Elanur Aydın

Antioxidative, anticancer and genotoxic properties of α-pinene on N2a neuroblastoma cells

Abstractα-Pinene, an organic monoterpene, is found in essential oils of pine and coniferous trees. To date, although various biological activities of α-pinene have been demonstrated, its neurotoxicity has never been explored. Therefore in this study, we aimed to describe in vitro antiproliferative and/or cytotoxic properties by 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, genotoxic damage potentials by single cell gel electrophoresis, and oxidative effects by total antioxidant capacity (TAC) and total oxidative stress (TOS) analysis of α-pinene. Statistical analysis of MTT assay results indicated significant (p < 0.05) decreases of the cell proliferation rates in healthy neurons treated with α-pinene at only 400 mg/L, while significant decreases were observed in N2a cells at 100, 200 and 400 mg/L. On the other hand, the mean values of the total scores of cells showing DNA damage were not found significantly different from the control values on both cells. In addition, our results indicated that 10 and 25 mg/L of α-pinene treatment caused increases of TAC levels in primary rat neurons without any alterations of its level in N2a cells. However, α-pinene treatments at higher doses led to increases of TOS levels in both cell types. Overall our results suggest that α-pinene is of a limited therapeutic use as an anticancer agent.

Anticancer and antioxidant properties of terpinolene in rat brain cells.

Abstract Terpinolene (TPO) is a natural monoterpene present in essential oils of many aromatic plant species. Although various biological activities of TPO have been demonstrated, its neurotoxicity has never been explored. In this in vitro study we investigated TPO’s antiproliferative and/or cytotoxic properties using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test, genotoxic damage potential using the single-cell gel electrophoresis (SCGE), and oxidative effects through total antioxidant capacity (TAC) and total oxidative stress (TOS) in cultured primary rat neurons and N2a neuroblastoma cells. Dose-dependent effects of TPO (at 10 mg L-1, 25 mg L-1, 50 mg L-1, 100 mg L-1, 200 mg L-1, and 400 mg L-1) were tested in both cell types. Significant (P<0.05) decrease in cell proliferation were observed in cultured primary rat neurons starting with the dose of 100 mg L-1 and in N2a neuroblastoma cells starting with 50 mg L-1. TPO was not genotoxic in either cell type. In addition, TPO treatment at 10 mg L-1, 25 mg L-1, and 50 mg L-1 increased TAC in primary rat neurons, but not in N2a cells. However, at concentrations above 50 mg L-1 it increased TOS in both cell types. Our findings clearly demonstrate that TPO is a potent antiproliferative agent for brain tumour cells and may have potential as an anticancer agent, which needs to be further studied. Sažetak PROTUTUMORSKA I ANTIOKSIDATIVNA SVOJSTVA TERPINOLENA U MOŽDANIH STANICA ŠTAKORA Terpinolen (TPO) prirodni je monoterpen prisutan u esencijalnim uljima mnogih aromatskih biljaka. Premda su otprije poznate razne biološke aktivnosti TPO-a, dosad nije ispitana njegova neurotoksičnost. Svrha je ovog istraživanja in vitro bila utvrditi antiproliferacijska i/ili citotoksična svojstva TPO-a pomoću testa 3-(4,5-dimetiltiazol-2-yl)-2,5 difeniltetrazolijeva bromida (MTT), njegov genotoksični potencijal pomoću komet-testa te oksidativno djelovanje kroz ukupni antioksidativni kapacitet i ukupni oksidativni stres u uzgojenim primarnim neuronima štakora i N2a stanicama neuroblastoma. U objema staničnim linijama ispitani su učinci TPO-a u skladu sa sljedećim dozama: 10 mg L-1, 25 mg L-1, 50 mg L-1, 100 mg L-1, 200 mg L-1 i 400 mg L-1. Značajni (p<0.05) pad stanične proliferacije u primarnim neuronima štakora zamijećen je pri dozama od 100 mg L-1 naviše, a u N2a stanicama neuroblastoma pri dozama od 50 mg L-1 naviše. Niti u jednoj staničnoj liniji TPO se nije pokazao genotoksičnim. Usto se primjenom TPO-a pri dozama od 10 mg L-1, 25 mg L-1 i 50 mg L-1 povećao ukupni antioksidativni kapacitet primarnih štakorskih neurona, ali je takvo djelovanje izostalo u N2a stanica. Međutim, pri koncentracijama višim od 50 mg L-1 TPO je povećao ukupni oksidativni stres u objema staničnim linijama. Naši rezultati nedvojbeno pokazuju da je TPO snažan antiproliferacijski agens u tumorskih stanica mozga, a njegovu potencijalnu ulogu kao protutumorskog lijeka trebalo bi dalje istraživati.

