Protective effect of lycopene against cytotoxic and genotoxic effects induced by mixture of two pesticides (dimethoate and lambda-cyhalothrin) on human peripheral blood lymphocytes

Abstract

Lycopene is an acyclic isomer of β-carotene and it is mainly found in tomatoes. It is a potent singlet oxygen-quenching carotenoid and one of the most potent antioxidants. In addition to its antioxidant properties, lycopene shows various biological effects including anti-mutagenic and anti-carcinogenic activities. Pesticides form an important group of man-made noxious chemicals. Their potential synergistic or antagonistic side effects in humans have not yet been extensively investigated. In this study, the protective effect of lycopene against cytotoxic and genotoxic effects of two pesticide mixtures (lambda-cyhalothrin (LCT) and dimethoate (DIMET)) on human peripheral blood lymphocytes have been investigated using the Trypan blue exclusion and comet assays. For this purpose, human lymphocytes have been treated with 5 μg ml⁻¹ and 10 μg ml⁻¹ lycopene concentrations for 1 h. After lycopene treatment, lymphocytes were treated with pesticides DIMET (1 and 2 μl ml⁻¹) and LCT (0.15 and 0.3 μl ml⁻¹) and mixtures of 1 μl ml⁻¹ DIMET + 0.15 μl ml⁻¹ LCT and 2 μl ml⁻¹ DIMET + 0.3 μl ml⁻¹ LCT (1:1 v/v) for 2 h. DMSO was used in 0.1% as negative control, and 0.1 mM H₂O₂ was used as positive control. As a result, it was found that lycopene has a protective benefit against cytotoxic and genotoxic effects of pesticides and pesticide mixtures. In this context, 5 μg ml⁻¹ lycopene concentration was found to be more effective than 10 μg ml⁻¹ lycopene concentration (p < 0.05, ANOVA).

Keywords:

• Comet assay
• cytotoxicity
• genotoxicity
• lycopene
• lymphocytes
• dimethoate
• lambda-cyhalothrin

1. Introduction

For centuries, several hundred pesticides have been used in agricultural practice in order to enhance food production by eradicating unwanted insects and controlling disease vectors (Anwar 1997). Unfortunately this situation has resulted in some negative side effects on human health and on the environment. Many people are potentially at risk of becoming intoxicated when using pesticides in agriculture, in health programs and even at home. Moreover, people trying to commit suicide by using pesticides, and some consumers eating pesticide-contaminated foods are at risk, due to easy accessibility of pesticides and high levels of pesticide residues found in, for example vegetables on the market (Konradsen et al. 2003; Hamilton et al. 2004; Maroni et al. 2006). The World Health Organization (WHO 1992, 1999) reported that around three million pesticide poisoning cases occur annually and result in 220,000 deaths worldwide. Many of these chemicals are mutagenic (Galloway et al. 1987; Garaj-Vrhovac and Zaljezic 2000).

Chemical pesticides are widely used in modern agriculture alone or in combination and they represent an important group of environmental pollutants. Recently, it has been demonstrated that pesticides and many other industrial chemicals may induce free radical production into experimental animals, which possibly play an important role in causing toxicity. Occupational or environmental exposure to mixtures of pesticides has been associated with increased genotoxicity in human lymphocytes (Rupa et al. 1989; De Ferrari et al. 1991; Carbonell et al. 1995; Gómez-Arroyo et al. 2000; Shaham et al. 2001; Pastor et al. 2001; Bolognesi et al. 2002, 2004). Although there are many studies on the genotoxicity of individual insecticides, very limited data are available on the genotoxicity of insecticide mixtures in the literature. Unfortunately, predicting the toxicity of pesticide mixtures based on the results of the toxicity of a single component is not possible (Marinovich et al. 1996). Some pesticides individually induce genotoxicity and some other pesticide mixtures have much greater genotoxic effects. Exposure to known genotoxic compounds can induce DNA damage not only directly but also through other mechanisms such as oxidative stress or inflammatory processes (Lebailly et al. 1998, Shadnia et al. 2005; Hernández et al. 2013). It has been shown that pesticides may induce oxidative stress by producing free radicals, enhancing lipid peroxidation, causing drastic increase in the activities of antioxidant enzymes and by forming a pool of vital antioxidant components in mammalian systems (Banerjee et al. 1999; Nordberg and Arner 2001; Etemadi-Aleagha et al. 2002; Rai and Sharma 2007). On the other hand, some epidemiological studies have shown the association between chronic exposure to pesticides and recorded cases of human malignancy (Safi 2002).

