Anti-oxidative and anti-genotoxic effects of carnosine on human lymphocyte culture

Abstract

The aim of this study was to investigate the effects of carnosine, a biological antioxidant, on the oxidative stress and genotoxicity by a single dose of carbon tetrachloride (CCl₄; 5 mM) in the human lymphocyte culture. We studied the anti-genotoxic effects of carnosine by using sister chromatid exchange (SCE) test system. Also, the anti-oxidative effects of carnosine were evaluated by using superoxide dismutase (SOD), glutathione peroxidase (GPx), total glutathione (GSH) and malondialdehyde (MDA) assay. The SCE frequency was increased when treated with CCl₄. Carnosine at 10 and 20 mM reduced SCE frequency in the human lymphocyte (p < 0.001). In addition, CCl₄ treatment significantly depleted the level of GSH, reduced the activity of SOD and GPx and elevated the level of MDA (p < 0.001). Carnosine treatment led to significant attenuation of CCl₄-induced oxidative stress by normalization of the activities of SOD and GPx and the level of GSH and MDA (p < 0.05 or 0.001). These results suggest that carnosine could provide anti-oxidative and anti-genotoxic protection for the oxidative and genotoxic agents that cause many diseases including cancer and neurodegenerative disease.

Introduction

Organochlorides include a series of compounds (chloroform, carbon tetrachloride [CCl₄]) with effects that have been proven to be carcinogenic to animals and that are suspected of being carcinogenic to human beings.

International Agency for Research on Cancer (IARC) classify CCl₄ as a group 2B carcinogen, indicating that there is sufficient evidence of CCl₄ carcinogenicity in animals, and it is considered as ‘possibly carcinogenic to humans.^1,2 CCI₄ can induce lipid peroxidation and the production reactive oxygen species (ROS) that can be explained based upon the formation of radical metabolites and compromised antioxidant status. It is proposed that a mechanism of action for CCI₄ involves the induction of oxidative stress and secondary genotoxicity. ³ ROS generated during CCl₄-induced oxidative stress are capable of inducing DNA strand breaks and pro-mutagenic DNA adducts, such as 8-oxodeoxyguanosine (8-oxodG) and malondialdehyde-deoxyguanosine. ⁴ Exposure to CCl₄ has been linked to increased incidences of lymphosarco mas and lymphatic leukemia in workers in the rubber industry ⁵ and exposure to dry cleaning solvents, including CCl₄, results in increased levels of lung and cervical cancers and slight excesses of leukemia and liver cancer. ⁶

The dipeptide carnosine is synthesized from its component amino acids β-alanine and histidine by the enzyme carnosine synthase. β-Alanine, a non-proteinogenic amino acid, is produced mainly by the liver as the final metabolite of uracil and thymine degradation.^7,8 Carnosine is present in muscle and brain tissue in concentrations up to 20 mM and is suggested to be an important physiological antioxidant.^9,10 It may also regulate glycolysis, muscular contraction and oxidative phosphorylation, stimulate the immune system, bind copper, zinc and calcium ¹¹ and purines, ¹² which suggests potential involvement in gene regulation or signal transduction. ⁷ Carnosine and related dipeptides have been postulated to have numerous biological roles including pH buffering, regulation of enzyme activity and inhibition of oxidative reactions. Among antioxidant mechanisms reported for carnosine are its ability to inactivate ROS, scavenges free radicals and chelate prooxidative metals.^9,13–16 It also affects superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and glutathione (GSH) and malondialdehyde (MDA) levels.^14,17,18

There are a few studies about the anti-genotoxic effect of carnosine. Previous studies show that carnosine has the protective effect on DNA damage and chromosomal aberration in rats and in vitro studies.^10,19–21

To our knowledge, anti-genotoxic and anti-oxidative effects of carnosine have not been evaluated using human cell culture. In our study, we aimed to determine the anti-genotoxic and anti-oxidative effects of carnosine on human lymphocyte culture.

Materials and methods

Chemicals

The chemicals (BrdU, Hoechst, Colcemide, Carnosine, SOD, NBT, NADPH, GSH, Xantine oxidase, NaN₃, SDS and TBA) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). RPMI-1640, FBS, streptomycin, glutamine, CCl₄ and EDTA were obtained from Merck.

