Effect of temperature, salinity, pH and naphthalene on ethoxyresorufin-O-deethylase activity of Oreochromis mossambicus

Abstract

Hepatic ethoxyresorufin-O-deethylase (EROD) activity of Oreochromis mossambicus was examined in response to naphthalene, a polycyclic aromatic hydrocarbon (PAH) as a bioindicator of exposure. This study also examined the effects of varying water parameters such as salinity, temperature, and pH on this fish model. Temperature, salinity, and pH produced cyclic changes in EROD activity which increases and/or decreases. After exposure to lower naphthalene concentrations up to 6 ppm, no marked change in activity was noted, whereas at higher concentrations EROD activity was increased. Data suggest that EROD measurement may be useful as a potential biomarker for the detection of hydrocarbon pollution.

Keywords:

• ethoxyresorufin-O-deethylase
• Oreochromis mossambicus
• pH
• salinity
• temperature
• ethyl naphthalene
• enzyme
• substrate

Introduction

Currently aquatic systems are being used as dumping grounds for a variety of pollutants. Such xenobiotics accumulate within tissues of aquatic organisms due to either, direct exposure from water and sediments or, indirectly through the food chain, and result in different levels of impairments (Subramanian and Amutha 2006a). Fish, as a bioindicator species, play an increasingly important role in the biomonitoring of water pollution because they respond to more sensitive changes of the aquatic environment. Sudden fish mortality is indicative of the acute level of toxicity due to pollutants. Sublethal levels of pollutants following chronic exposure produce adverse physiological effects and tissue damage in vital organs including gill, liver, intestine, and kidneys, which can be measured by biochemical, physiological, or histological measures in fish (Mondon, Duda, and Nowak 2001; Subramanian and Amutha, 2006). Cytochrome P450 (CYP450) are the diverse multigene family of heme-containing proteins that oxidize, hydrolyze, or reduce compounds by inserting an oxygen atom into the substrate during the reaction cycle (Nebert, Puga, and Vasiliou 1993; Nelson et al. 1996). CYP 1A1 is the reliable biomarker for the polycyclic aromatic hydrocarbons (PAH) (Amutha et al. 2009) and ethoxyresorufin is a substrate for this enzyme. PAH are derived from petroleum, spilled fuel, street run-off, and coal tar from coal gasification, creosote treatment of wood, or coke ovens at steel plants (Ringuette et al. 1993). The metabolism of xenobiotics mainly occurs in liver (Daly et al., 1993; Guengerich et al. 1995).

The monooxygenase enzyme system as well as the conjugating enzymes serves to detoxify and eliminate many pollutants from the body. The monooxygenase enzymes decrease the lipid solubility of organic contaminants and thereby help to facilitate excretion of toxicants. Most of these enzymes are synthesized or activated in the liver on exposure to certain organic contaminants and thus may be used as bioindicators of specific contaminant exposure. There are two phases in the detoxification process. A polar reactive group is introduced into the organic contaminant during Phase I and thereby increasing its solubility in water to excrete or make it a more suitable substrate for Phase II conjugation reaction enzyme (Adams 1990; Arun and Subramanian 2007). Over 90% of Phase I reactions are mediated by CYP450 (Lewis, Loannides, and Parke 1998) The CYP450 gene responsible enzymes activate the oxidative metabolism of compounds including drugs and environmental pollutants. CYP450 formerly termed the “mixed function oxidases” are important oxidative enzymes in Phase I metabolism of the xenobiotics (Lewis 2001). Metabolism or biotransformation through Phase I (CYP450 dependent monooxygenase enzymes) and Phase II (conjugating enzymes) pathways is an important requisite for detoxification and excretion of lipophilic chemicals (Andersson and Förlin 1992; Arun et al. 2000). CYP4501A dependent monooxygenase ethoxyresorufin-O-deethylase (EROD) has an important function in the biotransformation of many xenobiotics and its specific induction is considered as a molecular marker in fish for exposure to PAH and planar halogenated hydrocarbons (PHH), which are usually aryl hydrocarbon receptor agonists (Stegeman and Hahn 1994; Bucheli and Fent 1995; Jewett et al. 2002).

