Mutagenic biomonitoring of pirethroid insecticides in human lymphocyte cultures: Use of micronuclei as biomarkers and recovery by Rosa canina extracts of mutagenic effects

Abstract

Context: Insecticides are used to control pests. Cypermethrin and fenvalerate are widely used pirethroid insecticides in the world. Rosa canina L. (Rosaceae) is used as a traditional medicinal plant against viral infections and disorders of the kidneys and urinary tract due to its high vitamin C level.

Objective: The genotoxic effects of cypermethrin and fenvalerate were examined with the micronucleus (MN) test. Then, we determined the ability of the water (RC_wtr) and ethanol (RC_eta) extracts of rosehip (R. canina) to overcome the possible genotoxic effects of the insecticides.

Materials and methods: Preliminary studies determined that the application concentrations were 20, 30, 40, and 50 ppm for cypermethrin, 25, 50, 75, and 100 ppm for fenvalerate, and 100 ppm for rosehip extracts. DMSO (dimethyl sulphoxide) (1%) and 1 mM EMS (ethyl methanesulfonate) were used as negative and positive control groups, respectively. The application groups belonging to insecticides and plant extracts were added to culture tubes including chromosome B medium and peripheral blood for MN test.

Results: The MN frequencies were found 0.725 in the negative control group, 2.700 in the positive control groups, 1.275 in the highest application group of cypermethrin, and 1.600 in the highest application group of fenvalerate. The MN frequencies in cypermethrin + RC_wtr, cypermethrin + RC_eta, fenvalerate + RC_wtr, and fenvalerate + RC_eta application groups were, respectively, determined as 1.000, 1.075, 1.225, and 1.275.

Conclusion: According to the results, cypermethrin and fenvalerate have genotoxic effects, the water and ethanol extracts of rosehip reduced the genotoxicity of the both insecticides.

• Insecticide
• micronuclei
• mutagenic effects
• pirethroid
• Rosa canina

Introduction

Although there are several advantages in agricultural activities, pesticides cause various problems such as cancer and environmental pollution. The group used most widely is insecticides. Insecticides are used to control harmful insects. However, they do not only damage insects but also harm people. Even sub-lethal doses of some insecticides can be mutagenic (Abd-Alla et al., 2003) and may lead to diseases such as cancer (Atamanalp & Cengiz, 2002). There are various investigations available on toxic and genotoxic effects of cypermethrin and fenvalerate, which are pirethroid insecticides. Mukhopadhyay et al. (2003) reported that cypermethrin may be genotoxic even at low concentrations. Bhunya and Pati (1988) observed that cypermethrin increased micronucleus (MN) formation depending on the dose in mice. Giri et al. (2003) reported that cypermethrin increased the frequency of SCE in mouse bone marrow cells. According to Puig et al. (1989), cypermethrin led to chromosomal structural defects and sister chromatid exchange (SCE). Furthermore, cypermethrin was reported to cause toxicity at Drosophila melanogaster development stages (Karataş & Bahçeci, 2008) and increase free radical formation in rats (Giray et al., 2001). Fenvalerate was also reported to be genotoxically effective (Giri et al., 2002). Xia et al. (2004) noted that fenvalerate and its metabolites caused morphological abnormalities and genotoxic defects in human sperm. Pati and Bhunya (1989) reported that fenvalerate significantly increased chromosomal aberrations (CA) and MN formation in mice. In a study conducted by Prasanthi et al. (2005), it was determined that fenvalerate caused oxidative damage in male rats.

Various plants are used against different effects of chemicals at present. Rosa canina L. (Rosaceae) is one of the plants that is used for alternative treatment purposes and have antioxidant properties. According to various researchers (Cemeroglu, 1992; Ozcan, 2000; Serteser et al., 2008), the rosehip plant (R. canina) has antioxidant properties and can eliminate DNA damage associated with oxidative stress (Kılıçgün & Dehen, 2009). Several studies which have been made in this direction are available. A study conducted by Kızılet et al. (2013) showed that the ethanol extract of rosehips decreased the genotoxic effects of EMS in D. melanogaster. Westhuizen et al. (2008) reported that Rosa roxburghii Tratt (chestnut rose) plant, which is located in the same family with rosehips, significantly showed anti-mutagenic effect against mutagens, such as alfatoxin B1. In this study, anti-mutagenic effects of the water and ethanol extracts of rosehip were investigated against genotoxic effects of pirethroid insecticides in human peripheral lymphocytes.

