Abstract
One of the useful and most commonly cultivated commercially species, migratory locust (Locusta migratoria; Orthoptera), was investigated in light of genotoxic damage potentials. For this aim, we evaluated the genotoxic potentials of water soluble extracts of L. migratoria on cultured human blood cells. The micronucleus, sister chromatid exchange and structural chromosome aberration assays were applied to assess DNA and chromosomal damage produced by aqueous extracts in vitro. The extracts were added to the cultures at different concentrations ranging from 0 to 1000 mg/L. Our results indicated that these extracts did not exhibit genotoxicity at tested concentrations. We conclude that this in vitro approach for biomonitoring genotoxicity assessment is useful for comparing the potential health risks of edible insects.
Introduction
More than 1000 species of insects, mainly in developing countries, that are edible at a certain stage of their life cycle have been identified worldwide (Cerritos, 2009). Of these, grasshoppers are widely studied mainly because of their economic importance in many parts of the world. The famous member of the grasshoppers, the migratory locust (Locusta migratoria) Linnaeus (Orthoptera: Acrididae), is widely distributed in many part of the world like Africa, Asia, Australia and New Zealand (Cerritos, 2009; Metcalf and Metcalf, 1993). L. migratoria is one of the useful and most commonly reared species in the laboratory (Hink and Erlandso, 1994) and is also cultivated in some European countries such as Czech Republic, Poland and Turkey in order to sell alive to many countries. We estimate that L. migratoria will also be produce massively for human consumption in very near future, and therefore, we suggest that it would be useful to determine the genotoxic potential of L. migratoria before massively produced.
On the other hand, edible insects constitute a very common and important food source in many developing countries, although these insects contain powerful pharmacologically active substances, which are known vertebrate toxins (Akinnawo et al., 2002). So eating of these insects may cause serious harmful effects on humans. At this context, the potential toxic effect of these popular edible insects needs to be investigated in more detail. One of the limited numbers of genotoxicity studies on some edible aquatic insects was conducted by Incekara and Turkez (2009). In this investigation, extracts of three edible aquatic insects, Hydrophilus piceus, Dytiscus marginalis and Cybister sp., on sister chromatid exchange (SCE) frequency of cultured human blood lymphocytes was examined and found their nonmutagenic potentials. A similar cytogenetic study was enforced on the two edible grasshopper species, Saga ephippigera ephippigera and Callimenus dilatatus by Turkez et al. (2010b). The result of this study clearly revealed that these species were also nongenotoxic.
To our best knowledge, there is no report on the cytogenetic effects of this locust on human blood cells. In present study, we aimed to assess the genotoxic potential of L. migratoria on cultured human lymphocytes by SCE, chromosome aberration (CA) and micronucleus (MN) assays. In fact, SCE is considered to be a very sensitive, rapid and simple cytogenetic end point for evaluating the genotoxic potential of a variety of mutagenic and carcinogenic agents (Das, 1998). Genetic alterations, mainly CAs and formations of MN in cell cytoplasm, are the early biological effects of mutagenesis and/or carcinogenesis (Hagmar et al. 1998; Geyikoglu and Turkez, 2005a, 2005b; Geyikoglu et al., 2005, 2007; Turkez et al., 2007; Geyikoglu and Turkez, 2008; Turkez, 2008; Turkez and Geyikoglu, 2010). The MN test is also a very sensitive and useful method that can detect both clastogenic and aneugenic activity (Buschini et al. 2004; Sisman and Turkez, 2010; Turkez et al., 2010a, 2010c, 2010d, 2011, 2012). Moreover, high levels of SCE and MN frequency have been observed in persons at higher cancer risk due to exposure to a wide variety of carcinogens (Kumar and Panneerselvam, 2008).