The endogenous antioxidant defenses are inadequate to reduce the pesticide-induced free radical overproduction. Antioxidant intervention seems to inhibit or reduce free radical toxicity and therefore offers protection against pesticides. A number of dietary antioxidants have been reported to decrease free radical attack on biomolecules (El-Habit et al. 2000).

Lycopene is an acyclic carotenoid with 11 linearly arranged conjugated double bounds (Clinton 1998). A naturally occurring carotenoid in tomatoes and tomato products, it has attracted considerable attention as a potential chemo-preventive agent. Recently, lycopene has become the focus of much interest because of its highly efficient antioxidant scavenging activity against singlet-oxygen and free radicals (Cohen 2002; Heber and Lu 2002; Jonker et al. 2003; Michael and Bausch 2003; Stahl and Sies 2003; Tapiero et al. 2004; Velmurugan et al. 2004; Wertz et al. 2004). This antioxidant activity is a potential mechanism by which lycopene may contribute to the prevention of a range of oxidative damage, toxicity, and diseases. Lycopene has an antioxidant activity due to its free radical-scavenging ability (Di Mascio et al. 1989; Srinivasan et al. 2007; Alshatwi et al. 2010). Experimental evidence suggests that lycopene can quench singlet oxygen, scavenge free radicals of nitrogen dioxide (NO₂), thiyl (RS) and sulphonyl (RSO₂). Because of its antioxidant properties, lycopene has gained attention and become one of the most studied chemo-preventive agents (Scolastici et al. 2007). The available reports have shown that little or no work has been carried out on the protective effects of lycopene against pesticide-induced toxicity in human lymphocytes.

The aim of this study, in view of the known antioxidant effects of lycopene, is to evaluate the possible protective effects of lycopene on different concentrations of two pesticide mixtures, dimethoate (DIMET), which is an organophosphate pesticide (OP); and lambda-cyhalothrin (LCT), which is a Type II synthetic pyrethroid, against the cytotoxic and genotoxic effects induced in human peripheral blood lymphocytes.

2. Materials and methods

2.1. Chemicals

The chemicals dimethylsulfoxide (DMSO), RPMI 1640 medium, Histopaque 1077, phosphate-buffered saline (PBS), Trypan blue, Triton X-100, low melting point agarose, normal melting agarose and ethidium bromide were purchased from Sigma Aldrich (St Louis, MO, USA). Korumagor 40 EC (dimethoate EC 400 g l⁻¹, Koruma, İzmit, Turkey) and Karate Zeon (lambda-cyhalothrin 50 g l⁻¹, Syngenta, İzmir, Turkey) were purchased from a local agricultural drug store in Aydın, Turkey. Lycopene (Lyc-O-Mato Lycored, Soft Gel Technologies, Inc., LA, CA, USA) was purchased from Vega Natural Products, Konya, Turkey.

2.2. Preparation of the lycopene solution

We bought the lycopene as soft gels (30 soft gels in a package) and each soft gel contained 10 mg lycopene. Lycopene (5 mg) was dissolved in 12.5 μl dimethylsulfoxide (DMSO) and made up to 5 ml sterile distilled water which was preheated to 37°C. The stock solution of lycopene was 1 mg ml⁻¹. From this stock, 5 and 10 μg ml⁻¹ concentrations were used in 1 ml lymphocyte culture (RPMI). The final concentration of DMSO in the lymphocyte culture was 0.1%. In the experiment, 0.1% DMSO was used as solvent control.

