Biologia.2013 Oct;68(5):1004-1009

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

Elanur Aydin1, Hasan Türkez2 & Fatime Geyikoğlu1

1 Department of Biology, Faculty of Science, Atat¨urk University, 25240 Erzurum, Turkey; e-mail: elanuraydinn@gmail.com

2 Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, Erzurum, Turkey

 

Abstract

α-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.

Key words: alpha-pinene; comet assay; MTT assay; neurotoxicity; N2a neuroblastoma cell line; oxidative status.

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Brain cancers, such as glioblastoma, neuroblastoma (NB), and others are among the most devastating tumours in humans and are often rapidly fatal despite aggressive treatments (Maidment & Pilkington 2001; Clarke 2004). NB is a pediatric cancer originated from primitive cells of the sympathetic nervous system and is the most common and deadly solid tumour in childhood (Maris & Matthay 1999; Brodeur 2003). Results studies have examined a range of familial, lifestyle and environmental risk factors for NB (Heck et al. 2009).

α-Pinene (2,6,6-trimethylbicyclo[3.1.1]hept-2-ene) is an organic monoterpene compound, which is present naturally in the essential oils of coniferous and pine trees, rosemary, eucalyptus, camphor and psidium (Simonsen 1957; Nijholt & Mullen 1980; Rupar-Gadd et al. 2006; Bae et al. 2012). α-Pinene was reported to have a broad spectrum of biological activities, i.e. anti-inflammatory (Bae et al. 2012), antibacterial (Dorman et al. 2000) antioxidant (Wang et al. 2008), anticancer (Wang et al. 2012) and antinociceptive (Him et al. 2008) activities. α-Pinene has high potential for human exposure since it was applied in various industries, such as pulp and paper industry, forest production, pharmaceutical industry, food industry, colour printing and paint process (Linares et al. 2009; Montes et al. 2010; Rene et al. 2010; Wu et al. 2012).

In the current study, we evaluated the antiproliferative effects of α-pinene and we found that α-pinene only at 400 mg/L significantly reduced the cell viability on cultured primary rat neurons, while α-pinene at concentrations above than 50 mg/L significantly reduced the cell viability on N2a NB cells (Figure 2). Our findings also indicate that α-pinene is neither genotoxic nor mutagenic on healthy neurons and N2a NB cells using the comet assay since the observed mean values of the total scores of cells showing DNA damage (for SCGE assay) was not found significantly different from the control values on both cells.

The study of some antioxidants use in cancer treatment is a rapidly improving area. Antioxidants have been extensively studied for their ability to prevent or treat cancer in humans (Singh & Lippman 1998). Also, regular intake of natural antioxidants is associated with reduced risks of cancer (Cai et al. 2004). To appraise the antioxidative activity of α-pinene, TAC and TOS assays were performed in this study. α-Pinene applications at its low concentrations (10 and 25 mg/L) caused increases of TAC levels in cultured primary rat neurons. On the other hand, α-pinene (at concentrations of 200 and 400 mg/L) caused significant decreases in TAC levels and increases in TOS levels on healthy neurons. And high concentrations of α-pinene (higher than 50 mg/L) caused significant decreases in TAC levels and increases in TOS levels on cancer cells in vitro (Table 1).

In conclusion, our results showed difference between rat N2a NB cells and cultured primary rat neuron cells. This paper also demonstrates that the α-pinene possesses weak antioxidant and cytotoxic activity in cultured primary rat neurons. In addition, α-pinene has a weak antioxidant properties and little anticancer potentials on rat N2a NB cell line. The efficacy of anticancer chemotherapy is, however, limited by the cytotoxic effect on healthy cells due to a lack of selectivity of α-pinene and poor uptake of the therapeutics by N2a NB cells.

Elanur Aydın-fig1Figure 1. Preparation of primary rat neuron cultures.

 

Elanur Aydın-fig2Figure 2. Cytotoxic effect of α-pinene on cultured primary rat neurons and N2a NB cells. (An asterisk represents statistical difference from the control value at the level of 0.05).

 

Table 1. TAC and TOS levels on cultured primary rat neurons and N2a NB cells exposed to α-pinene (mg/L) for 24 h.aElanur Aydın-tab1a TAC in mmol of trolox equivalent per litre, TOS in mmol of H2O2 equivalent per litre.

 

Acknowledgments

The authors thank to Mrs. Büşra Nur Akgül and Mr. Alihan Doğan for their contributions.

 

 

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