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Components of antioxidative system in Allium cepa as the toxicity monitor of trichloroethylene (TCE)

Shams Tabrez^a; Masood Ahmad^a

a Faculty of Life Sciences, Department of Biochemistry, AMU, Aligarh 202 002, India First published on: 16 July 2010

To cite this Article Tabrez, Shams and Ahmad, Masood(2011) 'Components of antioxidative system in Allium cepa as the toxicity monitor of trichloroethylene (TCE)', Toxicological & Environmental Chemistry, 93: 1, 73 — 84, First published on: 16 July 2010 (iFirst)

To link to this Article: DOI: 10.1080/02772248.2010.498375 URL: http://dx.doi.org/10.1080/02772248.2010.498375

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Vol. 93, No. 1, January 2011, 73–84

Components of antioxidative system in Allium cepa as the toxicity monitor of trichloroethylene (TCE)

Shams Tabrez and Masood Ahmad*

Faculty of Life Sciences, Department of Biochemistry, AMU, Aligarh 202 002, India

(Received 19 March 2010; final version received 25 May 2010)

Trichloroethylene (TCE) is a potent hepatocarcinogen (National Cancer Institute, USA) and is widely used as a metal degreasing agent. Concentration- dependent effect of TCE exposure on Allium cepa (onion) bulbs for 48 h was determined on the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GPx), ascorbate peroxidase (APx), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR). Levels of hydrogen peroxide (H2O2), ascorbate (ASC), and glutathione (GSH) were also measured in onion homogenates to monitor TCE-induced oxidative stress.

At 200 ppm TCE concentration, the activities of GST and GR in exposed onion bulbs exhibited roughly three-fold induction compared with control, whereas reduction in activities at higher levels of TCE concentration was recorded. A two- fold induction of CAT with a simultaneous and significant induction in activities of APx, GPx, and MDHAR at increasing TCE concentrations was observed at 150 ppm of TCE exposure. Minimum level of induction was observed for SOD (79%) and DHAR (63%) at 250 and 300 ppm of TCE, respectively. Results indicate that GST, GR, APx, and CAT inA. cepasystem could serve as potential indicators of TCE-induced enzymatic perturbations.

Keywords: Allium cepa; trichloroethylene; antioxidant enzymes; metabolites;

cycloheximide; indicators

Introduction

An increase in industrialization and urbanization over the past four decades resulted in increased demands of chemicals, which eventually caused deterioration of environment.

With particular reference to trichloroethylene (TCE) vis-a-vis Aligarh, India, a small survey through Mitra Nagar locality in this city revealed that several ailments in the local population could be attributed to TCE exposure (Dua 2003; Saxena 2009). This chemical is used as a cleansing agent by the city’s lock and brass industry. According to the estimates of Aligarh office of Uttar Pradesh Pollution Control Board (UPPCB), there were at least 125 units using this chemical in Aligarh (Dua 2003). TCE has been shown to exhibit various types of toxic responses in animal and plant kingdoms. Several workers have described the effects of TCE on microsomal mixed function oxidase system in animal models (Kawamoto et al. 1988; Bloemen et al. 2001; Kumar et al. 2001; Vidal, Basseres, and Narbonne 2001).

*Corresponding author. Email: [email protected]

ISSN 0277–2248 print/ISSN 1029–0486 online ß2011 Taylor & Francis

DOI: 10.1080/02772248.2010.498375 http://www.informaworld.com

Downloaded By: [Ahmad, Masood] At: 06:39 19 November 2010

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Allium cepais the common onion and has been widely used in all parts of the world as a flavoring vegetable. Allium cepa-root-length inhibition bioassay has been recommended for the routine monitoring of water pollution since it is quite sensitive and a valid indicator of toxicity (Fiskesjo¨ 1985). The primary aim of this study was to evaluate the potential of the antioxidant/detoxification enzymes ofA. cepaas a model of TCE pollution in water.