2.3. Isolation of lymphocytes

The peripheral blood of one volunteer was taken after free informed consent. A 5 ml blood sample was aseptically collected in heparinized sterile glass tube from one 30-year-old non-smoking healthy female individual not exposed to radiation or drugs. The volunteer did not present disease symptoms and reported not having used prescription drugs for at least 30 days prior to collection. Lymphocytes were isolated from blood using Histopaque-1077 (Sigma) and cultured as described previously (Boyum 1968). Blood was diluted 1:1 with phosphate-buffered saline (PBS) and layered onto the Histopaque in the ratio of 4:3 (blood + PBS: Histopaque). The blood was centrifuged at 400 ×g for 30 min at room temperature. The lymphocyte layer was removed and washed twice in PBS at 250 ×g for 10 min. Then, the cells were washed with RPMI-1640 media. Each culture contained 1 × 10³ cells ml⁻¹ culture medium.

2.4. Treatment of lymphocytes with lycopene and pesticides

After 24 h following the culture initiation, lymphocytes were treated with 5 and 10 μg ml⁻¹ concentrations of lycopene at 37°C for 1 h. After 1 h lycopene treatment, the lymphocytes were treated with two concentrations of pesticides (1 and 2 μl ml⁻¹ DIMET, 0.15 and 0.30 μl ml⁻¹ LCT) and pesticide mixtures (PM) – 1 μl ml⁻¹ dimethoate + 0.15 μl ml⁻¹ LCT and 2 μl ml⁻¹ dimethoate + 0.30 μl ml⁻¹ LCT – at 37°C for 2 h. In the experiment, 0.1% DMSO was used as solvent control; and 0.1 mM hydrogen peroxide (H₂O₂) (a direct acting mutagen) was used as positive control.

2.5. Cytotoxicity determination by Trypan blue dye exclusion assay

For the determination of cytotoxicity in lymphocytes, viable cell numbers were determined using the Trypan blue exclusion assay (Son et al. 2003; Lee et al. 2005). Trypan blue was added to all lymphocyte cultures in a ratio of 1:1 after the lycopene and pesticide treatments. The preparations were examined under a standard light microscope at 100× magnification. The ratio of live cells to dead cells (cell viability) was determined and the results were expressed as a percentage.

2.6. Single cell gel electrophoresis (comet assay)

The DNA damage in lymphocytes was determined by alkaline comet assay (Singh et al. 1988). After the lycopene and pesticide treatments, lymphocytes were suspended in 150 μl of molten 0.5% low melting-point agarose (LMPA) in PBS (phosphate buffered saline) without calcium and magnesium. Then 150 μl aliquots of the cell suspension were rapidly spread on three slides pre-coated with 85 μl of 1% normal-melting agarose (NMA) and cover-slipped (24 × 50 mm). After the agarose was allowed to solidify for 5 min at 0°C the coverslips were gently removed and a third layer of 75 μl LMPA was added. The slides were then placed in a tank fiLMPA with lysis solution (2.5 M NaCl, 0.1 M EDTA, pH 10, 10% DMSO and 1% Triton X-100 both freshly added) at 4°C. After 1 h, the slides were removed from the lysis solution and incubated in fresh electrophoresis buffer (0.3 M NaOH and 1 M EDTA, pH > 13) for 20 min at room temperature for unwinding of DNA. Electrophoresis was then carried out at room temperature in the same electrophoresis buffer for 30 minutes at 0.7 V/cm and 300 mA. After electrophoresis, the slides were gently washed twice for 5 min in fresh neutralization buffer (0.4 M Tris-HCL, pH 7.59) followed by dehydration in absolute methanol. The slides were stained with 75 μl 1× ethidium bromide and cover-slipped. For each treatment, 100 randomly selected cells from each of the three slides (300 cells per concentration) were evaluated for DNA damage visually using a 40× objective on a flbromide a microscope (Olympus BX 51; Olympus, USA). The DNA damage was quantified by visual classification of cells into fiof categories of “comets” corresponding to the tail lengths (Anderson et al. 1994): Type 0, undamaged; Type I, low-level damage; Type II, medium-level damage; Type III, high-level damage; and Type IV, complete damage. From the arbitrary values assigned to the different categories (from Type 0 = 0 and Type 4 = 4), a genetic damage index (GDI) was calculated for each concentration (Pitarque et al. 1999). GDI values were calculated based upon differential weightings given to the different grades of damage for five arbitrary categories from Type 0 (undamaged) to Type IV (highly damaged). This categorization was used to obtain quantitative measures for the slide which was based on a weighting which was applied to the number of cells with the different grades of damage where the GDI= (Type I + 2 × Type II + 3 × Type III + 4 × Type IV) / (Type 0 + I + II + III + IV).

