Susceptibility of cord blood antioxidant enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase to different antibiotics: in vitro approach

Abstract

Cord blood has numerous facilities for life and used in many different areas. Cord blood contains many different catalytic proteins including antioxidant enzymes. Here we purified human cord blood glutathione reductase (hcbGR), glutathione S-transferase (hcbGST) and human cord blood glutathione peroxidase (hcbGPx) from human cord blood erythrocytes and analyzed the inhibition effects of the antibiotics incorporating cefuroxime, ceftriaxone, ceftizoxime and cefoperazone, on these enzymes. K_I values for the drugs ranged from 10.42 to 28.72 µM for hcbGR, 32.7 to 244.8 µM for hcbGPx, and 32.39 to 267.3 µM for hcbGST. Cefuroxime caused the highest inhibition on all enzymes with K_I values of 10.42, 32.39, 32.7 µM for hcbGR, hcbGST, and hcbGPx, respectively. All drugs displayed non-competitive inhibition regardless of their structures. Since these drugs are often used during pregnancy, identification of possible undesired impacts on various parameters has a great importance for pharmacological and medical applications.

Keywords::

• Glutathione
• antibiotic
• cord blood
• inhibition

Introduction

After a baby is born and the umbilical cord is cut, some blood remains in the blood vessels of the placenta and the portion of the umbilical cord that remains attached to it. After birth, the baby no longer needs this extra blood. This blood is called placental blood or umbilical cord blood: “cord blood” for short¹.

Cord blood contains red blood cells, white blood cells, platelets, and plasma, like blood. In addition, cord blood is a rich source of stem cells that may have potentially lifesaving benefits for baby and family¹. It is collected because of its stem cell contents, including hematopoietic cells, which can be used to treat hematopoietic and genetic disorders².

The tripeptide reduced glutathione (GSH: l-γ-glutamyl-cysteinyl-glycine) is the most important intracellular nonprotein thiol in mammalian cells. It is found at concentrations of 1–3 mM in human brain cells³, and it possesses reducing and nucleophilic properties. It can exist in either a reduced (GSH) or oxidized (GSSG) form. Glutathione reductase (EC 1.8.1.7, GR) is an ubiquitous flavoenzyme that catalyzes the NAD(P)H-dependent reduction of GSSG to GSH. This enzyme maintains adequate levels of the cellular GSH pool, which is critical not only for maintaining the cellular redox status by keeping sulfhydryl groups of cytosolic proteins in their reduced form^4,5 but also because numerous toxic or potentially toxic compounds, including some metals, are either taken up by or removed from the cells by GSH-mediated pathways⁶. Glutathione peroxidase (EC 1.11.1.9, GPx) catalyzes the reduction of hydrogen peroxide by GSH. Different groups demonstrated the presence of glutathione peroxidase in erythrocytes⁷. Glutathione S-transferases (EC 2.5.1.18, GSTs), are members of a multigene family of isoenzymes ubiquitously expressed in most living organisms. It was subsequently shown that these enzymes catalyze the conjugation of GSH to a variety of electrophilic compounds, thus establishing the now widely accepted role of GSTs as cell housekeepers involved in the detoxification of endogenous as well as exogenous substances⁸.

Antibiotics are powerful drugs that fight against bacterial infections and save lives when used properly. They either kill bacteria or keep them from reproducing. Antibiotics are widely used to deal with various disorders, but there are few reports of their effects on enzyme activities. Some studies found either increases or decreases in mammalian enzyme activities, and the inhibitor or activator effects of some antibiotics have been rarely investigated. Nevertheless, a few recent investigations regarding the inhibitory actions of different type of drugs and chemicals on GR have been reported by our group^9–15.

Cefuroxime is a second-generation cephalosporin antibiotic whereas ceftriaxone, ceftizoxime and cefoperazone are third-generation cephalosporin antibiotics. Third-generation cephalosporin antibiotics have broad spectrum activity against gram-positive and gram-negative bacteria¹⁶. As known, all of these drugs are commonly used during pregnancy.

A number of investigations focus on the property of umbilical cord. For instance, the immunologic properties of cord blood were reported to differ from mature bone marrow or peripheral blood stem cells. Also, cord blood cells were shown to produce increased amounts of anti-inflammatory cytokine interleukin-10, which may downmodulate graft-versushost disease¹⁷. However, there are not sufficient data on the inhibition of cordblood enzymes by various factors such as drugs, nutritients or etc. It is important to identify the enzyme activities during pregnancy and characterize the factors increasing or decreasing those activities. Therefore, in an effort to provide beneficial data for further investigations, the present study aimed to evaluate the in vitro effects of commonly used antibiotics on hcbGR, hcbGPx, and hcbGST enzymes.

