Nurşen Başaran

Antioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol

The leafy parts of thyme and its essential oil have been used in foods for the flavour, aroma and preservation and also in folk medicines. In the present study the genotoxicity of thymol and carvacrol was examined using comet assay. In V79 Chinese hamster lung fibroblast cells treated with 1, 5, 25 microM thymol and carvacrol, only 25 microM thymol caused some clastogenic DNA damage. For detection of oxidative DNA damage, the comet assay with formamido pyrimidine glycosylase (Fpg) protein was used: When V79 cells were treated with 1, 5, 25 microM thymol and carvacrol and post-treated with Fpg enzyme, no significant increase of Fpg-sensitive sites was observed at all concentrations studied. Reactive oxygen species (ROS) generation decreased slightly in the presence of thymol (1-100 microM) and carvacrol (5 microM) between 1 and 4h, yet increased at the highest 100 microM concentration of carvacrol after 24h. Thymol and carvacrol displayed a concentration dependent antioxidant capacity, whilst gamma-terpinene which lacks a phenolic group did not show any antioxidant capacity in the trolox equivalent antioxidant capacity (TEAC) assay. The results of this study indicate a lack of clastogenic activity for thymol and carvacrol at biologically relevant concentrations, and a moderate antioxidant activity in vitro.

Modulating effects of flavonoids on food mutagens in human blood and sperm samples in the Comet assay

The flavonoids silymarin, myricetin, quercetin, kaempferol, rutin, and kaempferol-3-rutinoside have been examined in combination with the food mutagens 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp) and 2-amino-3-methylimidazo-4,5-f)quinoline (IQ) in the Comet assay in human lymphocytes from donors A and B and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in human lymphocytes and sperm over a similar dose range in the absence of metabolic activation. Only quercetin and kaempferol were examined in blood with metabolic activation, but there was no difference in response to that obtained without activation. In the blood there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm this was also the case with silymarin and myricetin. With kaempferol there was no antigenotoxic effect and quercetin protected below baseline levels. Since the effects were observed in lymphocytes and sperm over a similar dose range, it would suggest that the Comet assay responses occur in somatic and germ cells in a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

Flavonoids modulate Comet assay responses to food mutagens in human lymphocytes and sperm

The flavonoids, silymarin, myricetin, quercitin, kaempferol, rutin and kaempferol-3-rutinoside have been examined in combination with the food mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-3-methylimidazo-(4,5-f) quinoline (IQ), in the Comet assay in human lymphocytes from donor A and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in lymphocytes and sperm. The assays were performed in the absence of metabolic activation, since when quercetin and kaempferol were examined in blood with metabolic activation, there was little or no difference in response to that obtained in its absence. In the blood, there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm, with silymarin, myricetin and quercitin, antigenotoxic effects only were observed, but with kaempferol, in general, there were no protective effects. The food mutagen, 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), was also examined in addition to Trp-P-2 and IQ in combination with silymarin and myricetin in donors A and C in human lymphocytes only. Similar exacerbation of effects were found at low doses of these flavonoids with antigenotoxic effects at high doses. This was confirmed in the Ames test. There were slightly different profiles in lymphocytes and sperm, but antigenotoxic effects were observed over a similar dose range. This would suggest that effects occur in somatic and germ cells on a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

Effect of various genotoxins and reproductive toxins in human lymphocytes and sperm in the Comet assay

There have been conflicting reports as to whether the mean sperm count in some men has diminished over the last 50 years. The downward trend has been suggested to coincide with an increase in exposure to estrogen-like compounds. These estrogenic substances are ubiquitous in the environment. We have examined the effect of such substances (diethylstilbestrol, beta-estradiol, daidzein, genestein, and nonylphenyl) in the single cell gel electrophoresis assay (Comet assay) in human sperm and compared responses with those from human peripheral lymphocytes in the same donor and in peripheral lymphocytes from a female donor. In addition, effects from the estrogens have been compared to those from known reprotoxins and genotoxins. These include lead sulfate, nitrate and acetate, dibromochloropropane, ethylene glycol monoethyl ether, 1,2-epoxybutene, and 1,2,3,4-diepoxybutane. All compounds produced positive responses, but ethylene glycol monoethyl ether only produced positive responses in sperm cells in the male and not in peripheral lymphocytes, and similarly the phytoestrogens (genistein, daidzein) were less responsive in the peripheral lymphocytes in the male than in the sperm. This may be due to greater sensitivity of sperm cells because of their lack of repair. However, since damage was generally seen over a similar dose range, a one-to-one ratio of somatic and germ cell damage was observed and has implications for man for risk assessment purposes.

Assessment of DNA strand breakage by the alkaline COMET assay in dialysis patients and the role of Vitamin E supplementation.

Although the role of reactive oxygen species (ROS) in chronic renal failure (CRF) is not definitely demonstrated, a consistent number of observations has provided evidence for the presence of oxidative stress in uremic patients undergoing maintenance dialysis. In order to investigate this hypothesis further and to understand the role of antioxidant supplementation, peripheral blood lymphocytes were taken from 36 dialysis patients before and after Vitamin E supplementation in a dosage of 600 mg per day (2x300 mg) for 14 weeks and examined in the alkaline Comet assay for DNA strand breakage. The results were also compared with those of 36 controls with comparable age, sex, and smoking habits, and with no history of renal disease. The DNA breakage observed in the lymphocytes of patients before Vitamin E supplementation was significantly higher than in the controls (P<0.001) but a clear protective effect of Vitamin E supplementation were observed after 14 weeks of therapy.