Materials and methods

Chemicals

Cypermethrin (CAS no. 52315-07-8), fenvalerate (CAS no. 51630-58-1), ethyl methanesulfonate (EMS, CAS no. 62-50-0), cytochalasin (CAS no. 14930-96-2), methanol (CAS no. 67-56-1), ethanol (CAS no. 64-17-5), and glacial acetic acid (CAS no. 64-19-7) were purchased from Sigma (St. Louis, MO). Giemsa (Cat no. 109204) supplied by Merck, Mumbai, India. Dimethyl sulphoxide (DMSO, CAS no. 67-68-5) and chromosome medium B (Cat no. F 5023) were, respectively, purchased from Riedel (Wuppertal, Germany) and Biochrom (Berlin, Germany).

Plant extract

Berries of the rosehip plants were collected from natural areas at an altitude of 2000–2200 m in the highlands of Erzurum, Turkey, in September 2012, during the maturation period. The plant was identified by Meryem Şengul Koseoglu (Atatürk University, Turkey). Voucher specimens are deposited in the Herbarium of Atatürk University’s Faculty of Science (Erzurum, Turkey). Rosehip berries were dried in indirect light and a clean environment. Then, both the berries and the seeds in the fruit were milled with the help of a blender. An ethanol extract of rosehips was prepared according to Cakir et al. (2005). To prepare the ethanol extract, the milled rosehip samples were kept in ethanol (95%) for 4 d. The plant–ethanol mixture was filtered at the end of each day and ethanol was removed from the released solution. The remaining extract was used as the ethanol extract (RC_eta). The water extract of rosehips was prepared on the basis of the method applied by Halici et al. (2005). According to this, 100 g of the milled rosehips was placed in 200 ml of distilled water. Rosehip–water mixture was afflicted in the water-bath which was adjusted 50 °C for 2 h and subsequently filtered. After the released solution was passed lyophilizator, the water extract of rosehip plant (RC_wtr) was obtained. As a result of the preliminary studies, application doses of both RC_wtr and RC_eta were determined as 100 ppm.

Selection of application doses for insecticide

Application doses were designated as 20, 30, 40, and 50 ppm for cypermethrin and 25, 50, 75, and 100 ppm for fenvalerate with the pre-trials. DMSO (1%), which was a solvent for cypermethrin and fenvalerate, was the negative control; ethyl methane sulfonate (EMS) (1 mM), was used as a positive control group.

MN test

For the MN test, human peripheral lymphocytes cells were cultured. Heparinized blood that was received from four different non-smoking, healthy individuals was added to culture tubes containing chromosome B medium. These tubes were incubated at 37 °C for 3 d. Application groups belonging to insecticides and plant extracts were added to the culture tubes after 24 h. Cytochalasin was added at 48 h. The culture was harvested at 72 h. Culture tubes were passed from the hypotonic solution (0.075 M) and the fixing solution consisting of a mixture of glacial acetic acid–methanol (1/3) and centrifuged. After the supernatant was discarded, slides were prepared. After drying, the slides were stained with Giemsa (Bochum, Germany). About 1000 bi-nucleated cells for each donor were examined under the microscope (400×). MN frequency and the number of cells containing MN were determined according to Fenech et al. (2003). The nuclear division index (NDI) was calculated according to Kocaman and Topaktas (2009), (NDI = 1 × M1 + 2 × M2 + 3 × M3 +4 × M4/N). In this formula, M1–M4 represents the number of cells with one to four nuclei and N is the total number of viable cells scored. The Duncan test was used for statistical evaluation.

Results

While the average MN frequency and NDI were, respectively, 0.725 and 1.62 in the negative control group (1% DMSO), these values were, respectively, 2.700 and 1.52 in the positive control group (1 mM EMS) (Tables 1 and 2). In application groups of cypermethrin, the average MN frequency was determined as 0.775 in the lowest application group (20 ppm) and 1.275 in the highest application group (50 ppm). NDI decreased from 1.53 to 1.33 in the lowest and highest application groups (Table 1). These differences between the experimental and DMSO control groups were found to be statistically significant (p < 0.05).

