nandeesha, 2002; Lee et al., 2010). With the rapid population growth and increasing urbanization wastewater reuse in aquaculture and agriculture is considered to play an important role in reducing the waste product, saving the water, particularly when fresh water resources are fast depleting and closing the nutrient cycle (WHO, 1989). The massive amounts of nutrients in sewage serve as an ideal fertilizer for planktons and algae to flourish and enhance the productivity of the aquatic ecosystem, which serves as valuable food

q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address: Department of Biology, Faculty of Science, UAE University, P.O. Box 17551, Al Ain, United Arab Emirates. Mobile: +971 50 6513513/3735522. E-mail address: alghais@emirates.net.ae

source for fish and other aquatic organisms (WHO, 1989; FAO-AL-COM, 1994; Lee et al., 2010).

However, in today's industrialized society sewage water, raw or even treated, contains a vast numbers of deleterious xenobiotics including heavy metals, pesticides and industrial chemicals, and pathogens, that bio-accumulate in marine organisms and may cause toxicity to fish, handlers and eventually the consumers (Hej-kalet al., 1983; WHO, 1989; Almrothet al., 2008; Stoliarand Lush-chak, 2012). One potential solution to farming fish in sewage water, without residual foul odor and with acceptable levels of harmful chemical toxins and pathogens in the fish body, is a cleaning/detoxification process called "depuration", in which toxins and pathogens are allowed to flush out by keeping the fish in clean water for at least 2-3 weeks before harvest (WHO, 1989; FAO-AL-COM, 1994; Lee et al., 2010). There is a need to further investigate the effectiveness of short-term depuration in removing the chemical and biological toxins from the fish and thus the potential use of depuration as a protective health strategy in aquaculture.

Stress indicators at cellular and tissue levels have been developed in fish and other aquatic organisms in the recent past

0025-326X/$ - see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2013.07.005

to monitor environmental contamination (Al-Ghais and Ali, 1999; Al-Ghais et al., 2000; Lam and Gray, 2003; Facey et al., 2005; Mde-gela et al., 2010; Stoliar and Lushchak, 2012). Tissue cholinesteras-es and non-protein reduced glutathione (GSH), which protects cell against oxidative injury and detoxicates xenobiotics and/or their metabolites, have been validated as pollution biomarkers in fish and other aquatic animals (Otto and Moon, 1996; Al-Ghais and Ali, 1999; Lam and Gray, 2003; Stefano et al., 2008). Recently, attempts made to investigate cholinesterase/AChE activity in fish tissues as early-warning biomarker for the assessment of pollution in ponds/lakes receiving sewage wastewater revealed site- and tissue-specific variations in AChE responses (Lopez-Lopez et al., 2006; Mdegela et al., 2010). Moreover, organ-level biomarkers, liver size (hepatosomatic index, HIS) and macrophage aggregates in the spleen of rock bass, were found to be useful in monitoring harbor contamination with the effluent from sewage treatment plant (Facey et al., 2005). However, much less is known about the responses of cellular biomarkers to aquatic environment contamination with sewage and their potential usefulness in monitoring the depuration of marine organisms grown in the sewage-fed aquaculture.

The current study was, therefore, undertaken to evaluate the of status of cholinesterase(s) active towards acetylcholine, referred to as AChE (Prasada Rao and Ramana Rao, 1984; Rodriguez-Fuentes and Gold-Bouchot, 2004), and non-protein GSH in the liver and muscle, and hepatosomatic index in Mozambique Tilapia (Tilapia mossambica, Peters), a commercially important and relatively resistant species well adapted to grey water aquaculture (De Silva et al., 2004), exposed to fresh water, treated sewage water and follow-up depuration in fresh water in order to validate these cellular bio-markers for monitoring the potential fish toxicity that may be caused by culturing the fish in treated sewage water and the effectiveness of depuration process in sewage-fed aquaculture.

2. Materials and methods

Acetylthiocholine, thiocholine, Ellman's reagent (5,5'-dithio (2-nitrobenzoic acid), DTNB), reduced glutathione (GSH), bovine serum albumin and (Tris[hydroxymethyl]aminomethane) were obtained from Sigma Chemical Co., a division of Sigma-Aldrich Corporation, USA.

Samples (n = 16) of T. mossambica (Peters) raised in the fish farm containing fresh water as a reference group (Group I/Clean, 166 ± 54 g), treated sewage water of Ras Al Khaimah Emirates of the UAE (Group Il/Sewage, 164±22g) and treated sewage water followed by depuration in an aquarium containing fresh water (100 gallons) for 6 weeks (Group Ill/Depurated, 140±22g) were collected using a fish trap during the period November 2009-March 2010. After collection the fish samples were immediately placed in ice bucket and later stored at -20 °C till further investigation.