Materials and methods

Chemicals

2,′ 5′-ADP Sepharose-4B was obtained from Pharmacia. Glutathione-sepharose 4B, Sephadex G-200, l-chloro-2,4-dinitrobenzene (CDNB), t-butyl hydroperoxide, protein assay reagents, GSH, GSSG, NADPH were obtained from Sigma-Aldrich Co. All other chemicals were analytical grade and obtained from Merck.

Glutathione reductase activity

Enzymatic activity was measured by Beutler’s method¹⁸ with a Shimadzu Spectrophotometer UV-(1208), at 25°C. The assay system contained 100 mM Tris-HCl buffer pH 8.0, including 0.5 mM EDTA, 3.3 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 at 25°C.

Glutathione peroxidase activity

Glutathione peroxidase was assayed in a l-ml system containing 0.1 M potassium phosphate buffer, pH 7.0, 0.2 mM NADPH, 1 i.u. glutathione reductase, 4 mM GSH, 4 mM EDTA, 4 mM sodium azide. The reaction mixture was incubated at 37°C for 10 min after which 10 μl of 10 mM t-butyl hydroperoxide were added to start the reaction. No t-butyl hydroperoxide was added to the blank cuvette. The rate of reaction was measured at 37°C by following the decrease in the absorbance at 340 nm using a spectrophotometer⁷.

Glutathione S-transferase activity

Enzymatic activity was determined spectrophotometrically by measuring the conjugation of CDNB with GSH⁸. The 1 ml assay mixture contained 0.5 mM CDNB, 1 mM GSH, and 100 mM potassium phosphate buffer, pH 6.5. The rate of increase in absorbance at 340 nm was measured for 5 mm at 37°C against a blank containing the reaction mixture without enzyme.

Preparation of the hemolysate

Erythrocytes were purified from fresh human cord blood obtained from the Blood Centre of Erzincan Hospital. From normal pregnant women ad term, following the birth by caesarian section or normal delivery, 2 mL of umbilical cord blood was withdrawn by syringe just before the separation placenta and transferred into K₃EDTA anticoagulated blood tubes and mixed. The blood samples were stored at −40°C until analysis. The blood samples were centrifuged at 2250g for 15 min and the plasma and buffy coat were removed. The packed red cells were washed three times with KCl (0.16 M) and hemolyzed with 5 volume of ice-cold water and then centrifuged (4°C, 10,000g, for 30 min) to remove the ghosts and intact cells^5,11

Ammonium sulphate precipitation

The hemolysate was subjected to precipitation with ammonium sulphate at different intervals depending on the enzymes. Enzyme activity was determined both in the supernatant and in the precipitate for each respective precipitation. The precipitate was dissolved in phosphate buffer (50 mM; pH = 7.0). The resultant solution was clear, and contained partially purified enzyme. Then it was dialysed at 4°C in 1 mM EDTA + 10 mM K-phosphate buffer (pH 7.5) for 2 h with two changes of buffer¹¹. Partially purified enzyme solution was kept at 4°C.

Partial purification of hcbGPx

The purification steps are given in Tables 1–3. The hemolysate was centrifuged for 30 min at 10,000g to remove the ghosts. Solid ammonium sulfate was added to the supernatant with constant stirring and the precipitate formed between 25 and 50% saturation of (NH₄)₂SO₄ was separated by centrifugation and suspended in 10 mM potassium phosphate buffer, pH 7.0, with 0.7 mM β-mercaptoethanol and dialyzed against the same buffer to remove the (NH₄)₂SO₄⁷.

Table 1.  Purification scheme of hcbGR.

Purification step	Total volume (ml)	Activity (U/ml)	Total activity (U)	Protein (mg/ml)	Specific activity (U/mg)	Yield (%)	Purification factor
Hemolysate	30	0.30	9	32	0.0094	100	1
Ammonnium sulfate precipitation (30–70%)	9	0.71	6.39	30	0.0226	71	2.40
2,′ 5′-ADP Sepharose 4B affinity chromatography	5	1.12	5.6	0.42	2.666	62.22	283.62
Sephadex G-200 gel filtration chromatography	4	0.74	2.96	0.06	12.333	32.89	1312

Table 2.  Partial purification scheme of hcbGPx.

