DETECTION OF GENETIC DAMAGE INDUCED BY PESTICIDES USING CYTOGENETIC AND BIOMARKERS ASSAYS IN Allium AND Pisum

urrently, due to an increase in the population worldwide, there has been an urge for increasing the productivity of crops and agricultural by-products. This yield increment has been obtained mainly by a massive use of pesticides to control the impacts of noxious insects, pyhtopathogens and weeds in agriculture. Pesticides are a multimillion dollar market corresponding to an estimated value of 25.6 million dollars per year (Cardoso et al., 2010) and their application is still the most effective and accepted means for the protection of plants from pest. However, a series of deleterious effects on environment safety and human health have become apparent, where teratogenic, carcinogenic, and mutagenic effects have received special attention (Giacomazzi and Cochet, 2004; Nguyen-Ngoc et al., 2009). It is also known that certain pesticides are promutagens which are metabolized to mutagens (Cardoso et al., 2010). Because of the undesirable side effects of pesticides, there has been an increase in consumer awareness to avoid the use of these compounds (Zucchi et al., 2008) and/or public pressure to enhance their regulation for applications in preor postharvest. The majority of pesticides have been tested in a wide variety of mutagenicity assays covering gene mutation, chromosomal alteration and DNA damage (Inceer and Beyazoglu, 2000; Soliman and Ghoneam, 2004; Sousa et al., 2009; Cardoso et al., 2010; Lamsal et al., 2010). Although a number of biomarkers are available to assess transient and permanent genotoxic responses, biomonitoring studies on non target populations exposed to pesticides have essentially focused on cytogenetic end-points, namely chromosomal aberrations (CA), micronuclei (MN) frequency and sister-chromatid exchanges (SCE).

urrently, due to an increase in the population worldwide, there has been an urge for increasing the productivity of crops and agricultural by-products.This yield increment has been obtained mainly by a massive use of pesticides to control the impacts of noxious insects, pyhtopathogens and weeds in agriculture.Pesticides are a multimillion dollar market corresponding to an estimated value of 25.6 million dollars per year (Cardoso et al., 2010) and their application is still the most effective and accepted means for the protection of plants from pest.However, a series of deleterious effects on environment safety and human health have become apparent, where teratogenic, carcinogenic, and mutagenic effects have received special attention (Giacomazzi and Cochet, 2004;Nguyen-Ngoc et al., 2009).It is also known that certain pesticides are promutagens which are metabolized to mutagens (Cardoso et al., 2010).Because of the undesirable side effects of pesticides, there has been an increase in consumer awareness to avoid the use of these compounds (Zucchi et al., 2008) and/or public pressure to enhance their regulation for applications in pre-or postharvest.
The majority of pesticides have been tested in a wide variety of mutagenicity assays covering gene mutation, chromosomal alteration and DNA damage (Inceer and Beyazoglu, 2000;Soliman and Ghoneam, 2004;Sousa et al., 2009;Cardoso et al., 2010;Lamsal et al., 2010).Although a number of biomarkers are available to assess transient and permanent genotoxic responses, biomonitoring studies on non target populations exposed to pesticides have essentially focused on cytogenetic end-points, namely chromosomal aberrations (CA), micronuclei (MN) frequency and sister-chromatid exchanges (SCE).
Several investigators had studied the side effect of the pesticides on the heredity material of different plant cells (Soliman and Ghoneam, 2004;Lamsal et al., 2010).In particular, Allium cepa possess many advantages in the field of environmental mutagenesis for screening of genotoxic agents according to the standard protocol for the plant assays established by the International Program of Chemical Safety (IPCS) and the World Health Organization (Soliman and Ghoneam, 2004).Pisum sativum bioassay has been also

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shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis induced by chemicals, radiations and environmental pollutants (Grant and Owens, 2001).The use of plant root tips as a bioassay test system in the genotoxicity of pesticides has shown extremely good correlation with the bacterial and mammalian systems and could exhibits a good predictive value for human beings (Gopalan, 1999;Sadowaska et al., 2001;Soliman and Ghoneam, 2004).
In general, root development is initiated at the apex of the root tip by mitotic divisions in the meristematic regions (about 1 mm) in length above the root cap and the daughter F 1 cells about 1mm are moved upward to lengthen the root structure.A very small portion of the meristematic cells divide transversely to increase the girth of the root tip, whereas the majority divides longitudinally.Based upon this ontogenetic scheme (Ma et al., 1995), the majority of micronuclei should be in F1 except on some rare occasions, when there is a mitotic delay.In most of earlier studies, the Micronuclei (MN) frequencies were probably not scored from F1 cells.Thus MN frequencies obtained in this way would lose their fidelity and the efficiency of the test system would be reduced because the sensitivity of chromosomes to clastogens varies greatly throughout the mitotic cycle (Ma et al., 1995).Among the different cytogenetic assays in plants, the most effective and simplest indicator of cytological damage is micronucleus formation (Ma et al., 1995;Minissi and Lombi, 1997).Furthermore, the micronucleus test can also detect very weak mutagenic effects.
Among the pesticides used, herbicides play a crucial role in agricultural fields to avoid crop competition by weeds (Saladin et al., 2003).On the other hand, chemical insecticides are widely used in Egypt and other countries in the modern agriculture in order to minimize the loss in economic crops due to insect invading (Barakat, 1997).
In view of the mentioned reasons,