2.7. Statistical analysis

All the experiments were performed in triplicate form, and were analyzed by one way ANOVA (SPSS 11.5 software package). The statistically significant difference was considered to be p < 0.05. The normality of variables was evaluated using the Kolmogorov–Smirnov Z test. The statistical differences between the control and treatment groups were carried out using the non-parametric Mann–Whitney test (for independent samples). The correlations between different variables were determined using the Spearman rank correlation test.

3. Results

The protective effect of lycopene against the cytotoxic effects of DIMET and LCT and mixture of two pesticides (DIMET and LCT) on human peripheral blood lymphocytes were determined by the Trypan blue dye exclusion method. The viability percent of the cells decreased with the increase in the concentration of the pesticide. The results are presented in Table 1 and Figure 1.

Table 1. Cytotoxicity in human peripheral blood lymphocytes treated with pesticide mixtures and lycopene.

Groups	Cytotoxicity % ± SD
Solvent control	1.00 ± 1.00
H2O2	61.33 ± 0.58*
1 μl ml^−1 DIMET	12.66 ± 0.58*
2 μl ml^−1 DIMET	20.33 ± 0.58*
0.15 μl ml^−1 LCT	35.66 ± 0.58*
0.3 μl ml^−1 LCT	43.00 ± 1.00*
PM1	67.66 ± 0.58*
PM2	75.00 ± 1.00*
L1PM1	36.33 ± 0.58*
L1PM2	47.66 ± 2.00*
L2PM1	44.66 ± 2.51*
L2PM2	61.00 ± 2.08*

L₁: 5 μg ml⁻¹ lycopene; L₂: 10 μg ml⁻¹ lycopene;

PM₁: pesticide mixture (1 μl ml⁻¹ DIMET + 0.15 μl ml⁻¹ LCT);

PM₂: pesticide mixture (2 μl ml⁻¹ DIMET + 0.3 μl ml⁻¹ LCT).

*p < 0.05

Figure 1. Cytotoxicity in human peripheral blood lymphocytes treated with pesticide mixtures and lycopene.

Then, with 1 μl ml⁻¹ and 2 μl ml⁻¹ dimethoate treatment on lymphocytes, cell death rates were increased based on the concentration. As indicated in Table 1, especially after the 2 μl ml⁻¹ dimethoate treatment, the cytotoxic effects of the pesticides on lymphocytes was found to be significant in comparison with the solvent control (20.33% and 1.00%, respectively). LCT showed the cytotoxic effect on lymphocytes. This effect increased parallel to the increasing pesticide concentration. The cytotoxic effect of 0.15 μl ml⁻¹ and 0.3 μl ml⁻¹ concentrations of LCT on lymphocytes were found significantly high when compared with the solvent control (p < 0.05). Also, LCT was found more cytotoxic than dimethoate.

When lymphocytes were treated with various concentrations of the pesticide mixtures (1 μl ml⁻¹ dimethoate + 0.15 μl ml⁻¹ LCT and 2 μl ml⁻¹ dimethoate + 0.30 μl ml⁻¹ LCT) for 2 h, it was found out that cell survival rates were decreased significantly in comparison with the DIMET, LCT and solvent control (DMSO). Pesticide mixtures and H₂O₂ exhibited significant inhibition of cell viability (p < 0.05). As shown in Table 1, the cytotoxic effects of the pesticide mixtures was linearly correlated with the concentration (67.66% and 75.00%, respectively), and this effect was higher than the positive control H₂O₂ (61.33%), which was used as the positive control group. Furthermore, the cytotoxic effects of the pesticide mixtures was significantly higher than the individual cytotoxic effects of DIMET (12.66 and 20.33%), LCT (35.66 and 43.00%).

