Introduction

Contamination of the environment with anthropogenic compounds is a widespread phenomenon and occurs both from fixed and diffused source. Pesticides carried away by rains and floods to larger water bodies like ponds and rivers alter the physico-chemical properties of water. Cypermethrin, the alpha-cyano-3-phenoxybenzyl ester of 2,2-dimethyl-3-(2,2-dichlorovinyl) cyclopropane carboxylic acid is a widely used pesticide based on pyrethroids and account for almost over 30 % of the global pesticide use (Dahamna et al. 2011). Although synthetic pyrethroids are less persistent and less toxic to mammals and birds, they are highly toxic to a number of non-target organisms such as bees, freshwater fish and other aquatic organisms even at a very low concentration (Oudou et al. 2004).

Biomarkers so called biological responses to xenobiotics have been studied as environmental pollution indicators and used as early warning systems of environmental contamination (Walker et al. 1996). The biochemical marker that is best studied so far is the stimulation of cytochrome P450-dependent monooxygenases (CYP 450) which is also considered the most dominant enzyme system responsible for oxidation processes in phase I biotransformation. Payne and Penrose (1975) were among the first to make use of this enzyme system as a biomarker, reporting elevated CYP 450 activity in fish from petroleum-contaminated sites in the aquatic environment and members of CYP 450 gene families 1–4 are considered prominent in xenobiotic metabolism. Thus, CYP 450 enzymes are important in the mechanisms underlying chemically induced toxicity or disease and chemical–ecological interactions. Fish species are useful as bioindicators, since they are promptly exposed to diverse anthropogenic water contaminants and in the fish, CYP 450 has primarily been studied as a biomarker indicating pollution of the aquatic environment by industrial or agricultural sewage. However, responses to xenobiotics in fish may differ from those in other species (Siroka and Drastichova 2004).

This work was aimed to evaluate the effects of cypermethrin on CYP1A, CYP2B, CYP2E1and CYP3A4 enzymes as biomarkers in Heteropneustes fossilis. Standardization of biomarkers for detection of sub-lethal effects of cypermethrin in this species under laboratory conditions will enable biomonitoring of cypermethrin in the environment.

Materials and Methods

Heteropneustes fossilis were collected from non-contaminated local ponds of Gossaipur of Darjeeling district. Fishes were acclimatized in the laboratory for a period of 2 weeks. After 2 weeks, healthy fish (male and female) weighing approximately 35 ± 5 g were transferred to glass aquarium with 50 l capacity in controlled light (12 h light/12 h dark), and aeration conditions. The fishes were fed regularly with small pieces of chopped fish during acclimatization and feeding stopped during the course of treatment.

The 96 h LC50 value was calculated using probit analysis (Finney 1971). Fish were randomly taken in 5 groups (10 fishes in each aquarium of 20 l capacity). The 96 h LC50 value for H. fossilis was determined as 3.783 μg/l (data not published). One group of the experimental fish (5–7 fish) was treated with sub lethal concentration (1.2 μg/l) of cypermethrin, Ripcord 10 % EC (1/3 of LC50 value) for 15 days. The water was renewed every 2 days and the desired concentration of the pesticide was maintained before introducing the treated group while only water was changed for the control groups.

Similarly, another group was administered with a single IP (intraperitoneal) injection of 0.03 mg/kg body weight of the pesticide. The 96 h IP LC50 value was determined as 0.090 mg/kg body weight (data not published). The pesticide was diluted with distilled water and the positive (+VE) control fishes received only an IP injection of distilled water in the same volume as the injected group. Homogeneity was maintained in all the groups by providing similar experimental conditions. Two treated and the +VE control groups were studied for the experiment to determine the stress level, specificity and interaction of the pesticide with respect to the control.

The fish were sacrificed by a single blow on the head and then livers were excised, weighed and the liver somatic index (LSI) was determined as percentage ratio of liver weight to body weight. The liver somatic index was calculated by using the formula: LSI = (liver weight/total live weight) × 100. As the liver samples were too small to be processed individually for enzyme activity, they were pooled before homogenization.