Immune alterations in lead-exposed workers.

Peripheral blood lymphocytes, serum immunoglobulins (IgG, IgA and IgM), C3 and C4 complement protein concentrations of 25 male lead-exposed workers from storage-battery plants were examined and compared to 25 healthy male controls. Lead exposure was assessed using blood lead levels measured by atomic absorption spectrophotometry and zinc protoporphyrin (ZPP) levels assayed by hematofluorometry. The absolute number and the percentage of functionally different subsets of peripheral blood mononuclear lymphocytes, i.e. T, T-suppressor, B and natural killer cells, were unchanged. However, T-helper lymphocytes were significantly lower in lead-exposed workers compared to healthy controls (P < 0.05). In addition, lead-exposed workers had a significant reduction in the IgG, IgM and C3, C4 complement levels (P < 0.05). These results suggest that human chronic exposure to lead may be detrimental to the immune system.

Effects of lead on immune parameters in occupationally exposed workers

BACKGROUND To assess the immune competence of workers occupationally exposed to lead, several subsets of peripheral lymphocytes, i.e., T, TCD4(+), TCD8(+), B, NK cells, serum immunoglobulin and complement protein concentrations, chemotaxis, and intracellular killing activity of neutrophils of 25 male storage battery workers have been analyzed and compared to 25 healthy males with no history of lead exposure. RESULTS The results of this study which indicated that industrial exposure to lead resulting in group mean blood lead concentrations of 75 +/- 18 microg/dl are associated with a significant depression of: T helper lymphocytes, Ig G, Ig M and C3, C4 complement levels, chemotaxis, and random migration of neutrophils. No correlation was found between the duration of exposure and the altered immune parameters. CONCLUSIONS The immune system can be a target for lead toxicity and elimination of lead hazard in working places is necessary.

Humoral and cellular immune parameters in untreated and phenytoin- or carbamazepine-treated epileptic patients

The peripheral blood lymphocyte subsets, serum immunoglobulins (Ig A, G, M), and C3 and C4 complement protein concentrations were determined in 40 healthy subjects, 30 phenytoin-treated, 22 carbamazepine-treated and 38 untreated epileptic patients. The levels of beta-lymphocytes, IgM and C3 complement proteins were found to be significantly higher in untreated epileptics than in healthy controls (P < 0.01, P < 0.02 and P < 0.05, respectively). The absolute number of beta-lymphocytes appeared to be unaffected by phenytoin or carbamazepine treatment; however, IgM levels were significantly lower in carbamazepine-treated patients than both epileptic (P < 0.01) and healthy (P < 0.05) controls. Phenytoin-treated patients had a significant reduction in the mean IgA and IgG levels compared to healthy and epileptic controls (P < 0.05). With both drug treatments, significantly lower T-suppressor lymphocyte counts and thus higher T-helper to T-suppressor lymphocyte ratios were observed with respect to healthy and epileptic controls. Our results demonstrate that while phenytoin decreases serum IgA and IgG levels, carbamazepine reduces IgM levels significantly, and untreated epileptics show immune profiles significantly different to those of healthy subjects, suggesting that epilepsy per se may be associated with certain immune aberrations induced by antiepileptic drugs.

Protective effects of curcumin against oxidative stress parameters and DNA damage in the livers and kidneys of rats with biliary obstruction

Curcumin, a most active antioxidant compound, has been suggested to have potential beneficial effects against most metabolic and psychological disorders, including cholestasis. In the present study, the effects of curcumin against oxidative stress and DNA damage induced by bile duct ligation (BDL) in Wistar albino rats for 14 days were investigated. The rats were divided into three following groups: Sham group, the BDL group and the BDL+curcumin group. A daily dose of 50mg/kg curcumin was given to the BDL+curcumin group intragastrically for 14 days. The biomarkers of hepatocellular damage were decreased in the BDL+curcumin group compared to the BDL group, indicating that curcumin recovered the liver functions. DNA damage as assessed by the alkaline comet assay was also found to be low in the BDL+curcumin group. Curcumin significantly reduced malondialdehyde and nitric oxide levels, and enchanced reduced glutathione levels and catalase, superoxide dismutase, and glutathione S-transferase enzymes activities in the livers and kidneys of BDL group. Curcumin treatment in BDL group was found to decrease tumor necrosis factor-alpha levels in the livers of rats. These results suggest that curcumin might have protective effects on the cholestasis-induced injuries in the liver and kidney tissues of rats.

The antioxidant and antigenotoxic properties of citrus phenolics limonene and naringin.

Phenolic compounds not only contribute to the sensory qualities of fruits and vegetables but also exhibit several health protective properties. Limonene and naringin are the most popular phenolics found in Citrus plants. In this study, we investigated the antioxidant capacities of limonene and naringin by the trolox equivalent antioxidant capacity (TEAC) assay and the cytotoxic effects by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in Chinese hamster fibroblast (V79) cells. The genotoxic potentials of limonene and naringin were evaluated by micronucleus (MN) and alkaline COMET assays in human lymphocytes and V79 cells. Limonene and naringin, were found to have antioxidant activities at concentrations of 2-2000 µM and 5-2000 µM respectively. IC50 values of limonene and naringin were found to be 1265 µM and 9026 µM, respectively. Limonene at the concentrations below 10,000 µM and naringin at the all concentrations studied, have not exerted genotoxic effects in lymphocytes and in V79 cells. Limonene and naringin at all concentrations revealed a reduction in the frequency of MN and DNA damage induced by H2O2.