The cytotoxic effects of individual pesticides and pesticide mixtures were decreased in the lymphocytes which were treated with lycopene before the pesticides and pesticide mixture treatments. The findings of this study suggest that the protective effects of a 5 μg ml⁻¹ concentration of lycopene against the cytotoxic effects of two pesticides and their mixtures was higher than at the 10 μg ml⁻¹ concentration (Table 1, Figure 1).

Genotoxicity assessments using the comet assay indicated that both pesticide mixtures could increase the DNA damage at concentrations much below the individual treatments. The results of the damaged nuclei, the genetic damage index and the arbitrary units as measured in the alkaline comet assay are shown in Table 2. Although the DNA damage could be monitored at higher concentrations of pesticides when used individually, the same extent of damage could also be obtained at very low concentrations of pesticide mixtures (Table 2).

Table 2. Analysis of DNA damage as measured by comet assay in human peripheral blood lymphocytes treated with pesticide mixtures and lycopene.

Groups	Total of damaged nuclei in 300 cells				% of damaged cells	Genetic damage index (GDI)	Arbitrary units (Au)
	Type I	Type II	Type III	Type IV
Solvent control	—	—	2	—	0.67 ± 1.15	6	0.06
H2O2	—	—	—	209	69.67 ± 0.57*	836*	2.79
1 μl ml^−1 DIMET	—	—	29	15	14.67 ± 0.57*	147*	0.49
2 μl ml^−1 DIMET	—	—	38	19	19.00 ± 2.00*	190*	0.63
0.15 μl ml^−1 LCT	—	28	39	50	39.00 ± 1.00*	373*	1.24
0.3 μl ml^−1 LCT	—	31	45	56	46.00 ± 2.00*	421*	1.40
PM1	—	37	62	90	63.66 ± 1.73*	620*	2.07
PM2	—	45	67	109	73.66 ± 1.52*	727*	2.42
L1PM1	—	18	40	46	34.66 ± 0.57*	340*	1.13
L1PM2	—	25	50	74	49.00 ± 2.00*	492*	1.64
L2PM1	—	21	49	71	46.34 ± 2.51*	467*	1.56
L2PM2	—	36	64	90	63.33 ± 2.08*	624*	2.08

L₁: 5 μl ml⁻¹ lycopene; L₂: 10 μl ml⁻¹ lycopene; PM₁: pesticide mixture (1 μl ml⁻¹ DIMET + 0.15 μl ml⁻¹ LCT); PM₂: pesticide mixture (2 μl ml⁻¹ DIMET + 0.3 μl ml⁻¹ LCT).

*p < 0.05.

Dimethoate at concentrations of 1 μl ml⁻¹ and 2 μl ml⁻¹ induced DNA damage in lymphocytes, and the DNA damage increased based on the concentration. The LCT also induced the DNA damage in lymphocytes, which increased with increasing pesticide concentration. The DNA damaging effect of 0.15 μl ml⁻¹ and 0.3 μl ml⁻¹ concentrations of LCT in lymphocytes was found to be significantly higher than with 1 μl ml⁻¹ and 2 μl ml⁻¹ concentrations of dimethoate.

It was found that all the concentrations of the pesticide mixtures induced significant DNA damage in lymphocytes compared to the solvent control group (p < 0.05) (Table 2, Figure 2). In particular, the DNA damage percentage in lymphocytes reached 73.66% after treatment with the 2 μl ml⁻¹ dimethoate + 0.30 μl ml⁻¹ LCT mixture. The genetic damage index and the arbitrary unit values also increased in pesticide treatment groups based on the concentration and in positive control group (H₂O₂) (69.67%) compared to the solvent control group, and reached statistically significant level, mainly by the increased percentage of type II, III and IV damages (p < 0.05). The DNA damaging effect of the pesticide mixtures on lymphocytes decreased lycopene treatment before the treatment of pesticide mixtures. It was found that the protective effects of a 5 μg ml⁻¹ concentration of lycopene against the DNA damaging effect of the two pesticide mixtures was higher than with 10 μg ml⁻¹ lycopene (Table 2).