Role of Peltigera rufescens (Weis) Humb. (a lichen) on imazalil-induced genotoxicity: analysis of micronucleus and chromosome aberrations in vitro.

Imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains, is suspected to produce very serious toxic effects on vertebrates. On the other hand, in recent years, a number of studies have suggested that lichens might be easily accessible sources of natural drugs that could be used as a possible food supplement. Extensive research is being carried out to explore the importance of lichen species, which are known to contain a variety of pharmacological active compounds. In this context, the anti-genotoxic effects of aqueous Peltigera rufescens (Weis) Humb. extracts (PREs) were studied against the genotoxic damage induced by IMA on cultured human lymphocytes using chromosomal aberrations (CAs) and micronucleus (MN) as cytogenetic parameters. Human peripheral lymphocytes were treated in vitro with varying concentrations of PREs (0, 5, 10, 25, 50 and 100 mg/L), tested in combination with IMA (336 mg/L). PREs alone were not genotoxic and when combined with IMA treatment, reduced the frequency of CAs and the rates of MNs. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of P. rufescens extract. The results of the present study indicate that this plant extract per se do not have genotoxic potential but can minimize the genotoxicity of IMA on human lymphocytes in vitro. In conclusion our findings may have an important application for the protection of human lymphocyte from the genetic damage and side effects induced by agricultural and medical chemicals hazardous in people.

The protective role of ascorbic acid on imazalil-induced genetic damage assessed by the cytogenetic tests.

Ascorbic acid (AA), known as vitamin C, has important antioxidant and metabolic functions, making its incorporation into the human diet essential. On the other hand, imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains is suspected to produce very serious toxic effects in vertebrates. In this study, the antigenotoxic effects of AA were studied against the genotoxic damage induced by IMA on cultured human lymphocytes using chromosomal aberration (CA) and sister chromatid exchange (SCE) as genetic end points. Human peripheral lymphocytes were treated in vitro with varying concentrations of AA (25, 50, 100, 200, and 400 μg/ml), tested in combination with IMA (336 mg/L). AA alone was not genotoxic and when combined with IMA treatment, reduced the frequencies of CAs and SCEs. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of AA. In conclusion, the preventive role of AA in alleviating IMA-induced DNA damage was indicated for the first time in the present study.

In vitro assessment of cytogenetic and oxidative effects of α-pinene

α-Pinene (α-pinene), a bicyclic monoterpene, is present in the oils of many species of coniferous trees, most notably the pine, and is known for its diverse biological properties such as antimicrobial, anti-inflammatory, antiproliferative and antioxidant. However, there are limited data on the cytogenetic and antioxidant effects of α-pinene in cultured human blood cells (n = 5) for the first time. The purpose of this study was to investigate the genetic, oxidative, and cytotoxic effects of α-pinene in cultured human blood cells (n = 5) for the first time. Human blood cells were treated with α-pinene (0 to 200 mg/L) for 24 and 48 h, and then cytotoxicity was detected by lactate dehydrogenase (LDH) release and (3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide) (MTT) assay, while DNA damage was also analyzed by micronucleus (MN) assay, chromosomal aberration (CA) assay and 8-oxo-2-deoxyguanosine (8-OH-dG). In addition, biochemical parameters (total antioxidant capacity (TAC) and total oxidative stress (TOS)) were examined to determine oxidative effects. The results of LDH and MTT assays showed that α-pinene (at 200 mg/L) decreased cell viability. In our in vitro test systems, it was observed that α-pinene did not cause any statistically important changes in the rates of studied genotoxicity endpoints but dose-dependent alterations were observed in TAC and TOS levels. α-Pinene treatment caused increases in TAC levels (at 25 and 50 mg/L) and decreases in TOS levels (only at 200 mg/L) on human lymphocytes. In conclusion, the findings of the present study confirm for the first time that α-pinene could be a significant source of natural antioxidant compound that may have beneficial health effects.