Microsomes were isolated using the procedure described by Chang and Waxman (1998), with minor modifications. Livers were perfused with a large volume of ice cold perfusion buffer (1.15 % KCl, 1 mM EDTA, pH 7.4) to get rid of unwanted tissues, fat bodies and blood and homogenized in four volumes of homogenization buffer (1.15 % KCl, 1 mM EDTA and 0.05 M Tris, pH 7.4) using a Teflon homogenizer. The homogenate was centrifuged in a cooling centrifuge at 12,000×g for 20 min. The supernatant was ultra centrifuged at 100,000×g for 60 min at 4 °C in a super speed vacuum centrifuge. The pellet were resuspended in two volumes of resuspension buffer containing 0.05 M Tris, 1 mM EDTA and 20 % Glycerol v/v, pH 7.4 to obtain the hepatic microsomal fraction.

Analysis of CYP 450 was based on the method described by Omura and Sato (1964) with minor modifications. The microsomal preparation was diluted in 0.1 M phosphate buffer and saturated with carbon monoxide for 40 s. The sample was equally divided between two cuvettes. The baseline (400–500 nm) was recorded and a few milligrams of sodium dithionite were added to only one of the cuvette in order to reduce CYP450 and the absorbance measured in a spectrophotometer.

Protein in the microsomal fraction was estimated as described by Lowry et al. (1951). The values of protein were determined from the standard curve prepared from OD values against different concentrations of bovine serum albumen standard.

EROD (ethoxyresorufin O-deethylase) was determined by spectrophotometric method of Klotz et al. (1984). The reaction mixture consisted of (0.1 M) NaCl, (2 µM) 7-ethoxresorufin, (0.1 M) tris buffer-pH 7.8 and 100–200 µg of microsomes. After pre-incubation at 30 °C for 5 min the reaction was initiated by the addition of (0.01 M) NADPH and the reaction was stopped following 10 min of incubation at the same temperature with 0.5 ml of ice cold trichloroacetic acid. After incubation, the amount of resorufin formed was measured at 572 nm.

Procedure of Schenkman et al. (1967) was adopted with minor modifications to determine the N,N-dimethylaniline (N,N-DMA) demethylase and aniline hydroxylase activity. The reaction mixture for N,N-DMA activity consisted of the (0.1 M) phosphate buffer-pH 7.4, (0.1 M) N,N-dimethylaniline, (0.15 M) MgCl2, (0.1 M) semicarbazide and microsomes (1.0 mg). The mixture were incubated for five minutes at 32 °C and the reaction started by adding (0.1 M) NADPH instead of NADPH generating system (NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and MgCl2). Following aerobic incubation for another 30 min the reaction was terminated by adding 0.5 ml each of 25 % zinc sulfate and saturated barium hydroxide. After centrifugation at 10,000×g for 10 min, 1 ml of the supernatant was mixed with 2 ml of double strength Nash reagent and incubated at 60 °C for 30 min. Formaldehyde formed as the end product of N,N-dimethylaniline demethylase activity was measured by the method of Nash (1953) at 412 nm.

The reaction mixture for aniline hydroxylase activity consisted of (0.1 M) aniline, (0.1 M) MgCl2, (0.12 M) tris-pH 7.4 and 1.0 mg of microsomal protein. The reaction mixture temperature was raised to 32 °C by incubation in a water bath for 5 min and the reaction was started by adding (0.1 M) NADPH. The reaction was terminated after 30 min by adding 0.5 ml of ice cold trichloroacetic acid. The precipitate was removed by centrifugation at 10,000×g for 10 min and 1 ml of supernatant was added to 1 ml of a solution containing 2 % phenol in (0.2 M) NaOH and 1 ml of (1 M) Na2CO3. After 30 min of incubation at 32 °C, the p-aminophenol formed as the end product of aniline hydroxylase activity was measured at 630 nm.