Plant materials
Seeds of Pisum sativum L. (Master B) and bulbs of Allium cepa (Giza 20) were kindly supplied by the Agricultural Research Center, Ministry of Agriculture, Giza, Egypt.

Pesticides
Two pesticides, commonly used in the Egyptian agriculture, were tested in the present study: the herbicide bentazone and the insecticide lannate.Both chemicals have been kindly supplied by the General Administration of Pesticides, Ministry of Agricultural of Egypt.

Laboratory assay
Onion bulbs and Pisum seeds were germinated as described by Fiskesjo (1988) and Grant and Owens (2001), respectively.When the roots of A. cepa L. and P. sativum were about 2 cm long, they were exposed to freshly prepared test solutions of different concentrations (500, 1000, 1500 ppm) at room temperature for six hours and maintained in tap water for 44 hours for root germination and recovery after treatment ( Ma et al., 1995).Tap water was used as a negative control.

In situ assay
The germinated seeds of Pisum were cultivated in pots (30 cm in diameter) containing equal amounts of mixed soil (sand: clay, 1:2 v/v).Ten seeds were sown in each pot and the irrigation was carried out as practiced by adding equal amounts of water to each pot.The pots were divided into five groups; the seeds of the first group were sprayed with tap water to serve as control.The other groups were sprayed twice with concentrations of 500 ppm and 1000 ppm of each pesticides; the spraying were done twice at both seedling and flowering stage before anthesis.

Micronucleus assay (MN)
The MN assay was conducted according to Ma et al. (1995).After treatments, the root tips were fixed immediately in aceto-alcohol (1:3) and then transferred to 70% alcohol and stored in refrigerator until use.For examination, the root tips were hydrolyzed in 1N HCL at 60°C for 3-8 min and stained in double stain of carbol fuchsin and acetoorcein.Root tips were then squashed in 45% acetic acid.Slides were made permanent and mounted in Canada balsam, examined and photographed.
Meristematic and F1 cell regions were prepared from each root tip separately.The mitotic indices of both the meristematic and F1 cell regions were determined (no. of dividing cells/total no. of examined cells X 100).The micronuclei were scored per 1000 interphase cells from 5-7 separate slides and expressed in terms MN/1000 cells.A treated/ control (T/C) ratio was used to compare the efficiency of the MN scoring from the meristematic and F1 cell regions of the root tips and also to compare the efficiency of MN scoring from the two plant tests (Ma et al., 1995).

Meiotic analysis
Six flowering small buds from each treatment were collected 24 hour after the last spray.Flower buds were fixed in (6:3:1) ethanol: glacial acetic acid: chloroform (Carnoy's fluid) and then preserved in 70% ethyl alcohol and stored at 4°C until cytological analysis.Staining and smearing were made according to Snow (1963).The various types of cells with normal and abnormal chromosomal behavior at both the 1 st and 2 nd meiotic divisions and tetrad stage were observed and counted.

Biochemical analysis
M1 seeds were used for biochemical analysis which included: storage protein banding patterns as well as measurement of both protein and nucleic acids content.

Estimation of total proteins and nucleic acids
Protein content was extracted from dry M1 seeds according to Bradford (1976).The protein content in the seed extract was determined by spectrophotometer.The quantity of protein in the M1 seeds of Pisum sativum samples was calculated from the standard curve using Bovine Serum Albumin solution as standard protein.
A method based on that of Shibho et al. (1967) was performed for estimation of both types of nucleic acids.RNA content was determined according to Ashwell (1957).The method of Burton (1968) was used to determine DNA content.

SDS-PAGE of seed protein
Total extracted proteins of M1 seeds of Pisum plants were separated by denatured polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970).Gels were stained with Comassie blue.The banding profile in the gel was photographed and analyzed using Gel documentation system.