Table 1. MN and the NDI values of human peripheral lymphocytes as a result of application of cypermethrin and rosehip (R. canina) plant extracts.

		Number of MN in binucleated cells
Application groups (ppm)	Number of binucleated cells	(1)	(2)	(3)	Average MN frequency (%)	Nuclear Division Index (NDI)
DMSO (1%)	4000	27	1	–	0.725^a	1.62^e
EMS (1 mM)	4000	85	10	1	2.700^d	1.52^d
Cypermethrin
20	4000	29	1	–	0.775^a	1.53^d
30	4000	36	2	–	1.000^b	1.47^c
40	4000	44	1	1	1.225^c	1.38^b
50	4000	47	2	–	1.275^c	1.33^a
Plant extracts
Cypermethrin 50 + RCwtr	4000	38	1	–	1.000^b	1.37^b
Cypermethrin 50 + RCeta	4000	41	1	–	1.075^b	1.35^a

MN, micronucleus.

The difference between the groups according to the Duncan test was found significant at the p < 0.05 level.

Each column followed by the same superscript letters are not different according to the Duncan test.

Table 2. MN and the NDI values of human peripheral lymphocytes as a result of application of fenvalerate and rosehip (R. canina) plant extracts.

		Number of MN in binucleated cells
Application groups (ppm)	Number of binucleated cells	(1)	(2)	(3)	Average MN frequency (%)	Nuclear Division Index (NDI)
DMSO (1%)	4000	27	1	–	0.725^a	1.62^e
EMS (1 mM)	4000	85	10	1	2.700^f	1.52^d
Fenvalerate
25	4000	35	–	–	0.875^b	1.54^d
50	4000	40	2	–	1.100^c	1.43^c
75	4000	53	1	–	1.375^d	1.32^b
100	4000	57	2	1	1.600^e	1.27^a
Plant extracts
Fenvalerate 100 + RCwtr	4000	45	2	–	1.225^d	1.31^b
Fenvalerate 100 + RCeta	4000	49	1	–	1.275^d	1.32^b

MN, micronucleus.

The difference between the groups according to the Duncan test was found significant at the p < 0.05 level.

Each column followed by the same superscript letters are not different according to the Duncan test.

In RC_wtr and RC_eta, application groups, which were administered together with the highest concentration of cypermethrin (50 ppm), decreases in MN frequencies were observed (Table 1). The MN frequency which was 1.275 in the highest application group of cypermethrin (50 ppm) regressed to 1.000 with RC_wtr and 1.075 with RC_eta. NDI values were found as 1.37 (RC_wtr) and 1.35 (RC_eta). After cypermethrin + RC_wtr and cypermethrin + RC_eta application, the decrease in MN frequencies and the increase in NDI values were found to be statistically significant (p < 0.05).

In the application group of fenvalerate, the average MN frequencies were identified as 0.875 in the lowest application group (25 ppm) and 1.600 in the highest application group (100 ppm). NDI which was 1.54 in the lowest applications group decreased to 1.27 in the highest application group (Table 2). The average MN frequency which was 0.725 in the DMSO control group rose to 1.600 in the highest application group of fenvalerate (100 ppm). NDI dropped from 1.62 to 1.27. These differences between the DMSO control and the treatment groups of fenvalerate were found to be statistically significant (p < 0.05).

In RC_wtr and RC_eta, application groups which were administered together with the highest application group of fenvalerate (100 ppm) decreases in MN frequencies were observed (Table 2). The average MN frequency that was 1.600 in the highest application group of fenvalerate (100 ppm) decreased until 1.225 with RC_wtr, 1.275 with RC_eta. NDI values were found as 1.31 (RC_wtr) and 1.32 (RC_eta). As a result of RC_wtr and RC_eta application, the observed reductions in the MN frequencies and the observed enhancements in NDI values were found to be statistically significant (p < 0.05).