The Effects of Taurine on Permethrininduced Cytogenetic and Oxidative Damage in Cultured Human Lymphocytes

The Effects of Taurine on Permethrininduced Cytogenetic and Oxidative Damage in Cultured Human Lymphocytes Permethrin (PM) is a common pyrethroid pesticide used to control pests in agriculture, forestry, horticulture, health care, homes, and textile industry. It is confirmed as a strong mutagen in animals and humans. Taurine (TA) is an amino acid found in mammalian tissues that protects the cell against DNA damage. In this study, we investigated whether supplementation of human lymphocyte cultures with TA (in the concentrations of 25 μg mL-1, 50 μg mL-1 and 100 μg mL-1) provided any protection against PM toxicity applied in the concentration of 200 μg mL-1. Genotoxicity was assessed using the micronucleus (MN) and sister chromatid exchanges (SCE) tests. In addition, we measured the total antioxidant capacity (TAC) and total oxidative stress (TOS) levels in the plasma to determine oxidative effects. PM increased SCE and MN levels and altered TAC and TOS levels. TA alone did not affect SCE and MN levels compared to controls, regardless of the concentration applied. In addition, it increased TAC levels without changing TOS levels. Moreover, it significantly buffered the negative cytogenetic and oxidative effects induced by PM in a clear dose-dependent manner. In conclusion, this study is the first to evidence the beneficial effects of TA against PM-induced DNA and oxidative damages in vitro. Djelovanje taurina protiv citogenetičkog i oksidativnog oštećenja u kulturama ljudskih limfocita uzrokovanih permetrinom Permetrin je piretroidni pesticid koji se često rabi za suzbijanje nametnika u poljoprivredi, šumarstvu, povrtlarstvu, zdravstvenoj zaštiti, domovima i tekstilnoj industriji. Poznat je kao snažan mutagen u životinja i ljudi. Taurin je aminokiselina koja se nalazi u tkivu sisavaca i štiti stanicu od oštećenja DNA. Svrha je ovog istraživanja bila saznati hoće li taurin (u koncentracijama od 25 μg mL-1, 50 μg mL-1 te 100 μg mL-1) zaštititi ljudske limfocite od toksičnoga djelovanja permetrina koji je dodan kulturama u koncentraciji od 200 μg mL-1. Genotoksično djelovanje ocijenili smo s pomoću mikronukleus (MN)-testa i testa izmjena sestrinskih kromatida (engl. sister chromatid exchanges, krat. SCE). Da utvrdimo oksidativno djelovanje, izmjerili smo ukupni antioksidativni kapacitet (engl. total antioxidant capacity, krat. TAC) i ukupni oksidativni stres (engl. total oxidative stress, krat. TOS) u plazmi. Permetrin je povećao učestalost SCE i MN te promijenio TAC i TOS. Sam taurin, bez obzira na koncentraciju, nije utjecao na učestalost SCE i MN u odnosu prema kontroli. Usto je podigao TAC, a da pritom nije utjecao na TOS. Štoviše, značajno je ublažio štetno citogenetičko i oksidativno djelovanje permetrina, a učinkovitost mu je bila izravno povezana s primijenjenom koncentracijom. Ovo je prvo in vitro istraživanje koje je pokazalo povoljno djelovanje taurina protiv oksidacijskoga djelovanja permetrina i oštećenja DNA koje on uzrokuje.

The evaluation of the genotoxic and oxidative damage potentials of Ulothrix tenuissima (Kütz.) in vitro