Sister Chromatid Exchangeassay

Peripheral blood lymphocytes were taken from four (two men and two women) nonsmoking healthy individuals. Lymphocyte cultures were prepared by adding 0.5 mL of heparinized whole blood to RPMI-1640 chromosome medium supplemented with 15% heat-inactivated fetal calf serum, 100 IU/mL streptomycin, 100 IU/mL penicillin, and 1%L glutamine. Lymphocytes were stimulated to be divided by 1% phytohemaglutinin. CCl₄ (in concentration of 5 mM) and Carnosine (in concentrations of 1, 5, 10 and 20 mM) were added to the cultures just before incubation. CCl₄ was used as an oxidative and genotoxic agent to test the anti-genotoxic and anti-oxidative potency of carnosine. ³ Also 100 μM ascorbic acid (AA) was used as positive control.^22,23 Experiments were performed on 7 groups.

For sister chromatid exchange (SCE) demonstration, cultures which are incubated at 37°C for 72 h, and 5-bromo 2-deoxyuridine at 8 mg/mL were added into cultures. All cultures were left in a dark room. Next, 0.1 mg/mL of colcemide was added 2 h before harvesting, in order to catch the cells at metaphase. Cells were harvested and treated for 30 min with hypotonic solution (0.075 M KCl) and fixed in a 1:3 mixture of acetic acid/methanol (vol/vol). Bromodeoxyuridine-incorporated metaphase chromosomes were stained with fluorescence plus Giemsa technique as described by Perry and Evans (1975). ²⁴ In SCE study, by selecting 30 satisfactory metaphases were recorded on the evaluation table. For each treatment condition, well-spread second division metaphases containing 44–46 chromosomes in each cell were scored, and the values obtained were calculated as SCEs per cell.

Biochemical analysis

The cultured human blood cell homogenates were prepared at a 1:10 (w:v) dilution in 10 mM potassium phosphate buffer, pH 7.4. Samples were centrifuged at 3000 rpm for 10 min at 4°C, and the supernatants were collected and immediately assayed for enzyme activities. All samples were measured in sixfold.

GPx assay

GPx activity in the cell culture supernatant was measured by the method of Paglia and Valentine (1967). ²⁵ Briefly, 50 μL of sample was combined with 100 μL of 8 mM NADPH, 100 μL of 150 mM reduced GSH, 20 μL of glutathione reductase (30 units/mL), 20 μL of 0.12 M sodium azide solution and 2.65 mL of 50 mM potassium phosphate buffer (pH 7.0, 5 mM EDTA) and the tubes incubated for 30 min at 37°C. The reaction was initiated with the addition of 100 μL of 2 mM H₂O₂ solution, mixed rapidly by inversion, and the conversion of NADPH to NADP was measured spectrophotometrically for 5 min at 340 nm. The enzyme activity was expressed as units per g protein using an extinction coefficient for NADPH at 340 nm of 6.22 × 10⁻⁶.

SOD assay

Cu, Zn-SOD activity in the cell culture supernatant was detected by the method of Sun et al. ²⁶ A total of 2.45 mL of assay reagent (0.3 mM xanthine, 0.6 mM Na2EDTA, 0.15 mM nitroblue tetrazolium [NBT], 0.4 M Na₂CO₃, 1 g/L bovine serum albumin) was combined with 100 μL of the sample. Xanthine oxidase (50 μL, 167 U/L was added to initiate the reaction, and the reduction of NBT by superoxide anion radicals, which are produced by the xanthine-xanthine oxidase system, was determined by measuring the absorbance at 560 nm. Cu, Zn-SOD activity was expressed in units of SOD per mg protein, where 1 U is defined as that amount of enzyme causing half-maximal inhibition of NBT reduction.