For estimations of biomarkers the fish were thawed out and their standard length and total body weight were recorded. All experimental manipulations, unless otherwise stated, were conducted at 0-4 °C. Whole liver and a piece of trunk muscle were dissected out, washed with ice-chilled normal saline, blotted dry and weighed. The hepatosomatic index (HSl = weight of liver x 100/total fish weight) was determined. A piece of liver or muscle was weighed, cut into small pieces and homogenized in chilled buffered-KCl (1.15% KCl buffered with 0.01 M Tris-HCl buffer, pH 7.4) with the help of Potter-Elvehjem homogenizer. The homogenate was used to determine AChE activity and GSH content separately in each fish.

The rate of AChE activity was measured photometrically by monitoring the appearance of thiocholine at 412 nm (Ellman

et al., 1961). The reaction mixture (3.0 ml) consisted of 0.05 M Tris-HCl buffer (pH 8.0), 0.34 mM DTNB, 1 mM acetylthiocholine and suitable amount of tissue homogenate. The reaction was followed by measuring the formation of thiocholine-DTNB complex at room temperature (25 °C). The AChE activity was expressed as nmole thiocholine (product) formed/min/mg protein. Protein was determined by the method of Lowry et al. (1951). The tissue content of GSH was measured as non-protein sulfhydryl group using Ellman's reagent, 5,5'-dithio (2-nitrobezoic acid), as described earlier (Sedlak and Linsay, 1968) and expressed as nmole GSH/g tissue. Sulfhydryl content was measured in the supernatant obtained after deproteinization of tissue homogenate with trichlo-roacetic acid and detected by reacting with the Ellman's reagent.

Data Analysis: For comparing and maintaining the uniformity and homogeneity, all the data were transformed into the same units and the results were expressed as mean ± SE. Differences between the groups were compared by Analysis of Variance (ANOVA) and p-values less than 0.05 were considered statistically significant.

3. Results and discussion

As shown in Fig. 1 the average hepatic AChE activity in T. mos-sambica (Peters) reared in treated sewage water (Group ll/Sewage) was significantly lower (26.6% p < 0.01) than that found in the control/reference fish procured from fish farm (Group l/Clean). The depressed hepatic enzyme activity in the fish exposed to TSW was only partially restored following depuration in fresh water for a period of 6 weeks (Group Ill/Depurated). The same trend was found for muscle AChE activity in the three groups of fish (Fig. 2). Muscle AChE activity in sewage-fed fish was also significantly depressed (30.3% p <0.01) as compared to that in control fish. However, depuration of the sewage-fed fish (Group lll/Depu-rated) completely restored the enzyme activity to the reference level observed in Group l fish. These findings are in line with an inhibition of AChE activity in the brain, liver and gill of Girardinich-thys viviparous (Bustamante) introduced into a lake in Mexico receiving untreated domestic wastewater, agricultural runoff and STP effluent (Lopez-Lopez et al., 2006). Likewise, low brain AChE activity was observed in grey mullet (Mugil cephalus) and grass goby (Zosterisessor ophiocephalus) collected from a highly eutrophic Orbetello Lagoon receiving town STP effluent in ltaly (Corsi et al., 2003). As suggested in earlier studies (Lam and Gray, 2003; Corsi et al., 2003; Stefano et al., 2008) these results indicate the presence

Fig. 1. Acetylcholinesterase activity in liver of Tilapia mossambica. Values are the mean ± SE of hepatic enzyme activity in fish (n = 16) raised in fish farm (Clean), treated sewage water (Sewage) and fresh water following sewage water (Depurated). p < 0. 01, Clean vs Sewage.

S.M. Al-Ghais/Marine Pollution Bulletin 74 (2013) 183-186

Fig. 2. Acetylcholinesterase activity in muscle of Tilapia. Values are the mean ± SE of enzyme activity of fish (n = 16) raised in fish farm (Clean), treated sewage water (Sewage) and fresh water following sewage water (Depurated). p <0. 01, Clean/ Depurated vs Sewage.

Fig. 4. Reduced glutathione level in muscle of Tilapia. Values are the mean ± SE of GSH content in muscle of fish (n = 16) raised in fish farm (Clean), treated sewage water (Sewage) and fresh water following treated sewage water (Depurated). p <0. 01, Clean vs Sewage.