This would further increase the efficacy of theA. cepa test in routine water monitoring studies. Monitoring studies on TCE exposure, in the strict sense of the term are quite insufficient, especially in the plant system despite the fact that plant-based studies are simple, cost effective, and sometimes more sensitive too compared with the animal system (Fatima and Ahmad 2005; Tabrez and Ahmad 2009a). Inhibitor studies with TCE were done in the presence of 50mg mL¹ cycloheximide (CHX), which is an inhibitor of eukaryotic protein synthesis involving 80Sribosomal particles. These studies were carried out to find out whether the increase in the activity of the test enzymes was at the synthesis or activity level. Chloramphenicol (50mg mL¹), another protein synthesis inhibitor acting on the ribosomal system of mitochondria and chloroplasts, was also included in our experiments to ascertain the location of these enzymes.

In our previous study, we recommended that the variation in the antioxidant enzymes ofA. cepacan serve as useful indicators for the detection of heavy metal pollution in water (Fatima and Ahmad 2005). In the present communication, we are reporting a plant-based model employingA. cepafor assessing the TCE pollution in wastewaters.

Materials and methods

TCE was obtained from Sisco Research Laboratories (SRL), India. The small bulbs of A. cepa(red variety) were purchased from the local market. Substrates and reagents for the enzyme analysis were obtained from SRL, India.

Moderate exposure ofA. cepato TCE

The basic protocol of Fiskesjo¨ (1985) was followed with slight modifications. Using a sharp knife, the yellowish brown scales and the bottom plates of the onion bulbs were removed. Test tubes (60 mL in capacity) were filled with different concentrations of TCE and one A. cepa bulb (without root) was placed on the top of each test tube.

The evaporated liquids of test tubes were refilled at 12 h interval. Aquaguard-purified water served as the negative control in all the experiments. The treatment was continued for 48 h in the absence of light in a dark chamber at 20C. After 48 h, the onion bulbs were taken out and used for enzymatic studies. Onion bulbs did not exhibit any visible change as a result of TCE exposure.

Enzyme extraction

Treated as well as untreated A. cepa bulbs were cut into small pieces and homogenized with two volumes of chilled sodium phosphate buffer (50 mol L¹, pH 7.0) containing 1 mol L¹EDTA. The homogenate was squeezed through muslin cloth and the extract thus obtained was centrifuged at 9000gfor 20 min at 4C. The clear supernatant was stored at 0–4C in 5 mL vials and was properly thawed prior to enzyme analysis.

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Enzymes analysis

(a) Superoxide dismutase (SOD): It was assayed by the method of Marklund and Marklund (1974). To 0.1 mL of supernatant 2.9 mL of 0.05 mmol L¹ Tris-succinate buffer, pH 8.2 was added, mixed well and incubated at 25C for 20 min. The reaction was started by adding 0.1 mL of 8 mmol L¹ pyrogallol solution. Change in absorbance per minute was immediately recorded for the initial 3 min at 420 nm. A reference set, containing 0.1 mL distilled water instead of supernatant solution, was also run simultaneously.

(b) Catalase (CAT): CAT was estimated as the decline in absorbance at 240 nm due to the decline of H2O2extinction at 25C as described by Aebi (1984). The reaction mixture contained 50 mmol L¹sodium phosphate buffer (pH 7.0), 30 mmol L¹ H2O2 and 0.1 mL enzyme extract. The decrease in absorbance at 240 nm was immediately noted after every 30 s for 3 min. Enzyme activity was calculated using the molar extinction coefficient of H2O2(436 mol L¹cm¹at 240 nm).

(c) Glutathione reductase (GR): Oxidized glutathione (GSSG) was used as the substrate. The reaction mixture contained 100 mmol L¹ phosphate buffer, 0.5 mmol L¹ EDTA, 1 mmol L¹ GSSG, 0.1 mmol L¹ NADPH, and 0.1 mL enzyme extract. Activity was measured as the decrease in NADPH concentration by recording the absorbance at 340 nm at 25C (Carlberg and Mannervik 1975).