Purification step	Total volume (ml)	Activity (U/mL)	Total activity (U)	Protein (mg/ml)	Specific activity (U/mg)	Yield (%)	Purification factor
Hemolysate	30	0.052	1.56	0.73	0.0071	100	1
Ammonnium sulfate precipitation (25–50%)	5	0.137	0.685	1.43	0.0958	43.9	13.5

Table 3.  Purification scheme of hcbGST.

Purification step	Total volume (ml)	Activity (U/ml)	Total activity (U)	Protein (mg/ml)	Specific activity (U/mg)	Yield (%)	Purification factor
Hemolysate	30	0.034	1.02	1.40	0.0024	100	1
GSH Sepharose 4B affinity chromatography	4	0.236	0.944	0.02	11.80	92.55	4916

Purification of hcbGR

Preparation of 2′, 5′-ADP Sepharose-4B affinity column was previously described^9,11,13. The dialyzed sample obtained previously was loaded onto the 2′, 5′-ADP Sepharose-4B affinity column and the column was washed with 25 ml of 0.1 M K-acetate + 0.1 M K-phosphate, pH 6, and 25 ml of 0.1 M K-acetate + 0.1 M K-phosphate, pH 7.85. Washing was continued with 50 mM K-phosphate buffer including 1 mM EDTA, pH 7.0, until the final absorbance difference became 0.05 at 280 nm. The enzyme was eluted with a gradient mixture of 0 to 0.5 mM GSH + 1 mM NADPH in 50 mM K-phosphate, containing 1 mM EDTA (pH 7.0). Active fractions were collected and dialyzed with equilibration buffer. All of the procedures were performed at 4°C^5,13.

After dried Sephadex G-200 (2 g) was used for a 165 ml column (2 × 50 cm) bed volume. The gel was incubated in distilled water at 90°C for 5 h. After removal of the air in the gel, it was loaded onto the column. Flow rate was adjusted to 15 ml/h by means of a peristaltic pump. Then the column was equilibrated with 50 mM Tris-HCl + 50 mM KCl buffer, pH 7.0, until the final absorbance difference became 0 at 280 nm. The dialyzed sample was mixed with 5% glycerol. The final sample was loaded onto the column and elutions were collected in 2 ml amounts. In each fraction, enzyme activity was determined at 340 nm. Active fractions were collected and stored at −20°C for testing the enzyme purity by electrophoresis^5,9,11,15.

Purification of hcbGST

The hemolysate supernate was passed through a 1 × 10 cm GSH sepharose 4B column at a flow rate of approximately 10 ml/h. The column was washed for 1 h with 20 mM potassium phosphate buffer (pH 7.2), followed by elution of 2 mM GSH with cold 50 mM Tris buffer (pH 9.5 at 4°C)^19,20. Fractions containing GST activity were pooled and dialyzed overnight at 4°C against 2 liters of 20 mM potassium phosphate buffer (pH 7.0).

Protein determination

Protein during the purification steps was determined spectrophotometrically at 595 nm according to the Bradford method using bovine serum albumin as the standard²¹.

SDS polyacrylamide gel electrophoresis

SDS polyacrylamide gel electrophoresis was performed after purification of the cord blood izo enzymes. It was carried out according to Laemmli procedure²².

In vitro drug effects

In order to determine the effects of some drugs on hcbGR and hcbGST, different concentrations of corresponding drugs were tested. These enzyme activities were measured and an experiment in the absence of drug was used as control (100% activity). The IC₅₀ values were obtained from activity (%) vs. drug concentration plots. The GSSG concentrations ranged from 0.015 to 0.15 mM for the determination of kinetic parameters with hcbGR. The GSH concentrations ranged from 0.25 to 2.0 mM for the determination of kinetic parameters with hcbGPx. The CDNB concentrations ranged from 0.01 to 0.075 mM for the determination of kinetic parameters with hcbGST. Inhibitors solutions were added to the reaction medium, resulting in 3 different fixed concentrations of inhibitors in 1 ml of total reaction volume. Lineweaver-Burk graphs²³ were drawn by using 1/V vs. 1/[S] values and K_I constant were calculated from these graphs. Regression analysis graphs were drawn for IC₅₀ using inhibition % values by a statistical package (SPSS-for windows; version 10.0) on a computer.