MDA assay

MDA levels in the cell culture supernatant were determined spectrophotometrically according to the method described by Ohkawa et al. ²⁷ A mixture of 8.1% sodium dodecyl sulphate, 20% acetic acid and 0.9% thiobarbituric acid was added to 0.2 mL of sample, and distilled water was added to the mixture to bring the total volume up to 4 mL. This mixture was incubated at 95°C for 1 h. After incubation, the tubes were left to cool under cold water and 1 mL distilled water with 5 mL n-butanol/pyridine (15:1, v/v) was added, followed by mixing up. The samples were centrifuged at 4000 × g for 10 min. The supernatants were removed, and absorbances were measured with respect to a blank at 532 nm. 1,1,3,3-Tetraethoxypropane was used as the standard. Lipid peroxide levels were expressed as μmol/l MDA. Protein concentrations in the cell culture supernatant were determined by Bradford method. ²⁸ All photometrical measurements were performed with an ELISA reader.

Total GSH assay

GSH levels in the cell culture supernatant were assessed according to the method of Tietze (1969) and Anderson (1996).^29,30 Briefly, 100 μL of sample was placed to a 3 mL cuvette and then 750 μL of 10 mM 5-5′-dithio-bis-2-nitrobenzoic acid (DTNB) solution (100 mM KH₂PO₄ plus 5 mM Na2EDTA, pH 7.5 and GSH-RD, 625 U/L) was added and incubated for 3 min at room temperature. Then, 150 μL of 1.47 mM β-NADPH was added, mixed rapidly by inversion and the rate of 5-thio-2-nitrobenzoic acid formation (proportional to the sum of reduced and oxidized GSH) was measured spectrophotometrically for 2 min at 412 nm. The reference cuvette contained equal concentrations of DTNB and NADPH but no sample, and values were presented as μmol per gram protein.

Statistical analysis

The statistical analysis of SCE values and biochemical parameters, Mann–Whitney U-test was used. A value of p less than 0.05 was accepted as statistically significant. Results were expressed as mean ± SE. For these procedures, SPSS 11.5 version for Windows (SPSS Inc, Chicago, Illinois, USA) was used.

Results

Anti-genotoxic effects of carnosine

The anti-genotoxic effects of carnosine are given in Figure 1. SCE frequency in CCl₄-treated group was higher than the frequency in the control group (p < 0.001; Figure 3). There was a significant decrease in the SCE frequency in CCl₄-treated group when compared with the groups receiving CCl₄ and carnosine. There is no significant effect of 1 mM concentration of carnosine on SCE frequency. The decrease in the SCE frequency was the highest 20 mM concentration of carnosine (p < 0.001). Also, this effect was more effective than control and ascorbic acid groups. This decrease in SCE frequency correlated with the increase in carnosine concentration (Figure 1).

Figure 1.

Effects of carbon tetrachloride (CCl₄) and different concentration of carnosine on the SCEs/Cell in the cultures of human peripheral lymphocytes in the study groups. ^ap < 0.001 compared with control group, ^bp < 0.05 compared with CCl₄-treated group, ^cp < 0.001 compared with CCl₄-treated group.

Figure 2.

Effects of carbon tetrachloride (CCl₄) and different concentration of carnosine on the SOD and GPx activities and level of MDA and GSH in the cultures of human peripheral lymphocytes in the study groups. ^ap < 0.001 compared with control group, ^bp < 0.05 compared with CCl4-treated group, ^cp < 0.001 compared with CCl₄-treated group.

Figure 3.

Representative examples of carbon tetrachloride (CCl₄)-treatment group (a), CCl₄ and carnosine (20 mM) treatment group (b) a cell with spontaneous sister chromatid exchanges.

The effects of carnosine on antioxidant enzymes activities

The activities of antioxidant enzymes such as SOD and GPx in the control and experimental groups are represented in Figure 2. Significant reduction in the activities of antioxidant enzymes were found in CCl₄ group when compared with the control (p < 0.001). On simultaneous supplementation with 1, 5, 10 and 20 mM concentrations of carnosine to CCl₄-treated group, significant increases in the activities of SOD and GPx were observed compared with CCl₄ alone−treated group, which shows the restoration of normalcy in carnosine-treated groups (Figure 2). This increase in SOD and GPx activities correlated with the increase in carnosine concentration. The most effective concentration of carnosine was 20 mM and the lowest effective concentration of carnosine was 1 mM (Figure 2).