(d) Glutathione-S-transferase (GST): GST activity was assayed by the method of Habig, Pabst, and Jakoby (1974) in 0.2 mol L¹phosphate buffer (pH 6.5) after adding 1 mmol L¹1-chloro, 2,4 dinitro benzene (CDNB) and 1 mmol L¹GSH in the reaction mixture, and following the increase in absorbance at 340 nm due to the formation of CDNB–GSH conjugate.

(e) Glutathione peroxidase (GPx): GPx activity was assayed according to the method described by Mohandas et al. (1984). The assay mixture consisted of phosphate buffer, EDTA, sodium azide, GR (three units), GSH, NADPH, H2O2, and 0.05 mL enzyme extracts. Oxidation of NADPH was recorded at 340 nm.

(f) Ascorbate peroxidase (APx): APx activity was measured by the method of Nakano and Asada (1981). The reaction mixture contained phosphate buffer, sodium ascorbate, and H2O2. The reaction was followed at 290 nm.

(g) Monodehydroascorbate reductase (MDHAR): MDHAR activity was assayed by the method of Hossain, Nakano, and Asada (1984). The reaction mixture contained Tris-HCl, NADH, ascorbate (ASC), and 0.15 units of ASC oxidase. The reaction was monitored at 340 nm.

(h) Dehydroascorbate reductase (DHAR): DHAR was assayed using the method of Hossain and Asada (1984). The reaction mixture contains phosphate buffer, reduced GSH, DHA, EDTA, and 0.1 mL of enzyme extracts. The reduction of DHA to ASC was followed at 265 nm at 25C.

Metabolite determination

(a) Hydrogen peroxide (H2O2): The amount of H2O2 was estimated by the horse radish peroxidase (HRPO)-mediated oxidation of phenol red by H2O2 (Pick and Keisari 1980). Reaction mixture of 200mL homogenates, 20 U mL¹HRPO, 0.28 mmol L¹ phenol red, and 10 mmol L¹ potassium phosphate buffer, pH 7.0 was incubated at 37C for 10 min in fully covered tubes. At the completion of incubation, the reaction mixture was centrifuged for 5 min at 2000g at 4C.

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The supernatant obtained after centrifugation was decanted into a fresh tube and 1 mol L¹NaOH was added to each tube. The samples were read at 610 nm.

(b) Ascorbate (ASC): ASC content was estimated using the bipyridyl method as described by Knorzer, Durner, and Boger (1996). An ASC standard calibration curve was previously run, and an extinction coefficient of 16.5 mol L cm¹ was obtained. For the determination of ASC contents,A. cepahomogenates (125mL) were neutralized with 25mL of 1.5 mol L¹ triethanolamine, and after mixing, 150mL of 150 mmol L¹ sodium phosphate (pH 7.4) and 150mL of water were added. For the determination of total ASC (ASCþDHA), the samples were neutralized, phosphate buffer and water were added, and then 75mL of 10 mmol L¹ dithiothreitol (DTT) was added and incubated for 15 min at room temperature.

The absorbance of the reaction mixture was recorded at 525 nm.

(c) Glutathione (GSH): GSH levels were estimated by the method of Jollow et al.

(1974) in 100mL of A. cepa homogenates using the classical thiol reagent 5,5⁰-dithiobis-2 nitro-benzoic acid (DTNB) [0.1 mol L¹phosphate buffer, pH 7.4]

at 25C. The yellow color developed by the reaction of GSH with DTNB was read at 412 nm.

Determination of protein concentration

Total protein was estimated by the method of Lowry et al. (1951).

Statistical evaluation

Three onions were used per treatment. Data was expressed as meanSD of three replicates from three independent experiments at least and analyzed by one-way ANOVA.

Differences among controls and treatment were determined using Student’st-test.Pvalues of less than 0.05 were considered statistically significant.

Results

Exposure ofA. cepabulbs to TCE resulted in the induction of detoxification enzymes that could be related to oxidative stress caused by TCE rather than its biotransformation.