<u a S S

= ■o

Fig. 3. Reduced glutathione level in liver of Tilapia. Values are the mean ± SE of hepatic GSH content in fish (n = 16) raised in fish farm (Clean), treated sewage water (Sewage) and fresh water following sewage water (Depurated). p < 0. 01, Clean/Depurated vs Sewage.

of AChE inhibitory neurotoxic chemicals like organophosphates and carbamate pesticides, heavy metals and/or industrial chemicals in the STP effluent investigated. These observations strongly support the importance of Tilapia tissue AChE activity as a bio-marker for the assessment of patho-physiological changes in fish caused by sewage pollution and its mitigation by depuration.

ln order to assess the status of oxidative stress, a pathological process, in T. mossambica exposed to complex mixture of chemicals and pathogens present in the TSW, the level of antioxidant GSH was determined in the liver and muscle of fish belonging to Group l/Clean, Group ll/Sewage and Group lll/Depurated (Figs. 3 and 4). The level of hepatic GSH was found to be significantly higher (31.9% p < 0.01) in the fish grown in TSW than that in the reference fish (Group l/Clean), but decreased following depuration in fresh water (Group lll/) to a level even lower that in the fish from a fish farm (Fig. 3). Notably, muscle GSH content was 4-fold higher in the fish exposed to STP effluent than that recorded in the fish procured from fish farm and remained unchanged following depuration (Fig. 4). An elevated intracellular GSH is probably a cellular adaptive response to protect against the deleterious effects of oxi-dative stress elicited by chemical/biological pollutants present in the sewage water and/or to cope with the increased GSH demand for xenobiotic detoxification. ln a study oxidative stress and anti-

Fig. 5. Hepatosomatic lndex (HSl) of Tilapia. Values are the mean ± SE of HSl of fish (n = 16) raised in fish farm (Clean), treated sewage water (Sewage) and fresh water following sewage water (Depurated). p < 0.01, Clean/Depurated vs Sewage.

oxidant enzyme activities were measured in Rainbow Trout (Oncorhynchus mykiss) caged for 14 days at different sites in a river in Sweden polluted by sewage treatment plant (STP) effluent and highly contaminated sediment from industries (Almroth et al., 2008). ln line with our observations, exposure of rainbow trout to STP effluent caused an increase in total (tGSH) and oxidized glu-tathione (GSSG) in liver as compared to the values recorded at reference site, while exposure to contaminated sediment caused no change in glutathione level indicating specificity in glutathione response to sewage pollution. The rise in hepatic glutathione content was attributed to an observed increase in the level of mRNA level of r-glutamylcysteine synthetase, the rate limiting enzyme in the biosynthesis of glutathione. lncreasing biological pollution in water bodies resulting from anthropogenic water eutrophication has also been implicated in an induction of oxidative stress. An increase in oxidative stress accompanied by marked elevated hepatic GSH was reported in Silver Carp and Zebra Fish exposed to toxin-producing bacteria e.g. enteric and cyanobacteria (Blaha et al., 2004). These studies suggest that the induction in GSH level, particularly 4-fold in Tilapia muscle, is caused by both chemical and biological pollutants present in sewage water and muscle GSH may be considered as a potential specific biomarker for sewage pollution.

Fig. 5 shows that Tilapia raised in treated sewage water exhibited a significantly greater (28.8% p <0.01) hepatosomatic index

(HSI) than that found in control fish procured from the fish farm and the increase in HSI reversed completely following depuration of sewage-fed fish. These findings support high efficacy of depuration process in reversing the hepatomegaly by flushing out of the fish the causative deleterious chemical/biological pollutants. Previous studies have recorded higher HSI values in Grey Mullet (M. cephalus) and Grass Goby (Z. ophiocephalus) from a Lagoon receiving STP effluent (Corsi et al., 2003) and in African Sharptooth Catfish (Clarias gariepinus) from sewage ponds (Mdegela et al., 2010) as compared to the values found in the reference fish collected from non-polluted sites and suggested its importance as a potential biomarker of chlorinated and aromatic hydrocarbons. In another comparative study a good correlation was observed between the HSI values in Rock Bass (Ambloplites rupestris) collected in 1992 and 1999 from Burlington Harbor, USA, receiving city's main STP effluent and high level of pollution in 1992 and low in 1999 following STP up-gradation in 1994 (Facey et al., 2005).

In summary these observations suggest the importance of Tilapia tissue AChE, GSH and HIS as potential biomarkers in monitoring the sewage pollution and its impact on the pathophysiology of fish. These results support that the depuration process might be a very effective practice for detoxification of fish raised in grey water culture.

Acknowledgements

Thanks are due to United Arab Emirates University, Al-Ain and Natural Resources Research Center (NRRC), Ras Al Khaimah for the support to this Research Project. All the persons, who have assisted and helped in this project, are thankfully acknowledged.

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