Statistical analysis
The data were statistically analyzed using a SPSS program (Nie et al., 1975).Linear regression dose-response curves were established and the coefficients of correlation and slope of the dose-response curve were calculated to mark the efficiency of the system as a clastogenicity bioassay.All experiments were repeated at least three times.

Cytogenetic analysis
The obtained data elucidated that, the two tested pesticides induced different mitotic changes on root tips of P. sativum and A. cepa appeared as a low mitotic index and a high frequency of induced micronuclei, suggesting the genotoxicity of the studied pesticides.Screening of pesticides in different bioassays systems revealed that some pesticides may have known or unknown mutagenic or carcinogenic effects (Tartar et al., 2006;Lamsal et al., 2010).The use of plants bioassays for studying the chromosome aberrations induced by different classes of pesticides is one of the oldest, simplest, most reliable and inexpensive methods available (Lamsal et al., 2010;Cardoso et al., 2010 ).

Micronucleus assay
Among the different cytogenetic assays in plants, the most effective and simplest indicator of cytological damage is micronucleus formation (Ma et al., 1995).Furthermore, the micronucleus test can also detect very weak mutagenic effects (Minissi and Lombi, 1997).In this study, all the concentrations of lannate and bentazone were capable to induce a significant increase in percentage of total MN in interphase cells of P. sativum and Allium cepa root tips relative to control (Plate 1 and Fig. 1).This increment of MN frequencies in F1 and meristem cells (M) of Pisum and Allium was dosedependent.On the other hand, Allium cells exhibited higher percentage of MN in both F1 (20-161%) and M (6-41%) cells compared to Pisum (15-117% in F1; 3-26% in M cells).
Since the MN are revealed in the subsequent generation in the interphase or prophase cells (F1 cells).Ma et al. (1995) suggested that efficient scoring should not be done in the meristematic mitotic cells and therefore a long recovery time covering two rounds of mitosis after treatment is required.This is designed to capture the maximum chromosome damage which was inflicted in the interphases (G1, S and G2) of the meristematic cells and later becomes nuclei in the F1 cell population (Ma and Xu, 1986).Some researchers scored MN from interphase cells and oth-ers scored MN together with chromosome abnormalities.The inconsistent use of one method over the others has reduced the efficiency of the test system.In order to increase the fidelity of the test system which scores MN in the F1 cells, data collection should be centered at the sensitive peak stage for the induction of chromosome breaks.According to Ma et al. (1995), the sensitive peak was represented by 44-h recovery period which gives the highest MN frequency.Based on the 24-h mitotic cycle in the roots, the 44-h recovery time captures the MN which were induced around the G1 or S stage of the parental meristematic cells and appeared in the interphase or prophase in the daughter cells.
Judging from the slope values of the dose-response curves (Fig. 2), it is clear that greater efficiency can be attained when the MN frequencies are scored from the F1 cell region rather than from the general meristematic region.F1 region showed both a higher slope value (0.031-0.1042) and correlation coefficient (R 2 = 0.8491-0.9976)compared to meristematic region (slope values = 0.0068-0.0256and R 2 = 0.7506-0.9537).There is a greater degree of synchrony of interphase cells in the F1 region than in the meristematic region, which facilitates the MN scoring.Scoring MN from F1 cells has the advantage of recording both chromosome breaks which are generated in the early G1 and the late interphase (G2) of the parental cells when conventional 44-48 h recovery time is used.On the other hand, the treated/control (T/C) value showed a higher fold increase in F1 cells than in M cells in both Pisum and Allium root tips (Table 1), confirming the high efficiency of using F1 cell populations for MN scoring.These findings were in accordance with that of Ma et al. (1995) where scoring MN was more efficient in F1 cells in both Vicia and Allium root tips.

Mitotic activity
Concerning the effect of lannate and bentazone treatments on cell division, the result showed that increase in the pesticide concentration decreases the mitotic indices significantly in both P. sativum and A. cepa root meristems compared to the control values (Fig. 1).It is clear that the reduction in mitotic activity in the root tips of A. cepa was more pronounced than in P. sativum.Such an inverse relation between the mitotic index and the dosage of treatment had been reported earlier by various workers studying different pesticides on various plant materials (El Nahas, 2000;Lamsal et al., 2010).The changes of mitotic activity in plant have been attributed to many factors (Kim and Bendixen, 1987;Lamsal et al., 2010): 1) the inhibition of certain types of nuclear proteins essential in the mitotic cycle, 2) the inhibition of DNA synthesis and 3) the reduction of various necessary metabolites which are important for transition from G1 to S and from G2 to mitosis.