CYP1A induction was observed with benzene, naphthalene, anthracene, acenaphthene, benzo [g,h,i,a] perylene, and fluorene, but low induction was found with fluoranthene and phenanthrene (Kennedy and Jones 1994; Fent 2003; Wang et al. 2006). CYP4501A dependent enzymes were found to be induced by polychlorinated biphenyls (PCB), PAH, dibenzofurans, and dibenzodioxins in mammals (van der Weiden et al. 1989; Lemaire-Gony and Lemaire 1992; Ueng et al. 1997). In the light of the above, this study was designed to investigate the induction of EROD by one of the hydrocarbons, naphthalene, under different dynamic aquatic conditions such as salinity, pH, temperature, and duration of exposure using Oreochromis mossambicus as a fish model.

Materials and methods

Animal selection

The exotic, edible euryhaline, omnivore fish, O. mossambicus, is a sturdy fish known to tolerate a wide range of aquatic environmental parameters. In general, tilapia survive in pH ranging from 5 to 10 but do best at pH range 6–9 and Mozambique tilapia grow well at salinities near or at full strength seawater (Popma and Masser 1999). Immature O. mossambicus fish of approximate uniform size were segregated from the stock (collected from the River Cauvery and maintained in 2 ton cement cisterns) and their length and weight (length 9–12 cm and weight 18–24 g) were measured. Fish were acclimated to lab conditions for 2 days, divided into groups of 10 individuals each and placed in tubs of 25 L of experimental medium.

CYP1A1 dependent EROD assay

Cytochrome P450 1A1 enzyme activity was measured using the EROD activity assay. EROD activity was determined from the rate of formation of resorufin from ethoxyresorufin according to the method of Lester et al. (1993) and Lewis (2001). Frozen liver tissue was ground into fine powder and 100 mg of sample centrifuged for 5 min at 450 ×g. The sample was decanted, supernatant discarded, and pellet collected. Lysis buffer equal to five times the cell pellet volume was added, incubated for 15 min in ice and was subsequently transferred for homogenization. The homogenate was centrifuged at 700 ×g for 10 min at 4°C, supernatant collected, and centrifuged at 10,000 ×g in a microcentrifuge for 30 min at 4°C. The supernatant is the cytosolic fraction used for analysis.

EROD assay against environmental factors

The oscillating water parameters, such as salinity, temperature, and pH are the important factors that modulate both the biological and physiological activities of in situ organisms. These parameters may affect enzymatic reactions. Thus different groups of fish were subjected to varying predetermined ranges of salinity (1–10 ppt with an interval of 1 ppt), temperature (25–40°C with an interval of 5°C) pH (6–10 with an interval of 1 pH) and differing concentrations of naphthalene from 2 to 14 ppm over a time period of 12 and 24 h exposure. The experiments were run in duplicate with a positive and negative control for reference.

Results

Temperature

Fish liver EROD activity at 25°C (10.9 ± 0.51 pmol mg⁻¹ protein min⁻¹) peaked at 35°C (29.87 ± 0.94 pmol mg⁻¹ protein min⁻¹) then declined at 40°C (17.94 ±0.26 pmol mg⁻¹ protein min⁻¹) (Figure 1). Although water temperature increased hepatic EROD activity up to 35°C there was a decrease at 40°C.

Figure 1. EROD activity in the liver cytosol of O. mossambicus exposed to different temperatures.

pH

In the case of pH, lower EROD activity was noted at pH 6 (7.6 ±0.42 pmol mg⁻¹ protein min⁻¹) and the activity gradually rose to attain a maximum at pH 10 (25.14 ± 2.11 pmol mg⁻¹ protein min⁻¹) (Figure 2).

Figure 2. EROD activity in the liver cytosol of O. mossambicus exposed to different pH.

Salinity

At 1 ppt salinity the EROD activity was very high (19.84 ±1.89 pmol mg⁻¹ protein min⁻¹), fell at 2–6 ppt (1.5 pmol mg⁻¹ protein min⁻¹) and then increased at 8 and 10 ppt (5.14 pmol mg⁻¹ protein min⁻¹) (Figure 3).

Figure 3. EROD activity in the liver cytosol of O. mossambicus exposed to different salinities.