Figure 1(a) depicts the level of SOD estimated inA. cepabulb after treatment with TCE.

SOD activity showed an increase of up to 79% (P50.001) at 250 ppm of TCE exposure.

However, at 300 ppm exposure a significant fall was observed. Figure 1(b) presents the induction in CAT activity inA. cepaas a result of TCE exposure. A rise of around 120%

(P50.001) in CAT level was observed at 150 ppm of TCE treatment. However, higher doses of TCE exposure showed a sharp decline in CAT activity. Figure 2(a) illustrates GST activity inA. cepabulbs exposed to different concentrations of TCE. The GST activity was also found to be maximum at 200 ppm and was 269 U min¹mg¹ protein compared with control, which was 88.7 U min¹mg¹protein. GST level was found to be inhibited at a higher concentration of TCE exposure. The induction in GR activity was witnessed until 200 ppm (Figure 2b). GR activity was recorded to be maximum at 200 ppm and was 25 U min¹mg¹ protein contrary to the control which exhibited a value of 8.7 U min¹mg¹ protein. Further increase in TCE concentration led to a decrease in GR activity with maximum inhibition at 300 ppm. Figure 3(b) presents the changes recorded in GPx activity consequent upon TCE treatment inA. cepabulbs. The level of

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GPx showed a continuous rise up to the maximum experimental concentration.

At 300 ppm, GPx activity rose up to 110% (P50.001) in comparison to control. APx activity showed roughly 180% (P50.001) induction at 300 ppm of TCE exposure compared to control (Figure 3a). A similar trend of enhancement was also witnessed in the activity profile of MDHAR (Figure 4a), which displayed an induction by 175% at 300 ppm of TCE exposure. DHAR too showed a gradual increase in activity but the rise was only 60% compared with the control value (Figure 4b). Figure (5a) shows the levels of H2O2recorded inA. cepa bulb after treatment with different concentrations of TCE.

H2O2 level showed a maximum decline of up to 70% compared with the control value.

Figure 5(b) and (c) depict the changes in ASC and GSH levels, respectively, inA. cepa treated with various concentrations of TCE. Both ASC and GSH levels also moderately

0 10 20 30 40 50 60

Trichloroethylene concentration

Enzyme activity (U/min/mg protein)

Control 50 ppm TCE 100 ppm TCE 150 ppm TCE 200 ppm TCE 250 ppm TCE 300 ppm TCE (b) CAT

(a) SOD

* **

***

** ** ***

***

**

Figure 1. Effect on (a) SOD and (b) CAT activities inA. cepabulb as a result of 48 h exposure to TCE.

Note: *, **, ***:P50.05,P50.01,P50.001, respectively, compared with control by ANOVA.

0 50 100 150 200 250 300

Control 50 ppm TCE 100 ppm TCE 150 ppm TCE 200 ppm TCE 250 ppm TCE 300 ppm TCE

***

*

** **

***

*** *** ***

(b) GR (a) GST

Figure 2. Effect on (a) GST and (b) GR activities inA. cepabulb as a result of 48 h exposure to TCE.

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dropped upon TCE exposure up to an extent of 50–55%. Figure 6 presents the changes recorded in the test antioxidant enzymes ofA. cepabulbs exposed to TCE in the presence of CHX (50mg mL¹). It is clear from the Figure 6 that CHX brought down the activities of all the test antioxidant enzymes back to the level of untreated controls.

Discussion

Several enzymes of detoxification machinery have been used as the indicators of xenobiotic pollution (Oesch and Arand 1999; Tabrez and Ahmad 2009b, c). As far as our knowledge

0 2 4 6 8 10 12 14 16

Control 50 ppm TCE 100 ppm TCE 150 ppm TCE 200 ppm TCE 250 ppm TCE 300 ppm TCE

**

*

***

*** *** *** *** ***

(a) APx

(b) GPx

Figure 3. Effect on (a) APx and (b) GPx activities in A. cepa bulb as a result of 48 h exposure to TCE.