Erythromycin demethylase activity was measured following the method of Werringloer (1978) at 412 nm. The reaction mixture consisted of (0.05 M) phosphate buffer-pH 7.25, (0.01 M) erythromycin, (0.15 M) MgCl2 and microsomes (1 mg). Reactions were initiated by the addition of (0.1 M) NADPH, and the mixture was incubated for 10 min at 32 °C. The amount of formaldehyde formed was measured follwing the method of Werringloer (1978) at 412 nm. The reaction was terminated by adding 0.5 ml of 20 % trichloroacetic acid. All assays were done in duplicate and repeated twice.

Differences between groups of animals or treatments were analysed using one-way analysis of variance (ANOVA) followed by Least Significant difference (LSD) test. The statistical significance was tested at 1 and 5 % levels.

Results and Discussion

Both the treated groups showed significant stimulation (p < 0.05) with respect to the control in the total CYP450 content (Table 1). The 15 days sub-lethal treated group showed higher stimulation (0.523 nmol/mg protein) than the 96 h IP treated group (0.509 nmol/mg protein). The +VE control group did not show any significant difference with the control group. The increase in CYP 450 content after the treatment positively correlated with the increase in the enzymatic activity.

Table 1 Total CYP 450 content and liver somatic index (LSI) of H. fossilis
Full size table

Both the treated groups and the +VE control group showed marginal increase in LSI with respect to that of the control but the means were not statistically significant. When all the groups were considered, the 96 h IP treated group showed higher LSI value than the 15 days sub-lethal treated and the control groups (Table 1).

Table 2 shows the enzymatic activity reflecting stimulation of CYP1A, CYP2B and CYP2E1 and inhibition of CYP3A4 isozymes. The treated groups, i.e. 96 h IP treated and the 15 days sub-lethal treated showed similar trend in the enzymatic activities when compared with the control in all the 4 enzyme activities studied—EROD, Aniline hydroxylase, N,N-dimethylalinile demethylase and Erythromycin demethylase. Similar results were also displayed by the control and the +VE control group. No significant difference in the enzyme activities was seen between the control and the +VE control (injected with distilled water alone) and also between 15 days sub-lethal and 96 h IP treated groups following injection with cypermethrin.

Table 2 EROD, N,N-dimethylaniline (N,N-DMA) demethylase, aniline hydroxylase (AH) and erythromycin demethylase (ERND) activity of hepatic microsomes in H. fossilis
Full size table

EROD activity was significantly stimulated (p < 0.01) by both the treatment regimes of cypermethrin. The 15 days treated group showed higher stimulation than the 96 h IP treated group. An increase of almost 1-fold in the activity was seen in both the treated group compared to the control and the +VE control (Table 2). Both the treated group showed stimulation in N,N-dimethylaniline demethylase activity compared to the control. The 15 days treated group of H. fossilis showed the highest stimulation with an increase of 225 % while 167 % increase was seen in 96 h treated group (Table 2). Aniline hydroxylase activity was significantly stimulated (p < 0.05) in both the treated group. The 96 h IP treated group of H. fossilis showed the highest stimulation with an increase of 260 % than that of 15 days treated group with 139 % when compared with the control. No stimulation in the CYP3A4 was shown by both the treated groups of fish studied. Also there was no significant difference in the activities of CYP 450 isozymes between the control and the +VE control.

The carbon monoxide difference spectra of dithionite reduced liver microsomes of H. fossilis were studied at 1 min interval over a period of 5 min. The maximum absorbance was noticed at 1–2 min and then a gradual decrease in absorbance was seen with increase in time interval. This time interval corresponds to the length of incubation required for maximum reduction of the enzyme by sodium dithionite. The CYP 450 converts to a stable denatured state CYP 420 under long periods of incubation thus adding to the absorbance at 420 nm. Two soret peaks were seen in the dithionite reduced spectra of liver microsomes (Fig. 1). These peaks consisted of the characteristic absorbance at 450 nm for the reduced CYP450-CO complex and the other of lesser magnitude at around 420–425 nm which may be possibly due to the absorbance of contaminating hemoglobin or CYP 420 (Klemz et al. 2010).