Levels of GSH

Figure 2 illustrates the levels of GSH in control and experimental groups. In CCl₄-treated group, the level of GSH was significantly decreased when compared with the control group. Upon exogenous supplementation with 1, 5, 10 and 20 mM concentrations of carnosine to CCl₄-treated group, a significant increase in the level of GSH was observed when compared with CCl₄ alone−treated group. At the same time, while comparing the supplemented concentrations of carnosine to CCl₄-treated group, the level of GSH was restored compared with control group.

Levels of MDA

The level of human lymphocyte MDA is presented in Figure 2. The data depict that CCl₄-treated group had significantly higher MDA level compared to control group. Carnosine-treated groups showed a trend to lower MDA level than CCl₄ alone-treated group. This decrease in MDA level correlated with the increase in carnosine concentration. The most effective dose was 20 mM.

Discussion

CCl₄ is metabolized primarily by cytochrome P450 (CYP) enzyme isoform 2E1 (CYP2E1) by reductive dehalogenation to yield a trichloromethyl (•CCl₃) radical which can add oxygen to form a trichloromethyl peroxy (•O-O-CCl₃) radical. The free radicals of CCl₄ abstract a hydrogen atom from a nearby polyunsaturated fatty acid (PUFA) in the endoplasmic reticulum or cell membrane producing fatty acid free radicals, which initiate an autocatalytic lipid peroxidation process generating more lipid hydroperoxides. ⁴ The present data shows that CCl₄ administration produced a marked oxidative impact as evidenced from the significant increase in LPO. The increase in lipid peroxides might result from increased production of free radicals and a decrease in antioxidant status. ³¹ Oxidative enzymes and the determination of MDA levels that are included among peroxidation final products are among the most widely used methods for determination of oxidative stress.^31,32

Oxidative stress occurs when the production of ROS exceeds the body’s natural antioxidant defense mechanisms, causing damage to macromolecules such as DNA, proteins and lipids. To counteract the damaging effect of ROS, aerobic cells are provided with extensive antioxidant defense mechanisms. The oxidative damage in a cell or tissue occurs when the concentration of ROS (O2^.−, H₂O₂ and OH^·) generated exceeds the antioxidant capability of the cell. ³³ Therefore, it could be due to significant decreases in the levels of non-enzymatic antioxidants (e.g., vitamin C, vitamin E, glutathione [GSH]) and enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx], and catalase [CAT]), which are the main determinants of the antioxidant defense mechanisms of the cell.^34,35 Carnosine has anti-oxidative effects. So far, many studies have been done to show the anti-oxidative effects of carnosine.^17,18,20,36

Carnosine gives electron to radical molecules reducing their damaging effects on lipids, proteins and DNA. Mozdzan et al. in their study showed that histidine and alanine separately do not have a reducing effect on Fe²⁺ and Fe³⁺ ions whereas carnosine has a reducing effect on these ions. ²⁰ In another research, it was shown that carnosine reduces the formation of free radicals and prevent lipid peroxidation in plasma of rats.^36,37 Other works on rats, it has been shown that carnosine reduces the oxidative stress that is caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxic substance and also increases the SOD, GPx activities and GSH level. ¹⁸ Carnosine decreases MDA level which is normal increasing and the GSH level which is normal decreasing with age in rats. ¹⁷ Our results are in agreement with the literature. Decreasing SOD and GPx activities and GSH level by CCl₄ treatment has been increased by carnosine addition. And also carnosine has decreased the MDA level.

There are very few studies related with the anti-genotoxic effects of carnosine. Mozdzan et al. showed in their etidium bromide binding assay: DNA damage induced by OH radicals produced by 10 mM H₂O₂, 200 μM FeSO₂ and 2 mM ascorbic acid has been decreased by carnosine addition. ²⁰ In another work with Chinese hamster ovary cells, hyperoxia-induced chromosomal breaks were reduced by 10 and 20 mM doses of carnosine. ¹⁰ In our work, especially the 10 and 20 mM concentration of carnosine has decreased the genotoxic effect of CCl₄ significantly.

Carnosine should have a decreasing effect on ROS and by this mechanism should be strong candidate as an anti-genotoxic natural substance.