Sensitivity test
It is evident from the slope values of the dose-response curves (Fig. 2), that Allium root MN was a more efficient test than the Pisum one.The relatively high efficiency of the Allium test is substantiated by the T/C values at all concentrations of both pesticides (Table 1).The T/C values showed higher fold increase in the percentage of micronuclei in F1 (4-32.2) and M (2-13.7) of Allium cells compared to T/C values of the MN frequencies in F1 (1.9-14.6)and M (1.5-13) of Pisum cells.Thus it was concluded that P. sativum was not as sensitive as A. cepa in detecting clastogenicity of the tested pesticides.The higher sensitivity of Allium could be attributed to its large size of chromosomes and this may led to greater susceptibility of Allium chromosomes to breakage (Grant and Owens, 2001).

Meiotic division
Statistical analysis of the results (Table 2) showed that all of the conducted treatments with both pesticides caused induction of significant percentage of abnormal pollen mother cells (PMC's)/plant in the first and second meiotic division (Plate 2).Bentazone treatments caused a higher percentage of abnormal PMC's as compared with lannate.Furthermore, the frequencies of total meiotic abnormalities were higher in the first division than in the second one in all treatments.These results indicate the potentiality of the investigated pesticides to induce meiotic irregularities.Similar results have been recorded by other authors (El-Nahas, 2000; Soliman and Ghoneam, 2004).
Stickiness, laggards, bridges, micronucleus and disturbed chromosomes were the main types of anomalies ob-served (Fig. 3 & Plate 2).The frequencies of these abnormalities depend on both the applied concentrations and the pesticide used.The most prominent anomaly observed, particularly in metaphase I was the sticky nature of chromosomes (Plate 2A).It is evident from Fig. (3) that the percentage of cells with sticky chromosomes reached a maximum frequency of 90% of the abnormal PMC's after treatment of lannate at 1000 ppm concentration compared with the bentazone value (75.20%).Stickiness has been suggested as a type of physical adhesion involving mainly the proteinacious matrix of chromatin material (Lamsal et al., 2010).On the other hand, laggards appeared in a considerable frequency in both the first and second meiotic divisions as one of the bivalents failed to reach the equatorial plan and remains free in the cytoplasm.Induction of laggards at metaphase I may be extended in all the subsequent meiotic stages, therefore laggards appeared at anaphase I and II (Plate 2 B & E).The highest percentage of laggards (20.20%) was observed at 500 ppm lannate.The induction of laggards could be attributed to the failure of the spindle apparatus to organize and function in a normal way (Patil and Bahat, 1992).Other type of abnormalities was the formation of anaphase bridges which appeared in a low significant percentage (Plate 2D).The percentage of bridges reached its maximum value of 4.50% at 500 ppm lannate (Fig. 3).The formation of bridges could be attributed to the general stickiness of chromosomes (Soliman and Ghoneam, 2004;Lamsal et al., 2010).A less common phenomenon was the ap-pearance of some PMC's with micronuclei (Plate 2C).The highest proportion of such abnormality recorded was 8.30% at bentazone 1000 ppm (Fig. 3).Fewer proportions of PMC's with disturbed poles were recorded at the 2 nd meiotic division, in which the equatorial planes at metaphase II and anaphase II (Plate 2F) disoriented in an abnormal fashion.The total frequency of disturbed cells reached a maximum value of 11.4% after spraying with 1000 ppm bentazone (Fig. 3).This abnormality is generally regarded to be the result of disturbance of spindle apparatus (Yuzbasioglu et al., 2003).

Changes in DNA and RNA contents
The present results of DNA and RNA content are confirmed by data obtained by other studies on different pesticides (Badr and Ibrahim, 1987;Topkatas and Rencuzogullari, 1996).Nucleic acid biosynthesis has been reported as a potential target sites affected by herbicides which in turn led to inhibition of protein synthesis (Soliman and Ghoneam, 2004).The mutagenic effect of the investigated pesticides on the genetic material of P. sativum M1 seeds is confirmed by its effect on the nucleic acids.Statistical analysis of the results revealed that all the conducted treatments caused significant reduction in the DNA and RNA contents in pea yield (Fig. 4).DNA concentrations ranged from 85.91-110.73µg/100g dry wt.compared with the control (120.72 µg/100g dry wt.), while the amounts of RNA ranged from 175.41 to 205.63 µg/100g dry wt as referred to control (219.40 µg/100g dry wt).In general the magnitude of decrease in the amounts of DNA and RNA was most pronounced at bentazone 1000 ppm, (85.91 µg/100g dry wt.and 175.41 µg/100g dry wt.) for DNA and RNA, respectively (Fig. 4).The inhibition of DNA and RNA synthesis by herbicides could be attributed to the inhibition of DNA replication, or due to the reduction of oxidative phosphorylation resulting in lower ATP level in the cell (Lamsal et al., 2010).On the other hand, herbicides that reduce ATP have been postulated to be strong inhibitors of RNA synthesis (Ashton and Crafts, 1981).

