Br in Vitro Genotoxic Effects of Four Helichrysum Species in Human Lymphocytes Cultures

Helichrysum sanguineum, Helichrysum pamphylicum, Helichrysum orientale, Helichrysum noeanum (Asteraceae) are medicinal plants. For centuries, they have been used as tea in Turkey because of their medicinal properties. So far no scientific evidence has been found in a literature survey regarding the genotoxic effects of these plants. This work evaluated the genotoxic effects on human lymphocyte cultures induced by methanol extracts of these plants, assayed in different concentrations (0.01, 0.05, 0.1, 0.5 and 1 mg/mL). According to the results, Helichrysum noeanum, Helichrysum pamphylicum and Helichrysum sanguineum induced the formation of micronuclei and decreased the mitotic and replication indexes. Helichrysum orientale did not affect these parameters, whereas Helichrysum noeanum, Helichrysum pamphylicum and Helichrysum sanguineum were clearly genotoxic. They should therefore not be used freely in alternative medicine, although their antiproliferative activity may suggest antimitotic and anticarcinogenic properties. Helichrysum orientale could be used in alternative medicine.


INTRODUCTION
The genus Helichrysum Mill, belonging to the family Asteraceae, is represented by approximately 300 species in the world.This genus is represented in Turkish flora by 26 taxa, of which 13 are endemic (Davis and Kupicha, 1975).Helichrysum species are aromatic plants, commonly known as everlasting, immortal flower and fadeless flower, grow wild in Anatolia and are widely used as tea.They are used for the treatment of kidney stones, urogenital disorders, stomach pain, jaundice, diarrhea and asthma (Baytop, 1997).
The micronucleus (MN), mitotic index (MI) and replication index (RI) analysis methods are cytogenetic tests that are used both in vivo and in vitro.An MN is a small extra nucleus separated from the main one, generated during cellular division by late chromosomes or by chromosome fragments.Because of its association with chromosomal aberrations, MN has been used since 1937 as an indicator of genotoxic exposure based on the radiation studies conducted by Brenneke and Mather (Heddle et al, 1983).The MI assay is used to characterize proliferating cells and identify compounds that inhibit or induce mitotic progression.RI measures cell division kinetics by counting the percentage of cells in first, second and third or more metaphase (Holland et al., 2002).
So far, no scientific evidence has been found in a literature survey regarding the genotoxic effects of these plants.This work evaluated the genotoxic effects of Helichrysum sanguineum, Helichrysum pamphylicum, Helichrysum orientale, Helichrysum noeanum, used in Turkey as folk medicine.Helichrysum pamphylicum and Helichrysum noeanum are endemic species of the Turkish flora.

Plant materials
The aerial parts of Helichrysum species were collected from Hatay (Helichrysum sanguineum), A n t a l y a ( H e l i c h r y s u m p a m p h y l i c u m ) , Ay d ı n (Helichrysum orientale) and Ankara (Helichrysum noeanum), Turkey, in the months of May and June, and were identified by Prof. Dr. Ergin Hamzaoğlu, Prof. Dr. Ahmet Aksoy and Dr. Ümit Budak.Voucher specimens (Budak 2034, Hamzaoğlu 3639, Budak 2001 andHamzaoğlu 3742) have been deposited in the Herbarium of the Department of Biology, Bozok University for future reference.

Preparation of the methanol extracts
The aerial parts of the plant material were dried in shade at room temperature and then ground to powder in a mechanic grinder.Then the powdered plant materials (10 g) were extracted in a Soxhlet extractor with 100 mL methanol (100%) at 60ºC for 6 h.After determining the yield, the extracts were filtered and concentrated to dryness under reduced pressure at 40ºC with a rotary evaporator.Finally, the extracts were kept at +4ºC until tested.

Subjects
The study involved six donors (three male and three female) at Yozgat, Turkey, in the age group 26 ± 45 years.The selection criteria for the subjects were based on a questionnaire intended to elicit information on the subject's age, smoking habits, alcohol consumption and health condition.Individuals selected for participation were nonsmokers and non-consumers of alcohol who had not been exposed to x-rays or gamma rays nor had had a viral infection.