Material and methods
Experimental design
The heparinized blood samples were obtained from two healthy male, nonsmoking donors, with no history of exposure to any genotoxic agent. Experiments including volunteer human subjects have also been performed according to the Declaration of Helsinki. Questionnaires were given to each blood donor to evaluate exposure history, and informed consent forms were signed by each of them. For all the volunteers, hematological and biochemical parameters were analyzed and no pathology was detected. Human peripheral blood lymphocyte cultures were setup according to a slight modification of the protocol described previously by Evans and O’Riordan (1975). The locust samples were purchased from a national company (Mira Co., Aksu, Antalya, Turkey) as alive, and killed without any chemical treatment. The different concentrations (1, 5, 10, 20, 25, 50, 100, 200, 250, 500, 750 and 1000 mg/L) of aqueous extracts of L. migratoria were treated with the cultures for 72 h. At the end of incubation period, the cultures were harvested. The lymphocytes were isolated and stained according to the protocols for SCE, CA and MN assays.
Genotoxicity testing
SCE assay
With the aim of providing a better visualization of SCEs, 5-bromo-20-deoxyuridine (Sigma®, St Louis, Missouri, USA; final concentration 20 mM) was added after culture initiation. Exactly 70 h and 30 min after beginning of the incubations, colcemid (Sigma®) was added to the cultures to achieve a final concentration of 0.5 mg/L. After hypotonic treatment (0.075 M KCl) followed by three repetitive cycles of fixation in methanol/acetic acid solution (3:1, v/v), centrifugation and resuspension; the cell suspension was dropped onto chilled and grease-free microscopic slides, air-dried, aged and then differentially stained for the inspection of SCE rate according to the fluorescence plus Giemsa (FPG) procedure (Perry and Wolff, 1974). For each treatment condition, 20 well spread second-division metaphases were scored and the values obtained were calculated as SCEs per cell.
CA assay
For analysis of structural chromosomal aberrations (chromatid or chromosome gap and chromatid or chromosome break), cultures were carried out for 72 h. Two hours prior to harvesting, 0.1 ml of colchicine (0.2 μg/mL) was added to the culture tube. To prepare slides, 3–5 drops of the fixed cell suspension were dropped on a clean slide and air-dried. The slides were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. Twenty well-spread metaphases were analyzed for each concentration to detect the presence of chromosomal aberrations. Criteria to classify different types of aberrations were in accordance with the recommendation of Environmental Health Criteria (EHC) 46 for environmental monitoring of human populations (IPCS, 1985).
MN assay
The MN test was performed by adding cytochalasin B (Sigma®; final concentration of 6 mg/mL) after 44 h of culture. At the end of the 72-hour incubation period, the lymphocytes were fixed with ice-cold methanol:acetic acid (3:1). The fixed cells were put directly on slides using a cytospin and stained with Giemsa. All slides were coded before scoring. The criteria for scoring micronuclei were as described by Fenech (1993). At least 2000 binucleated lymphocytes were examined per concentration (two cultures per concentration) for the presence of one, two or more micronuclei.
Statistics
The statistical analysis of was performed by Student’s t test was used in this study to assess experimental values in the SCE, CA and MN analysis The level of 0.05 was regarded as indicative of statistical significance.
Results
The observed SCE and CA rates of control and experimental groups are shown in Figures 1 and 2. Our results indicated that various concentrations of L. migratoria did not change the rates of SCEs and CAs when compared with control values (p > 0.05). In accordance with the SCE and CA results (Figures 2 and 3), all the L. migratoria extracts did not induce any MN formation when compared with control group in the present study (Figure 4).
Locusta migratoria.
Frequency of sister chromatid exchanges in cultured human lymphocytes exposed to various concentrations of Locusta migratoria extracts.
The rates of chromosome aberrations in cultured human lymphocytes exposed to various concentrations of Locusta migratoria extracts.
The rates of micronucleus (%) in cultured human lymphocytes exposed to various concentrations of Locusta migratoria extracts.
Discussion
Our in vitro findings clearly indicated that the aqueous extracts of this locust do not show genotoxic effects. However, the toxicokinetics of the locust extracts need to be investigated in more detailed on different in vivo mammalian models for certain suggestions for healthy consumption. Because, we estimate that L. migratoria will be produced much for human consumption in near future.
In another view, this toxicity research will also serve to biomedical productions because it is well known that animal toxins may even become important in curing diseases such as cancer. We also suggest that this in vitro approach that includes the collaborative use of several genetic endpoints such as SCE, CA and MN tests will serve to compare the potential health risks of edible insects related with mutagenesis or carcinogenesis.
Footnotes
Acknowledgments
The author thanks three volunteers for the blood samples.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.