Note: **, ***:P50.01,P50.001, respectively, compared with control by ANOVA.

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Enzyme activity (U/min/mg ptrotein)

Control 50 ppm TCE 100 ppm TCE 150 ppm TCE 200 ppm TCE 250 ppm TCE 300 ppm TCE (b)DHAR

(a) MDHAR

** **

* **

***

**

Figure 4. Effect on (a) MDHAR and (b) DHAR activities in A. cepa bulb as a result of 48 h exposure to TCE.

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0 0.2 0.4 0.6 0.8 1 1.2

Trichloroethylene concentration (ppm) H2O2 level (micromolar)

Control 50 ppm 100 ppm 150 ppm 200 ppm 250 ppm 300 ppm

***

*

***

(a)

0 1 2 3 4 5 6 7 8 9

Trichloroethylene concentration (ppm)

Ascorbate level (micromolar)

*

**

** ***

***

(b)

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Trichloroethylene concentration (ppm)

GSH level (nmoles/mg protein)

*

**

*** ***

*

***

(c)

***

Figure 5. Patterns of H2O2(a), ASC (b) and GSH (c) levels inA. cepa bulbs as a result of 48 h exposure to increasing the concentrations of TCE.

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on the indicators studies for the TCE is concerned, nobody seems to have done such work in theA. cepasystem.

SOD is an endogenous enzymatic scavenger and constitutes the first line of defense against oxygen-derived free radicals converting the superoxide anion (O₂ ) into H2O2

(Briganti and Picardo 2003; Sezer et al. 2007). Three classes of SODs have been observed in plants; cytosolic and chloroplastic Cu–Zn/SODs, a chloroplastic Fe/SOD, and a mitochondrial Mn/SOD, of which the former two have been shown to be regulated by oxidative stress (Azevedo et al. 1998; Kliebenstein, Monde, and Last 1998). In the present investigation, the rise in the level of SODs inA. cepatissue homogenate was only around 80% compared with control (Figure 1a). Watanabe and Fukui (2000) also reported an increase in SOD activity in mouse liver after TCE treatment. However, a concentration- dependent inhibition in SOD activity in human epidermal keratinocytes was reported by Zhu et al. (2005) as a result of TCE exposure.

CAT and peroxidases are the major enzymes involved in H2O2 detoxification. CAT exhibited a concentration dependent increase up to 150 ppm of TCE treatment (more than two fold) inA. cepa system. After that the activity dropped drastically and attained the level close to that of control at 300 ppm (Figure 1b). Vidal, Basseres, and Narbonne (2001) found an increase in the CAT level in fresh water clams exposed to TCE in aquarium for 5 days. An increase in CAT activity in the livers of mice as a result of TCE intake was also reported by several workers (Elcombe, Rose, and Pratt 1985; Goel et al. 1992; Watanabe and Fukui 2000). A decrease in CAT activity as a result of TCE treatment in rats was also reported by some authors (Elcombe, Rose, and Pratt 1985; Tabrez and Ahmad 2009c).

The GST and GR activities in the TCE exposed A. cepa bulbs have been shown in Figure 2 a and b, respectively. GR participates not only in H2O2 scavenging, but also favors a high GSH/GSSG ratio to maintain a proper cellular redox (Srivastava, Tripathi, and Dwivedi 2004). The role of GST in detoxification is well-documented (Rees 1993;

Fernandes et al. 2002; Ferrat et al. 2003; Tabrez and Ahmad 2009b, c). Interestingly both the GR and GST activity attained a peak at 200 ppm of TCE treatment followed by decline at higher doses. Such pattern of xenobiotic metabolizing enzymes is expected in view of typical tolerance and toxicity of this pollutant. Tabrez and Ahmad (2009c) also reported a rise in GR level as a result of TCE intake in rat. The increasing trend of the GR and GST activities up to 200 ppm of TCE could serve as indicator of TCE pollution. The dose-dependent activity of these enzymes seems to be a better index of TCE pollution,

0 50 100 150 200 250 300

TCE TCE-CHX

Control

Activity (U/min/mg protein)

GPx MDHAR APx GR CAT GST

Figure 6. Effect of CHX on GPx, MDHAR, APx, GR, CAT, and GST activities inA. cepabulb as a result of 48 h exposure to TCE.

