The effect of some antibiotics on glutathione reductase enzyme purified from liver and erythrocyte of Lake Van pearl mullet

Abstract

Context: The effect of antibiotics (amikacin, cefazolin, ivermectin, and kanamycin) on glutathione reductase (GR) isoenzymes activity in liver and erythrocyte of the fish, Chalcalburnus tarichi (Lake Van pearl mullet, Pallas 1811) (Cyprinidae) were investigated.

Objective: This study determined the biochemical characterization of GR purified from the liver and erythrocytes of C. tarichi and the inhibition effect of the antibiotics on the GR isoenzymes.

Materials and methods: GR was purified by affinity chromatography from the tissues of C. tarichi. The biochemical characterization of GR such as optimum temperature, optimum pH, and ionic strength were determined. The inhibition effects of the antibiotics on the isoenzymes were evaluated as IC₅₀ and K_i values. K_i constant and 50% inhibitory concentration (IC₅₀) value for antibiotics were determined by Lineweaver–Burk graphs and plotting activity % versus [I], respectively, at five different concentrations of antibiotics.

Results: Optimum temperature, pH, and ionic strength were determined for isoenzymes as 40 °C, 60 °C; 8.0, 8.0, and 50, 50 mM, respectively. Subunit molecular weights of the isoenzymes were estimated as 55 kDa by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). In addition, IC₅₀ and K_i values were calculated for amikacin, cefazolin, ivermectin, and kanamycin. The antibiotics showed non-competitive inhibition effects. IC₅₀ values were calculated as 16.3, 36.6, 0.504, and 18.8 mM for liver and 20.0, 30.4, 0.787, and 31.8 mM for erythrocyte, respectively. K_i constants were 13.9, 18.4, 0.654, and 11.2 mM for liver and 23.2, 46.4, 1.19, and 36.4 mM for erythrocyte, respectively.

Discussion and conclusion: The results indicated that the antibiotics displayed non-competitive inhibition.

• Enzyme purification
• inhibition
• Lake Van pearl mullet liver and erythrocyte

Introduction

The undesirable biologic effects of oxidative agents, such as free radical and reactive oxygen species, are eliminated by enzymatic and non-enzymatic antioxidant defense systems. Enzymatic defense is provided by many enzyme systems, such as glutathione reductase, glutathione peroxidase, glutathione S-transferase, superoxide dismutase, catalase, aldoketoreductase, and DNA repair enzymes. Particularly, glutathione reductase is essential for the maintenance of cellular glutathione in its reduced form, which is highly nucleophilic for many reactive electrophils (Carlberg & Mannervic, 1975).

GR participates in reduced state of glutathione (GSH), a ubiquitous thiol-containing tripeptide, in several functions of vital importance to the cell. In animal tissues and non-photosynthetic organisms, it maintains the thiol groups in the proper redox state, and it detoxifies a variety of potentially harmful electrophilic compounds. The oxidized disulfide form (GSSG) is reduced by the enzyme GR, which employs as a reductant NADPH generated by the hexose monophosphate pathway (Arias & Jakoby, 1976). This enzyme catalyzes reduction of GSSG to GSH in the presence of NADPH and maintains a high intracellular GSH/GSSG ratio of about 500 in red blood cells (Kondo et al., 1980; Senturk et al., 2008a,b). GSH is the most abundant low-molecular weight thiol in almost all cells and is involved in a wide range of enzymatic reactions (Meister, 1981). Since GSH has an important role in detoxification of xenobiotics, such as carcinogens, toxins, and drugs, inhibition of GR may lead to low concentration of GSH and concomitantly a high concentration of exogenous compounds in the tissues, resulting in severe pathological conditions, such as hemolytic anemia, diabetes, and neurologic disorders (Beutler, 1984; Gul et al., 2000; Jacobasch & Rappoport, 1996; Knapen et al., 1999).

A member of the Cyprinidae family, Chalcalburnus tarichi (Lake Van pearl mullet, Pallas 1811) is a fish species that only inhabits the Lake Van Basin. The Lake Van represents an interesting ecosystem in the world, known as the biggest soda lake in the world; its water is highly alkaline with a pH of 9.8. Chalcalburnus tarichi has bright-silver color, its back is grayish green, and the abdominal region is silver. Its body is covered with small scales, and its eyes are large. It feeds on phyto- and zooplanktons. Its average life span is 7 years, and the fish reaches reproductive maturity at 3 years old. Chalcalburnus tarichi is a diadrom fish that lives in the lake, but during the reproduction period, it immigrates to the surrounding freshwater rivers returning after the reproduction period of April–July (Sari, 2008).