Discussion

In our studies, both cypermethrin and fenvalerate were observed to increase the average MN frequency (p < 0.05). The obtained data showed genotoxic effects of both insecticides (Tables 1 and 2). The findings we have obtained are similar to the findings of various researchers in the literature. Mukhopadhyay et al. (2003) reported cypermethrin may be genotoxic even at low concentrations. Bhunya and Pati (1988) observed that cypermethrin increased MN formation depending on the dose in mice. Cypermethrin was reported to cause MN formation in polychromatic erythrocytes of mice (Bakhitova & Pashin 1988; Sankar et al., 2010) and changes in the number of chromosomes in bone marrow cells (Institoris et al., 1999). Surrales et al. (1990) found that fenvalerate at rates ranging 10–50 mg/ml significantly increased MN frequency in human lymphocytes. Puig et al. (1989) observed that fenvalerate affected the cell cycle in human lymphocyte cells, caused CA, and a significant increase in the SCE frequency. Ghosh et al. (1992) reported that fenvalerate caused a significant increase in frequency of CA in Swiss albino mice.

The water and the ethanol extracts of rosehip reduced the average MN frequencies which were caused by both insecticides (Tables 1 and 2). These decreases were found statistically significant (p < 0.05). As a result of the data obtained, it was observed that rosehip extracts reduced the genotoxic effects of both insecticides (p < 0.05). Several previous studies show that the antigenotoxic effects of rosehip are available. Kızılet et al. (2013) reported that the ethanol extract of rosehip reduced genotoxic effects of EMS in Drosophila melanogaster. Ascorbic acid which was abundantly found in R. canina decreased the genotoxic effects of mutagen compounds such as EMS, MMS, and ENU was identified (Kaya, 2003). Anter et al. (2011) reported that protocatechuic acid which was found in R. canina (Khadem & Marles, 2010) showed an antigenotoxic effect. Methyl gallate which was another compound in R. canina (Hvattum, 2002) potently inhibited the formation of micronucleated reticulocytes in the mouse peripheral blood induced by a KBrOP₃ treatment in vivo was reported (Lee et al., 2005). In a study conducted by using the Ames test by Westhuizen et al. (2008) it was reported that R. roxburghi Tratt (chestnut rose) plant located in the same family with R. canina significantly reduced mutagenic effects of aflatoxin B1. Studies which used different plants as curative are available. It was observed in a study by Uysal et al. (2012) that the methanol extract of Echium amoenum Fisch. and Mey (Boraginaceae) decreased the genotoxic effects of EMS. It has been reported that an extract of Panax ginseng C.A.Meyer (Araliaceae) has an anti-recombinogenic effect in D. melanogaster (Pereira et al., 2008). Agrawal and Pandey (2009) determined that a plant extract of Bauhinia variegata L. (Fabaceae) reduced the MN frequency in mice and showed an anti-mutagenic effect. In addition, a study conducted using the MN test in human peripheral lymphocytes by Kong et al. (1995) reported that extracts of the Pine tree seeds (pine needle) have an anti-mutagenic effect. According to Kılıçgün and Dehen (2009), R. canina has a protective effect against DNA damage.

The anti-genotoxic effects of rosehip are thought to arise from oxidative stress-relieving properties. There are several studies related to rosehip plant reducing oxidative stress caused by free radicals. Özcan (2000) stated that the methanol extracts of R. canina showed antioxidant properties and antioxidant activity of rosehip reached to the highest value at 0.4% concentration. According to Serteser et al. (2008), rosehip is a potent-free radical scavenger and a natural source of antioxidants. Rosehip plants have various phenolic compounds such as methyl gallate (Hvattum, 2002), protocatechuic acid (Khadem & Marles, 2010), and flavonoids (Daels-Rakotoarison et al., 2002). It was reported that there was a strong correlation between the phenolic compounds in R. canina and antioxidant activity (Daels-Rakotoarison et al., 2002).

Conclusion

As a result, it has been concluded that both cypermethrin and fenvalerate showed genotoxic effects in human peripheral lymphocytes and the water and the ethanol extracts of rosehip reduced the genotoxicity of these insecticides.

Declaration of interest

The authors report no conflicts of interest.