Results and discussion

GR from human cord blood erythrocytes was purified in this study by ammonium sulphate precipitation, 2′, 5′-ADP Sepharose 4B affinity chromatography and Sephadex G 200 gel filtration chromatography. The purified enzyme was characterized with a specific activity of 12.333 EU/mg proteins, a yield of 32.89% and a purification coefficient of 1312 (Table 1). GPx was partially purified by hemolysate preparation, ammonium sulphate precipitation and dialys. The purified preparation was characterized with a specific activity of 0.0958 EU/mg proteins, a yield of 43.9% and a purification coefficient of 13.5 (Table 2). GST was purified by GSH Sepharose 4B affinity chromatography. The purified enzyme had a specific activity of 11.80 EU/mg proteins, a yield of 92.55% and a purification coefficient of 4916 (Table 3). Inhibition effects of the drugs were tested under in vitro conditions; IC₅₀ values were calculated by Activity (%)-[Inhibitor] graphs and are given in Table 2. K_I values were calculated from Lineweaver-Burk graphs²³ and are given in Table 4.

Table 4.  Ki and IC50 values obtained from regression analysis graphs for hcbGR, hcbGST and hcbGPx in the presence of different drugs.

Inhibitor	hcbGR		hcbGST		hcbGPx
	IC 50 (μM)	Ki (μM)	IC50 (μM)	Ki (μM)	IC50 (μM)	Ki (μM)
Cefuroxime	18.8	10.42	56.4	32.39	44.3	32.7
Ceftriaxone	21.3	17.34	61.8	35.41	48.6	34.1
Ceftizoxime	39.7	22.76	278.3	164.73	186.3	121.2
Cefoperazone	48.4	28.72	483.8	267.35	313.4	244.8

The undesirable biologic effects of oxidative agents, such as free radical and reactive oxygen species (ROS), are eliminated by enzymatic and nonenzymatic 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^24–28. The products of oxidative damage initiated by hydroxyl radicals like 4-hydroxyalkenals (membrane lipid peroxides) and base propenals (products of oxidative DNA degradation) are highly cytotoxic. Glutathione S-transferases (GSTs) detoxify such endogenously produced electrophiles by conjugation with glutathione (GSH) as well as by acting as glutathione peroxidases to detoxify toxic base propenals like thymidine hydroperoxide²⁹. Particularly, GR is essential for the maintenance of cellular glutathione in its reduced form, which is highly nucleophilic for many reactive electrophils^30,31.

Numerous studies have demonstrated that oxidative stress is a key pathogenic factor in the development of illness complications. Antioxidant enzymes constitute a supportive team of enzymes which provide defense against the reactive intermediates of dioxygen reduction. These enzymes are cooperative in several aspects^32,33. Our groups recently investigated the interaction of human erythrocyte GR enzyme with several types of analgesic, anaesthetic drugs and antibiotics as well as metal ions^5,9–15.

It is well-known that many chemicals and drugs affect the metabolism via enzyme activities and the results may be hazardous. Thus, drug enzyme interactions should be well-characterized. Although there is a lack of studies regarding drug-enzyme interactions, the investigations on this issue is increasing day by day. Here we purified the GR and GST enzymes with simple chromatographic methods and GPx partially; and determined inhibitory potentials of commonly used drugs. As seen in Table 4, all drugs showed inhibitory action on these enzymes. Cefuroxime was the most powerful inhibitor for these enzymes. The second most potent inhibitor was ceftriaxone with the K_I values of 17.34, 34.1, and 35.41 µM for hcbGR, hcbGPx and hcbGST, respectively. Ceftizoxime caused relatively lower inhibition on hcbGR but much lower on hcbGST and hcb GPx. Cefoperazone displayed very low inhibition with K_I values of 28.72 µM for hcbGR, 267.3 µM for hcbGST and 244.8 µM for hcbGPx, respectively. Overall data indicates that cefuroxime and ceftriaxone showed relatively higher inhibitory effect as compared with others and that these drugs inhibit the enzymes in non-competitive manner. This result is not surprising because molecular structures of the drugs are not similar to physiological substrates of the enzymes (Figure 1). The drugs are most likely interacting with the regions other than the active site, probably with the sulphydril groups as they are quite bulky. As mentioned above, small molecule (especially drug)-enzyme interaction studies have gained a great interest over the recent years³⁴. Our study is thus a contribution to the literature data on the inhibiton of GR, GPx and GST. Our study also constitutes a starting point for the enzymatic evaluation of cord blood and provides useful information for further kinetic investigations. Moreover, findings of this investigation help to identify possible undesired impacts of these drugs, in the case of uncontrolled usage, for pharmacological and medical applications.

Figure 1.  Structures of tested antibiotics.

Declaration of interest

The authors report no declarations of interest