Most of the drugs affect the enzyme systems as an activator or inhibitor (Çiftçi et al., 2002; Erat et al., 2003; Edward & Morse, 1988; Jacobasch & Rappoport, 1996). Many drugs exhibit the same effects both in vivo and in vitro, but some of them may not show the same effects on enzymes (Beydemir et al., 2000).

This study purifies and characterizes GR isoenzymes from liver and erythrocyte of Lake Van pearl mullet by affinity chromatography compared to living species, and investigates the effects of the antibiotics on the isoenzyme activity.

Specifically, in this study, we characterized different properties such as optimum pH, temperature, and ionic strength of GR purified from liver and erythrocyte of the fish. We are not aware of any reports on the effects of amikacin, cefazolin, ivermectin, and kanamycin on the activity of GR purified from fish liver and erythrocyte.

Materials and methods

Chemicals

2′,5′-ADP Sepharose 4B was purchased from Pharmacia (Piscataway, NJ). Sephadex G-200, NADPH, GSSG, and protein assay reagents and chemicals for electrophoresis were purchased from Sigma Chemical-Co. (St. Louis, MO). All other chemicals used were of analytical grade and purchased from either Sigma or Merck (St. Louis, MO). The fish’s liver and erythrocyte samples were obtained from C. tarichi. The fish authenticated by Mustafa Sarı were caught in April 2008 with the help of anglers from Lake Van. A fish specimen is kept at the Department of Biology, Faculty of Sciences, Yuzuncu Yil University, Van, Turkey for the future reference.

Preparation of the tissues homogenate

The liver was dissected and put in Petri dishes. After washing the tissues with physiological saline (0.9% NaCl), the liver sample was processed in a blender. The ground liver was homogenized for 5 min in solution contains 0.836 g Na₂HPO₄.2H₂O and 8.25 g glucose (1:5 w/v) using a glass-porcelain homogenizer (20 kHz frequency ultrasonic, Jencons Scientific Co., Bedfordshire, UK) and then centrifuged at 11 000 rpm for 60 min. After membrane and intact cells were removed, the pH of supernatant was adjusted at 7.3 by adding solid phosphate salts for column application.

Preparation of the hemolysate

Blood sample from C. tarichi was collected in a tube with ethylenediaminetetraacetic acid (EDTA). The blood samples were centrifuged at 1500 rpm for 20 min and the plasma was removed. The erythrocytes were washed three times with isotonic NaCl solution containing 1 mM EDTA. The washed erythrocytes were hemolyzed with five volumes of ice-cold distilled water containing 2.7 mM EDTA and 0.7 mM β-mercaptoethanol. The hemolysate was centrifuged at 12 000 rpm for 30 min and the ghosts and intact cells were removed (Beutler, 1984). After membrane and intact cells were removed, the pH of supernatant was adjusted at 7.3 by adding solid phosphate salts for column application.

Affinity chromatography on 2′,5′-ADP Sepharose 4B

Dried 2′,5′ ADP Sepharose 4B (2 g) was used for a small column (10 × 1 cm²). The gel was washed with 300 ml distilled water to remove foreign bodies and air, and suspended in 0.1 M K-acetate/0.1 M K-phosphate buffer (pH 6.0) and packed in column. After precipitation of the gel, it was equilibrated with 50 mM K-phosphate buffer including 1 mM EDTA, pH 7.3 by means of a peristaltic pump. The flow rate was adjusted to 20 ml/h. The dialyzed sample obtained from ammonium sulfate precipitation was loaded onto the column, and washed with 25 ml 0.1 M K-acetate + 0.1 M K-phosphate, pH 6 and 25 ml 0.1 M K-acetate + 0.1 M K-phosphate, pH 7.85. Then, the washing process was continued with 50 mM K-phosphate buffer including 1 mM EDTA, pH 7.3 until the final absorbance difference became 0.05 at 280 nm. The isoenzymes were eluted with a gradient of 0–0.5 mM GSH and 0–1 mM NADPH in 50 mM K-phosphate, containing 1 mM EDTA (pH 7.3). Active fractions were collected and dialyzed with equilibration buffer at 4 °C (Acan & Tezcan, 1989; Boggaram et al., 1979; Carlberg & Mannervik, 1981).

Activity determination

GR activity was determined spectrophotometrically with a Shimadzu Spectrophotometer UV-(1800) (Shimadzu Corporation, Shimadzu, Japan), at 25 °C, according to the method of Carlberg and Mannervik (1985). The assay system contained 435 mM K-phosphate buffer pH 7.3, including 1 mM EDTA, 1 mM GSSG, and 0.1 mM NADPH. One enzyme unit is defined as the oxidation of 1 μmol NADPH per min under the assay condition.

Protein determination

Protein concentration was determined at 595 nm according to the method of Bradford (1976), using bovine serum albumin as a standard.