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provided other pollutants are ruled out to enhance this activity. These findings also lead us to suggest that TCE might exert a sort of oxidative stress on the A. cepa. The reason for the decrease in activities of the antioxidant enzymes at higher doses of TCE exposure, in the present study, might be due to inactivation of the enzymes induced by excess production of ROS, or inhibition in the test-enzymes synthesis, or deleterious changes in enzyme subunits (Macrae and Ferguson 1985; Zaman and Zereen 1998). A gradual rise of up to more than two fold in GPx level was recorded in the test plant system. However, Watanabe and Fukui (2000) found a decline in GPx level as a result of TCE treatment in mice.

The ASC-GSH cycle also plays an important role in maintaining the ASC level (Noctor and Foyer 1998; Hancock and Viola 2005). In this cycle, ASC is oxidized to monodehydroascorbate (MDHA) radical while APx is using ASC as electron donor to scavenge H2O2. The MDHA can disproportionate spontaneously to ASC and DHA, or be enzymatically reduced to ASC by MDHAR. The DHA can be also reduced to ASC in a reaction mediated by DHAR, using GSH as the reducing substrate. The resulting oxidized-GSH is then reduced back to GSH by GR (Noctor and Foyer 1998; Davey et al.

2000). In this way, ASC and GSH are recycled when H2O2is scavenged in living cells.

ASC plays a role as the primary cellular antioxidant (Alscher, Donahue, and Cramer 1997). ASC and GSH are considered as the most important antioxidants involved in protection against ROS/free radicals through the ASC/GSH cycle (Gossett et al. 1996;

Kuzniak and Maria 2001; Drazkiewicz, Polit, and Krupa 2003).

MDHAR and APx activities were found to be approximately three fold enhanced at 300 ppm of TCE exposure compared with the control. Although DHAR also displayed a gradual rise in activity, the induction was only by 60%. Both ASC as well as GSH levels dropped upon exposure to TCE, which was found to be in the range of 50–55%.

Our group has also reported a dose-dependent depletion in GSH levels inA. cepaexposed to heavy-metal mixture (Fatima and Ahmad 2005). However, Goel et al. (1992) had found an increase in GSH level as a result of TCE intake in mice. H2O2level was also found to be reduced up to 70% at 300 ppm of TCE treatment, which is well-justified by the induction of APx and GPx.

In the presence of CHX, all the enzymes displayed the levels as those of untreated controls, while the enzymatic activities remained unchanged in the presence of chloramphenicol, thereby suggesting de novo synthesis of the cytosolic components of the antioxidant enzymes as a result of TCE exposure.

In view of the present findings, it is suggested that variations in the antioxidant enzymes ofA. cepacan serve as suitable model for the detection of TCE pollution in water.

In fact,A. ceparoot inhibition test is a cost-effective toxicity bioassay, routinely used in water monitoring studies (Fiskesjo¨ 1985). Therefore, it is our contention that its efficacy would be greatly increased if enzymatic studies are also carried out in the same onion bulbs exposed to the toxicant for the standardA. cepabioassay.

Conclusion

Allium cepa system can be successfully used for monitoring the changes in antioxidant enzymes as a result of TCE exposure. Based on this study, we conclude that GST, GR, APx, and CAT inA. cepasystem could serve as a potential indicator for assessing the TCE pollution and its hazards. Increase in the activities of these antioxidant enzymes was at the

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level of synthesis and none of them seem to have been synthesized in the mitochondria or chloroplasts ofA. cepacells.

Acknowledgments

Shams Tabrez is a senior research fellow of ICMR, New Delhi. The authors gratefully acknowledge the financial assistance to the department by the UGC, New Delhi under its DRS-II program.

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