In vitro mitotic index assay
After obtaining approval from Local Ethisc Committee, heparinized blood samples (0.4 mL) of six healthy donors were placed in sterile culture tubes containing 5 mL of PB karyotyping medium.Then, Helichrysum extract preparations were added to obtain the five final concentrations (0.01, 0.05, 0.1, 0.5 and 1 mg/mL).After mixing the contents of each culture tube by shaking gently, the culture tubes were incubated in a slanted position at 37°C for 72 h.After 70 h of incubation, a 0.1 mL colcemid solution (10 µg/mL) was added to each culture tube and mixed by shaking gently.After 72 h of incubation, cells were harvested by centrifugation, by hypotonic treatment (0.075 M KCl) and fixing in a fresh fixative solution (methanol:acetic acid, 3:1).The fixation step was repeated three times.Slides were air-dried and stained with Giemsa (Ozkul et al., 2005).MI was calculated as the proportion of metaphase for 2000 cells for each donor and concentration.

In vitro micronucleus assay
For MN analysis, the peripheral lymphocytes were incubated at 37 ºC for 72 h.The cells were treated with Helichrysum extract preparations at 0.01, 0.05, 0.1, 0.5 and 1 mg/mL concentrations.Cytochalasin B (Sigma) was added at 44 h of incubation at a final concentration of 5 µg/mL to block cytokinesis.At the end of incubation at 37 ºC, cells were harvested by centrifugation.The MN staining was performed according to Ozkul et al. (2005).
The slides were scored by a single observer.About 500 cells were examined at 600 check magnification from each slide and when MN cells were located they were examined under 1000 magnification.The criteria suggested by Scarpato and Migliore (1996) for recognizing MN was followed.
The extent and progression of nuclear division were measured by counting the number of cells with one, two, three and four or more nuclei in a total of 500 cells per sample (Figure 1).RI was calculated among 500 cells per culture, according to the following formula: RI = (1 x M1 + 2 x M2 + 3 x M3)/500, where M1, M2 and M3 are the number of cells in first metaphase, second metaphase and third or more metaphase, respectively (Holland et al., 2002).

Statistical analysis
The computer software program SPSS 10.0 was used to analyze the data.The statistical significance of the effects of Helichrysum species on MI, RI and MN was tested by repeated measures of the analysis of variance (ANOVA) and differences between groups were determined by the least significant differences (LSD) test and were considered significant for p<0.01.

Micronucleus
The results of MN test are given in Figure 2. The mean values of the MN rates of added different concentrations of plant extracts were between 7.83 ± 3.72 and 16.06 ± 3.84 for Helichrysum sanguineum, 12.33 ± 1.47 and 16.66 ± 1.53 for Helichrysum pamphylicum, 5.50 ± 3.02 and 6.90 ± 3.17 for Helichrysum orientale, 9.96 ± 1.85 and 14.06 ± 2.57 for Helichrysum noeanum.MN rates of 1 mg/mL added to plant extracts are 1.5-3 times more than that of in the control.When MN formation was analyzed after treatment with different concentrations of methanol extract of Helichrysum sanguineum (0.5 mg/mL), Helichrysum pamphylicum (0.01 mg/mL), Helichrysum noeanum (0.5 mg/mL), significant changes in the percentage of MN were detected (p<0.01).Helichrysum orientale did not induce any change in MN frequencies (p = 0.01).

Replication index
RI results are parallel to MI results.Increasing extract concentrations have caused a decreasing rate of RI.When RI was studied, no modifications could be detected for Helichrysum orientale (p = 0.01).As shown in Table II, changes in RI reflecting genotoxic effects were observed for Helichrysum sanguineum (0.05 mg/mL), Helichrysum pamphylicum (0.01 mg/ mL) and Helichrysum noeanum (0.1 mg/mL) (p<0.01).