Optimum pH determination

In order to determine the optimum pH, Tris–HCl (50 mM) and K-phosphate (50 mM) buffers were used in the pH range of 7.6, 7.8, 8.0, 8.2, 8.4, and 8.6.

Optimum temperature determination

For determination of the optimum temperature, enzyme activity was assayed 10 °C interval at different temperatures in the range from 20 °C to 80 °C. The desired temperature was provided by a Grant bath (model 6 G).

Optimum ionic strength determination

For the determination of optimum ionic strength, enzyme activity was determined using different concentrations in the range 10, 25, 50, 100, 200, 400, 800, and 1000 mM in 1 mM Tris-HCl buffer (pH 8.0)

Molecular weight determination – sodium dodecyl sulfate polyacrilamide gel electrophoresis

The subunit of the isoenzymes was determined by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). The molecular weight of the isoenzymes was determined according to Andrews (1965). First, to calculate the void volume, 10–200 kDa proteins were used as standard proteins.

SDS-PAGE

To check the isoenzymes purity, SDS-PAGE was performed by Laemmli’s method (Laemmli, 1970). The acrylamide concentration of the stacking and the separating gels were 3% and 8%, respectively, and 1% SDS was also added to the gel solution. Gel was stained for 2 h in 0.1% Coomassie Brillant Blue R-250 containing 50% methanol, 10% acetic acid, and 40% distilled water. The gel was washed with several combination of the same solvent without dye. Cleared protein bands were photographed (Figure 1).

Figure 1. SDS-polyacrylamide gel electrophoresis of GR purified by affinity chromatography.

Results

In the present study, the GR was first purified from liver and erythrocytes of C. tarichi. The purification steps comprise the preparation of supernatant and 2′,5′ ADP Sepharose 4B affinity chromatography (Senturk et al., 2008). Sepharose 4B was chosen as a matrix because of better flow properties than that of the other matrixes. At first, free hydroxyl groups of the matrix were activated with 2′,5′ ADP (Bell & Bell, 1988). The purification of liver homogenate and erythrocyte hemolysate was characterized with a specific activity of 122 EU/mg proteins for liver and 96 EU/mg proteins for erythrocyte, with a purification rate of 4552- and 7619-fold, respectively. The GR from liver homogenate and erythrocyte hemolysate was purified by using the affinity gel with elution buffer (50 mM K-phosphate/1 mM EDTA, 1 mM GSH, 0.5 mM NADPH, pH = 7.3). The elutes were plotted by arraying out protein determination at 280 nm and GR activity for liver homogenate (Figure 2) and erythrocyte hemolysate (Figure 3). Specific activities for tissues were calculated by using supernatant and purified enzyme solution. For the purification of supernatant GR by affinity chromatography, the column (1.3 × 60 cm²) was eluted by buffer at pH 7.3. It was eluted at 20 ml/h flow rate for fraction volumes of 6 ml (Boggaram et al., 1979; Carlberg & Mannervik, 1981; Le Trang et al., 1983).

Figure 2. Purification of liver homogenate GR by affinity chromatography and the columns (1.3 × 60 cm²) were eluted by buffer C at pH 7.3. It was buffer at 20 ml/h flow rate for fraction volumes of 6 ml.

Figure 3. Purification of erythrocyte hemolysate GR by affinity chromatography and the columns (1.3 × 60 cm²) were eluted by buffer C at pH 7.3. It was buffer at 20 ml/h flow rate for fraction volumes of 6 ml.

Optimal pH for liver and eythrocyte’s GR has been determined as equal volumes of the buffers (50 mM K-phosphate at pH of 6.0, 6.5, 7.0, 7.5, and 8.0; 50 mM Tris-HCl at pH of 7.5, 8.0, 8.5, and 9.0) and is shown in Figure 4. The plot temperature for GR isoenzymes activity was measured at 40 °C and is shown in Figure 5. The highest isoenzymes’ activity was seen in a concentration of 50 mM Tris-HCl as the ionic strength and is shown in Figure 6.

Figure 4. Optimal pH for liver and erythrocyte GR.

Figure 5. Optimal temperature for liver and erythrocyte GR.

Figure 6. Activity–ionic strength for liver and erythrocyte GR.

The effects of antibiotics, amikacin, cefazolin, ivermectin, and kanamycin, on GR isoenzymes activity purified from liver and erythrocyte of the fish were investigated. K_i and IC₅₀ values were obtained from regression analysis graphs for GR in the presence of five different drug concentrations (Table 1). The purification of GR isoenzymes was controlled with SDS-PAGE (Figure 1).

Table 1. K_i and IC₅₀ values obtained from regression analysis graphs for glutathione reductase in the presence of different drugs concentration.