Figure 2. DNA damage after the pesticide mixture treatments in the human lymphocytes visualized with flDNA damage microscopy. (A) solvent control (undamaged); (B) H₂O₂ treatment; (C) PM₁ treatment; (D) PM₂ treatment; (E) L₁PM₂ treatment; (F) L₂PM₂ treatment.

4. Discussion

Pesticides may induce the oxidative stress, leading to the generation of free radicals and causing lipid peroxidation, and may be the underlying molecular mechanism that gives rise to pesticide-induced toxicity (Agrawal et al. 1991; Khrer 1993; Almeida et al. 1997; Köprücü et al. 2008). The increased lipid peroxidation and oxidative stress can affect the activities of a number of protective enzymatic and non-enzymatic antioxidants that are known to be sensitive indicators of the increased oxidative stress.

Different pesticide formulations are often used simultaneously in complex mixtures, including a significant number of genotoxic compounds (Bolognesi 2003). Organophosphate and pyrethroid pesticides have been shown to pose a significant risk of adverse DNA effects (Bhali et al. 2009). Occupational exposure to mixtures of pesticides has been associated with an increase in genotoxic damage. The cytogenetic damage induced by pesticides appears to depend on the degree of exposure. A dose–response relationship can be hypothesized. Negative results have been associated with the low levels of exposure. By contrast, clear positive results were reported in populations which were subjected to high exposure levels. People suffering from severe intoxication as a result of attempted suicide or employees neglecting occupational safety measures during the spraying season are typical victims in this context.

Dimethoate is one of the most important OPs used extensively on a large number of crops against several pests (Gargouri et al. 2011; Ayed-Boussema et al. 2012). In acute exposure, organophosphate pesticides have the ability to induce toxicity in mammals through inhibition of acethylcholin esterase (AChE) (Hazarika et al. 2003; Ayed-Boussema et al. 2012). In chronic and sub-chronic exposures, in addition to acethylcholin esterase inhibition, the induction of oxidative stress has been reported as the main mechanism of OP toxicity (Banerjee et al. 2001; Ranjbar et al. 2002; Abdollahi et al. 2004; Gargouri et al. 2011). Other mechanisms responsible for the genotoxicity include,those related to the alkylating (Dedek et al. 1984; Mehl et al. 2000) and phosphorylating properties (Piña-Guzmán et al. 2005).

LCT is a third generation type-II synthetic pyrethroid containing 3-cyno group (Meister 1992). The genotoxic effects of synthetic pyrethroids depend on the test system or organism used in experiments. According to Bhunya and Pati (1988), cypermethrin causes micronuclei in mouse erythrocytes. Miadokova et al. (1992) also reported that supercypermethtin led to gene conversion in Saccharomyces cerevisiae and to increasing frequency of aberrant anaphase-telophase in root tips of Hordeum vulgare and Vicia faba.

The findings of this study suggest that the cytotoxicity and genotoxicity (i.e. the DNA damage) in positive control (H₂O₂) group, pesticides (DIMET and LCT) and pesticide mixtures (DIMET+ LCT) treatment groups increased significantly compared with the solvent control group. Organophosphates and pyrethroids have been reported to be genotoxic and as generating free radicals that react with the cell membranes and initiate the process of lipid peroxidation. The accumulation of these radicals can cause oxidative stress (Hérnandez et al. 2005; Salvador et al. 2008). Gargouri et al. (2011) suggested that dimethoate has cytotoxic effects on human lymphocytes, which was evidenced by increasing lipid peroxidation and protein oxidation as well as antioxidant enzyme activities such as superoxide dismutase (SOD) and catalases (CAT). Our study showed that DIMET, LCT and their mixture may disrupt the structure and function of SOD and CAT in lymphocytes. Thus, increased oxidative stress may cause cytotoxicity and DNA damage in lymphocytes.