DISCUSSION
Helichrysum species have been used in folk medicine for thousands of years throughout world.There are a lot of studies interested in properties of Helichrysum species and the use folk medicine derived from them.Some members of this genus have cholagogue and choleretic activity and stimulate the secretion of gastric juice (Litvinenko et al., 1992).Studies regarding Helichrysum species used in this study are summarized.The literature indicates the antibacterial, antiviral, antifungal (Aslan et al., 2006), antioxidant (Tepe et al., 2005) and anticancer (Topcu, 2007) effects of Helichrysum sanguineum, Helichrysum pamphylicum, Helichrysum orientale and Helichrysum noeanum.The major components of these species are flavonoids (Bingöl and Çubukçu, 1984;Çubukçu and Bingöl, 1981;Meriçli et al., 1984;Sezik and Akdemir, 1986).
The genotoxic effects of Helichrysum sanguineum, Helichrysum pamphylicum and Helichrysum noeanum may result from flavonoids.However, there are few reports about the cytotoxic, genotoxic and mutagenic effects of Helichrysum species and no information is available about the genotoxic effects of the Helichrysum species used in this study.For this reason, it is important to determine the genotoxic effects of these species.
There are many factors affecting the MN frequency in lymphocytes: age, gender, smoking and alcohol consumption, viral infection and X and gamma ray exposure (Müller, 1996).In this study, donors included only non-smokers and nonconsumers of alcohol who had not been exposed to X and gamma rays, nor had had a viral infection.An increase in MN may result from interactions of a great variety of cytotoxic and genotoxic agents with chromosomal damage.MN is an extremely valuable and highly relevant endpoint for the detection of potential carcinogens.Our results show an increase in the percentage of MN (Fig. 2), suggesting a strong interaction between extracts of Helichrysum sanguineum, Helichrysum pamphylicum, Helichrysum noeanum and chromosomal damage, which could be responsible for the observed genotoxicity.However, there are no significant effects of Helichrysum orientale on MN frequency.
MI and RI are used as indicators of adequate cell proliferation biomarkers.We detected a positive correlation between MI and RI; namely the lower the MI rates detected in exposed individuals, the lower the values of nuclear division progression expressed as RI.The decreases of MI and RI rates and the positive correlation between MI and RI could result from a cell cycle delay and cytotoxic effects of Helicheysum species.MI measures the proportion of cells in the M-phase of the cell cycle and its inhibition could be considered as delay in the cell proliferation kinetics (Rojas et al., 1993).Except for Helichrysum orientale, there are major differences among MI and RI rates of other Helichrysum species.
The present results also show that RI values were significantly lower in extract concentrations than in controls.This state can be explained with mitotic delay, which, by permitting the repair of genotoxic lesions or cells with greater chromosomal damage, may die before cell division (Santos-Mello et al., 1974).Multiple MN as the result of the loss of large part of the chromosome impairs or even prevents the cell division (Nath and Ong, 1990).
In the present study, we found that extracts of four Helichrysum species used in alternative medicine induced MN decreased MI and RI in human lymphocytes.We are of the opinion that Helichrysum sanguineum, Helichrysum pamphylicum and Helichrysum noeanum should not be used in high quantities in the general population because of their genotoxic properties (especially Helichrysum pamphylicum).Indeed this plant extracts could cause chromosomal damage (an increase in MN) and mitotic delay (decrease in MI and RI).According to our results, Helichrysum orientale is not genotoxic and thus it can be freely used in alternative medicine.The decrease in cell proliferation may indicate that Helichrysum sanguineum, Helichrysum pamphylicum and Helichrysum noeanum may also act as antimitotic and anticarcinogenic agents.In contrast, increasing MN rates showed that they could have a carcinogenic effect at high concentrations.Furthermore, there are a lot of compounds in the contents of Helichrysum species.The use of single constituents would allow more precise interpretation of data and the added effect of the numerous compounds will be lost.It is therefore likely that the mixture of Helichrysum compounds may result in synergistic effects.A further study will be needed to determine the effects of different compounds isolated from Helichrysum species and evaluate the synergistic effects on MI, RI and MN.Besides, this study should support with reports about the metabolisms of extracts of these species and others genotoxic studies in mammals.