Tissue	Antibiotics	IC50 (mM)	Ki (mM)	Inhibition type
Liver	Amikacin	16.3	13.9	Non-competitive
	Cefazolin	36.6	18.4	Non-competitive
	Ivermectin	0.504	0.654	Non-competitive
	Kanamycin	18.8	11.2	Non-competitive
Erythrocyte	Amikacin	20.0	23.2	Non-competitive
	Cefazolin	30.4	46.4	Non-competitive
	Ivermectin	0.787	1.19	Non-competitive
	Kanamycin	31.8	36.4	Non-competitive

Discussion

Living cells were exposed of various foreign chemicals such as drugs, food additives, and pollutants. These chemicals are called xenobiotics and are usually electrophilic compounds (Murray et al., 1996). The metabolism of xenobiotics usually involves two distinct stages, commonly referred to phases I and II. Phase I metabolism involves an initial oxidation, reduction, or dealkylation of the xenobiotic by cytochrome P-450 monooxigenases (Guengerich, 1991). This step is often needed to provide a molecule with hydroxyl- or amino-groups, which are essential for phase II reactions.

Phase II metabolism generally adds hydrophilic moieties, thereby making a toxin more water-soluble and less biologically active. Frequently involved phase II conjugation reactions are catalyzed by glutathione S-transferases (GSTs) (Beckett & Hayes, 1993), sulfotransferases (Falany, 1991), and UDP-glucuronyl-transferases (Bock, 1991). The GSTs catalyze the addition of GSH to a wide variety of exogenous compounds. GR, a flavo enzyme, is essential for the maintenance of cellular GSH in its reduced form, which is highly nucleophilic potent for many reactive electrophils (Carlberg & Mannervik, 1975).

Many chemicals and drugs affect the metabolism of living organisms by altering normal enzyme activity, particularly inhibition of a specific enzyme (Hochster et al., 1973). The effects can be dramatic and systemic (Christensen et al., 1982). Some chemicals and drugs inhibit GR enzyme activity, such as nitrofurazone, nitrofurantione, 5-nitroindol, 5-nitro-2-furoic acid, 2,4,6-trinitrobenzene sulfonate (TNBS) (McCallum & Barrett, 1995), and some polyphenolic compounds (Zihang et al., 1997). In a different study, dantrolene, a drug used to treat malignant hyperthermia, showed high inhibitory effect on human erythrocyte GR enzyme activity (Senturk et al., 2008). It was reported that nicotine causes competitive inhibition by binding the active site of GR. Nitro aromatic compounds can either be strong or weak inhibitors of erythrocyte GR (Grellier et al., 2001). Similar results were obtained from different studies (Erat & Ciftci, 2006; Senturk et al., 2008).

Many drugs are commonly used for the therapy of animal diseases. To our knowledge, the inhibition or activation effects of drugs on GR enzyme have not been investigated yet. Hence, the effects of some commonly used antibiotics on GR enzyme in liver and erythrocyte of the fish were investigated and IC₅₀ and K_i values were estimated for the drugs, and their inhibition effects were determined.

The present study shows that the GR activity was inhibited by amikacin, cefazolin, ivermectin, and kanamycin. In addition, the estimated IC₅₀ and K_i values showed that ivermectin is more effective inhibitor than the other antibiotics on the GR enzyme activity. Besides, inhibition types of all the antibiotics were determined to be non-competitive. If the antibiotics are given to animals, the dosages should be very well-controlled in order to prevent the adverse effects on GR activity in cells.

So far, studies on the examination of the GR isoenzyme characterization in liver and erythrocyte in the fish are not available; therefore, we could not compare our results with the previous results. In addition, it is difficult to compare data from different laboratories regarding the enzyme characterization because of high variability in analyzing enzyme in vitro and in vivo due to inconsistent factors like specific tissue differences. Our observations led us to conclude that the characteristics, the optimal pH, ionic strength, and temperature of GR isoenzymes purified from the various tissues of Lake Van fish, are in accordance with the other living species.

In conclusion, the antibiotics showed high inhibitory effect on GR isoenzyme activity in erythrocyte and liver of C. tarichi. According to the results obtained from this study, the antibiotics are determined to be potent inhibitors for GR and may cause some side effects in organisms. It was reported that the decrease in GR isoenzyme activity could lead to imbalance in the redox status due to decrease in GSH levels. For this reason, the antibiotics must be used carefully and the dosages should be very well-ordered to avoid the previously mentioned side effects.

Declaration of interest

The authors have declared that there is no conflict of interest. None of the authors has a commercial interest, financial interest, and/or other relationship with manufacturers of pharmaceuticals, laboratory supplies, and/or medical devices or with commercial providers of medically related services.