Moreover, we found decreased cytotoxicity and DNA damage in lymphocytes pretreated with lycopene before the treatments with pesticides and pesticide mixtures (DIMET+ LCT) in comparison with the pesticide mixtures treated lymphocytes. This might be due to the protective effect of the lycopene, because lycopene has the ability to act as a powerful antioxidant and singlet oxygen quencher, thereby inhibiting the destructive effects of the ROS (Rao and Agarwal 1998). Because of its antioxidant properties, lycopene has gained attention and has become one of the most studied chemo-preventive agents. The high number of conjugated dienes makes lycopene one of the most potent singlet oxygen quenchers among natural carotenoids (Di Mascio et al. 1989; Matos et al. 2006; Abdul-Hamid and Salah 2013). It has been suggested that lycopene can prevent carcinogenesis by protecting critical biomolecules including the DNA (Di Mascio et al. 1989). Lycopene may protect lipids, proteins, and DNA against oxidation in vivo (Reifen et al. 2004; Tapiero et al. 2004). Scolastici et al. (2007, 2008) added that lycopene is a suitable agent for preventing chemically induced DNA and chromosome damage. Weng Kong et al. (2010) reported that lycopene protected cells against DNA damage.

Matos et al. (2001) reported that lycopene supplementation decreased 77% of 8-oxo-dGuo levels in Fe-NTA/ascorbate-treated cells. These results indicate that lycopene provides strong protection against DNA-based oxidation. Furthermore, consumption of tomato products has been reported to reduce the susceptibility of lymphocyte DNA to oxidative damage (Riso et al. 1999). Our previous studies also suggested that pre- and post-treatment with lycopene reduced total chromosomal aberrations induced by ethyl methane sulfonate in Allium cepa (Aslantürk and Aşkın Çelik 2005, 2006).

It is known that mechanisms underlying the inhibitory effects of lycopene on carcinogenesis and mutagenesis may involve ROS scavenging, up-regulation of detoxification systems, interference with cell proliferation, induction of gap-junctional communication, inhibition of cell cycle progression, and modulation of signal transduction pathways (Bhuvaneswari and Nagini 2005). Among the effects of lycopene, reduction of DNA damage (Yeh and Hu 2000; Reddy et al. 2006), induction of apoptosis (Muller et al. 2002; Reddy et al. 2006) and inhibition of cell proliferation (Tang et al. 2005; Nahum et al. 2006) have been described.

In our present study, the higher concentration of lycopene (10 μg ml⁻¹) has been shown to be effective against pesticide mixture-induced cyto- and genotoxicity, but not as effectively as 5 μg ml⁻¹. This may be because this concentration may have resulted in byproducts which interfered with the antioxidant activity of lycopene, thus decreasing its effect. Hwang and Bowen (2005) studied the effect of lycopene on LNCaP human prostate carcinoma cell line and asserted that lycopene in low concentration showed only a lipid antioxidant effect, whereas at higher concentrations its inhibitory effect on cancer cells was mediated through increased DNA damage. Moreover, 10 μg ml⁻¹ lycopene could behave also as pro-oxidant. Yeh and Hu (2000) reported that lycopene and β-carotene could be pro-oxidant depending on their applied dosage and the type of the oxidant used in Hs68 cells. Srinivasan et al. (2009) also reported that 1, 5 and 10 μg ml⁻¹ lycopene had radio-protective effect against 1, 2 and 4 Gy γ-radiation-induced cellular damages in cultured human lymphocytes; however, they also added that 5 μg ml⁻¹ of lycopene was more effective than the other concentrations.

Our results show that pretreatment with lycopene protects the lymphocytes against cytotoxic and genotoxic effects induced by pesticides and pesticide mixture. Lycopene could be a suitable agent for preventing the toxicity of DIMET and genotoxicity-induced DNA damage. According to our results and to other reports about the potential health benefits of lycopene, consumption of lycopene-rich foods can help protect health against exogenous harmful effects. For more information and clear conclusions about the protective effects of lycopene against exogenous harm, further studies should be performed with different test systems.

Acknowledgments

This study was performed in Science and Technology Research Centre (BILTEM), Adnan Menderes University, Aydın, Turkey.

Disclosure statement

No potential conflict of interest was reported by the authors.