Fig. 1
figure 1

Carbon monoxide difference spectra of dithionite reduced liver microsomes of H. fossilis at 1 min interval over a period of 5 min

Full size image

LSI has frequently been used as a biomarker for examining fish exposed to environmental contaminants and also to determine the physiological status of the fish. LSI values are generally elevated in vertebrates experiencing stimulation of hepatic microsomal CYP 450 and the elevation in LSI may be due to the altered allocation of energy reserves for detoxification of organic compounds (Miller et al. 2009).

The biochemical responses assessed in the present study are the earliest indicators of exposure to stressors that can be detected in an organism. CYP 450 metabolizes and biotransforms most lipophilic xenobiotics including drugs, pesticides, carcinogens and environmental pollutants into more polar metabolites that can be easily excreted from the body and are useful as early warning indices of environmental alteration, so that remedial actions may be taken by resource protection managers in time to prevent permanent decline in fish populations (Thomas 1990).

It has been detected that variability of enzyme activity is higher if the organism in test is exposed for longer duration to the xenobiotic or pollutants as it brings about the physiological differences within the organism (Arellano-Aguilar et al. 2009). This may be the reason that the activities of CYP450 is slightly higher in 15 days sub-lethal treated compared to the 96 h IP treated group. Although an IP injection of test agents is not an environmentally relevant route of exposure, however, for pharmacological considerations, it is a common practice of exposure to chemical in animals as it allows a direct effect on the target organs (Rice and Roszell 1998). Fish can also be subjected to desired concentrations of pesticides through injection to cut down the time of exposure and also for understanding the effects on biotransformation enzymes (Assis et al. 2009).

CYP 450s, principally the CYP1A isoform have been used to detect the presence of pollutants in aquatic environment due to its high affinity to xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) and have been so far proved to be the most sensitive indicators (Schlenk and Di Giulio 2002). Although the evaluation of CYP1A stimulation by EROD activity is a common tool for quantifying environmental exposure to aryl hydrocarbon receptor (AhR) ligands, a number of studies have been conducted that establishes CYP1A stimulation in response to pesticides. Lemaire et al. (2010) have reported induced level of EROD activity in liver microsomes of roundnose grenadier in response to DDT exposure. Similar results have been confirmed in fishes exposed to deltamethrin (Assis et al. 2009) and chlorpyriphos (Rai et al. 2010).

Earlier studies have reported that Phenobarbital, specific inducer of CYP2B in mammals have not been found to induce CYP2B in fish (Bhutia et al. 2010), although CYP2B like activities do occur in fish as reported by Stegeman et al.(1997). It may be possible that pesticides do induce CYP2B like activities (Price et al. 2008) in fish. Though specific CYPs are necessary to metabolize xenobiotics, pesticides have the ability to induce one or more forms of CYP 450 simultaneously.

Aniline hydroxylation by cytochrome P450-dependent enzyme systems has been shown to be a possible way for metabolism in the liver of fish species. Aniline hydroxylase activity is suggested to be mainly catalyzed by enzymes belonging to the CYP450 2E1 sub-family in fish as in mammals and studies have reported an stimulation of CYP2E1 activity upon exposure to pesticides carbaryl (Tang et al. 2002) and parathion (Mutch et al. 2003).

Both the treated groups showed significant inhibition in erythromycin demethylase activity although CYP3A is reported to be induced in Labeo rohita exposed to chlorpyriphos (Rai et al. 2010). It is seen that CYP3A4 is the predominant isoform responsible for metabolizing pyrethroid pesticides in mammals (Scollon et al. 2009), thus, a potential explanation for the high pyrethroid toxicity in fish can be due to the inhibition of CYP3A4 isoform in the liver and other tissues.

The present study demonstrates evidence of CYP 450 related xenobiotic metabolism in the liver of H. fossilis exposed to cypermethrin. The CYP 450 mediated catalytic activity may also be a very efficient adaptive strategy of the species to increase tolerance to the pesticides and guarantee its survival. Although field trials were not done, based on the laboratory results it was concluded that at least the CYP450 mediated enzyme activities in H. fossilis could serve as a useful biomarker of cypermethrin pollution in the aquatic environment.