PAH (naphthalene) exposure

At 12 or 24 h naphthalene exposures, there was an increase EROD activity with lower concentrations (up to 6 ppm). Subsequently, at 12 h exposed fish liver showed a significant but gradual rise in EROD activity at higher (8–14 ppm) concentrations. In contrast the 24 h exposed fish exhibited a decrease at 8 ppm with subsequent mortality (Figure 4). Further, it was noted that in 12 h naphthalene exposure EROD activity rose with increase in concentration. Whereas in 24 h naphthalene exposure, the increase in EROD activity (up to 6 ppm) declined at 8 ppm, then fell further at 14 ppm and died.

Figure 4. EROD activity in the liver cytosol of O. mossambicus exposed to different concentration of ethyl-naphthalene after 12 and 24 h.

Discussion

Impact of water parameters on the enzyme activity

The use of CYP450 enzymes induction as an assessment tool for various stressors has increased in recent years. As with most biological phenomena, EROD in the tissues of an organism is influenced by a variety of internal (physiological), external (physicochemical), and temporal (duration) factors besides species specific variations (Addison et al. 1991; Bucheli and Fent 1995; Segner, Scholz, and Bohm 1995; Amutha 2008). Siroka et al. (2005) showed that CYP450 1A1 EROD is one of the most suitable biochemical biomarkers of aquatic hydrocarbon pollution. Based on the dose related inducible efficacy of organic xenobiotics, the CYP450 1A1 gene dependent enzyme, 7-EROD was activated to reduce lethality, and is thus often used as a biomarker for monitoring organic (hydrocarbon) pollution (Andersson and Förlin 1985; Payne et al. 1985; Haux and Förlin 1988; McMaster et al. 1991). Thus EROD activity in fish liver was measured in response to some environmental conditions.

EROD activity in response to temperature

EROD activity exhibited an increasing trend from 25 to 35°C exposure, then, declined in higher temperature which may be due to enhanced metabolic activity at higher temperatures. Similar strong positive EROD activity enhancement against temperature was reported in the dab fish Limanda limanda (Goksøyr et al. 1994). Hanson and Larsson (2007) also demonstrated increased EROD activity in response to a rise in water temperature in Perca fluviatilis. At higher temperature (30°C and 35°C), the heat stressed fish enhanced EROD for defense, but induction fails at higher temperature (40°C). This may be attributed to protein (enzyme) denaturation at 40–60°C (Lange et al. 1998; Van Laack and Lane 2000). Minchin, McIntosh, and Davies (2002) suggested that the optimum temperature for most fish is typically between 20 and 25°C and pH at 7.8.

EROD activity in response to pH

EROD activity increases with rise in pH which indicates that O. mossambicus species is able to tolerate higher alkaline conditions (Popma and Masser 1999) and is in agreement with Van Laack and Lane (2000). Since the environmental variables such as temperature and pH influence induction of EROD these need to be routinely measured throughout a study (Andersson and Koivusaari 1985; Willis, Edwards, and Addison 1991; Sleiderink et al. 1995; Arun et al. 2003).

EROD activity in response to salinity

Elevated EROD activity was observed at both low (1 ppt) and high (8–10 ppt) salinity which indicates that 2–6 ppt salinity was ideal for this species beyond which (both lower and higher side) is stressful. Subramanian, Sambasivam, and Krishnamurthy (1980) reported similar effects.

EROD activity in response to PAH (naphthalene) exposure

Studies on the leaping mullet (Liza saliens) and common sole (Solea vulgaris) found a relationship between hepatic EROD activities and the concentrations of PAH in the water (Arin and Sen 1999; Gravato and Santos 2002). A similar relationship was noted in this study between EROD and naphthalene concentrations and is in agreement with findings of Orrego et al. (2005). Wherein at low concentrations there is EROD induction but beyond the optimal level PAH are stressful and produce death. Thus, the concentration and duration of the toxin exposure are factors for EROD induction (Pohl and Fouts 1980; Shimada et al. 2003; Kirby et al. 2007).

Acknowledgments

The authors wish to record their thanks to Ministry of Earth Sciences, Government of India for the financial support, and DST-FIST and UGC-SAP for instrumentation facilities.