Several alga species are known to produce a variety of toxic metabolites that pose a threat to aquatic organisms, animals and humans. Moreover, these metabolites have been thought to cause serious diseases including certain cancers and neurodegenerative disorders. On the other hand, Ulothrix is a genus of filamentous green algae, generally found in fresh water and marine and abundantly available in some lakes and rivers of Turkey. To our best knowledge, no study has been performed to assess the genotoxic and biochemical effects of U. tenuissima on cultured human blood cells. Therefore, in order to determine clastogenic or aneugenic effects of aqueous alga extracts the micronucleus assay was carried out. Nuclear division index (NDI) in peripheral lymphocytes was also analyzed for cytotoxicity evaluations. In addition, biochemical parameters (total antioxidant capacity (TAC) and total oxidative stress (TOS)) were examined to determine oxidative effects. For this aim, we obtained heparinized blood samples from three healthy persons. The alga samples were collected from Porsuk Pond in Hasankale (Erzurum, Turkey) in summer period of the year 2010. The aqueous extracts of this species were added to cultures at different concentrations (0 to 5000 ppm) for 72 h. Our results showed that this alga did not cause any statistically important changes in the rates of studied genotoxicity endpoint. But dose-dependent alterations were observed in TAC and TOS levels and NDI rates. In conclusion, U. tenuissima was found to be non-genotoxic but caused sterility at higher concentrations due to oxidative stress.

Anti-genotoxic role of eicosapentaenoic acid against imazalil-induced DNA damage in vitro.

Eicosapentaenoic acid (EPA) is a polyunsaturated n-3 fatty acid and is essential to the health of mammals. Recent data show that EPA can act as anti-mutagenic agent. On the other hand, pesticides comprise a new and important class of environmental pollutants nowadays. Imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains is suspected to produce very serious toxic effects in vertebrates. The present study investigated the anti-genotoxic effect of EPA against the genotoxic damage induced by IMA on cultured human lymphocytes using chromosomal aberration (CA) and micronucleus (MN) tests as cytogenetic endpoints. Peripheral blood cells were treated in vitro with varying concentrations of EPA (2.5, 5, 10, 20 and 40 μg/ml), tested in combination with IMA (336 μg/ml). Our results revealed that the rates of CAs and MNs in lymphocytes were significantly (p < 0.05) increased by IMA as compared to the controls. The results also showed that EPA alone was not genotoxic. Moreover, when combined with IMA treatment, EPA reduced the frequencies of CAs and MNs. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of EPA. In conclusion, our data may have an important application for the protection of cultured human lymphocyte from the genetic damage and repercussions induced by agricultural and industrial chemicals hazardous in people.

The genoprotective activity of resveratrol on permethrin-induced genotoxic damage in cultured human lymphocytes

The aim of this work was to investigate the genetic effects of resveratrol (RSV) at concentrations of 10, 15, 25, 40, 75 and 100 µM and its activities on the genotoxicity induced by the permethrin (PM) (200 µM). After the application of PM and RSV, separately and together, cultured human lymphocytes were assessed by chromosome aberrations (CA) and sister chromatid exchange (SCE) tests. According to results, the frequencies of CA and SCE rates in the peripheral lymphocytes were significantly increased by PM compared with the controls. However, RSV had no genotoxic effect. Furthermore, the findings revealed that PM-induced increases in the mean frequencies of both genotoxic indices were diminished by RSV in a clear dose dependent manner, indicating its protective role towards the cells from PM exerted injury. In conclusion, these effects of RSV should be considered while evaluating the possible use of RSV as a therapeutic agent.

In vitro risk assessment of usnic acid

Lichens are symbiotic organisms composed of fungi and algae and are very common in Turkey. Lichen secondary metabolites are mainly phenolic compounds produced by fungal partner of lichen symbiosis. Usnic acid (UA) is one of the most common lichen metabolites, and it was reported that to be effective for a wide range of pharmacological purposes including antiviral, antitumor, and antiprotozoal. However, there are limited data on the genotoxic and antioxidant effects of UA in cultured human peripheral blood cells. Therefore, the aim of this thesis study was to investigate the genetic and oxidative effects of UA in cultured human blood cells (n = 5). The UA was added into culture tubes at various concentrations (0–200 μg/ml). Chromosomal aberrations (CA) and micronuclei (MN) tests were performed for genotoxic damage influences estimation. In addition, biochemical parameters (total antioxidant capacity (TAC) and total oxidative status (TOS)) were examined to determine oxidative effects. In our in vitro test systems, it was observed that UA had no mutagenic effects on human lymphocytes. Furthermore, our results indicated that low concentrations (1 and 5 μg/ml) of UA caused increases of TAC levels in cultured human blood cells. And, the TOS levels were not changed (p > 0.05) when all the concentrations (except for 200 μg/ml) of UA were applied. In conclusion, UA can be a new resource of therapeutics as recognized in this study with their nonmutagenic and antioxidant features.