Changes in protein contents
Soluble protein content in organisms is known to respond to a wide variety of stressors (Singh and Tewari, 2003).It was proposed that certain pesticides caused a significant reduction of protein level in plants (Mosleh et al., 2003;Singh and Tewari, 2003).In this experiment, changes in soluble protein content of Pisum seeds (M1), exhibited an inverse relationship with bentazone treatments.On the other hand, other investigators have recorded an increase in protein content in response to metal stress (Liu et al., 2007).Here, a positive correlation between protein content and lannate concentrations was remarkable (Fig. 5).Values of protein contents in both situations were statistically different from the control indicating its potential as a biomarker of reversible and irreversible changes in metabolism.The maximum value (22487.71µg/100g.dry wt.) was observed after treatment with lannate 1000 ppm while the minimum value (19779.25 µg/100g dry wt.) was observed after treatment with bentazone 1000 ppm (Fig. 5).

Changes in protein banding patterns
SDS-PAGE analysis was successfully used by some authors to establish biochemical genetic fingerprints of many plants (Badr, 1995;Ghareeb, 1998).The protein profile of M1 seeds of Pisum plants (treated and untreated) using SDS-PAGE gel is illustrated in Plate (3).Their molecular weights (MW), relative front, number of bands and average optical density (OD) are given in Table (3).The protein patterns showed qualitative and quantitative variations among the investigated samples.The variations included: the appearance of new bands, changes in band intensity, and changes in band relative mobility.Similar results were obtained by other authors (El-Nahas, 2000;Babaoglu et al., 2004).
The number of bands in the studied samples ranges between 8 and 10 bands in treated samples with molecular weights ranging between 273.652 and 88.254 KDa (Table 3).The most obvious changes in SDS-PAGE profile of M1 seeds of the treated plants were; the appearance of new band at lannate 500 ppm (94.606KDa ), three bands at lannate 1000 ppm (94.675,90.768 and 88.254 KDa) and other new band at both bentazone 500 and 1000 ppm (95.460KDa).The appearance of new bands may be explained on the basis of mutational events at the regulatory system of an unexpressed gene(s) that activate it (El-Nahas, 2000).Also, pesticides used in this investigation caused changes in band intensity which could be explained on the basis of induction of gene mutation at the regulatory system which modulate or increase transcription rate of a particular structural gene.This lead to the production of faint or over expressed protein bands (Barakat and Hassan, 1997).Moreover, the recorded changes in band intensity could be also attributed to the cytological abnormalities induced by pesticides (Soliman and Ghoneam, 2004).The presence of laggards and bridges support this conclusion.
Changes in bands relative mobilities could be also interpreted on basis of the occurrence of point mutation in the concerned structural genes that create stop codon prior or post the original.They gave rise to the production of shorter or longer polypeptide chains (Ghareeb, 1998;El-Nahas, 2000).
It is worthy to mention that gene mutation and changes in the gene expression have to be considered as the reasonable interpretation for the observed banding pattern changes.The correlation between the alteration in the protein banding patterns and mutational events has been demonstrated by other authors (Prasad and Zha, 1992;El-Nahas, 2000;Soliman and Ghoneam, 2004).Thus, the two investigated pesticides in the present work might be considered as strong mutagens and the observed changes in the banding patterns are heritable changes, as the treated par-ents were capable to transfer these mutational events to the next generation.
As a general conclusion, the two pesticides (bentazone and lannate) caused harmful effects at both cytogenetic and biochemical levels on the plants.However, the effect of bentazone was more pronounced in this regard than lannate.Therefore, it is suggested to test the mutagenic potential of pesticides on a more intensive and extensive basis, especially on non target systems before it is recommended for wider use in agricultural field.This study emphasizes that the programmes of pesticides application should also take into account the concentration of the pesticides and the nature of plant.
of Pisum sativum to assess the possible changes in nucleic acids and proteins.Laboratory assay included treatment of Allium and Pisum root tips with three concentrations (500, 1000 and 1500 ppm) of each pesticides for six hours followed by 44 h recovery period, for conducting Micronucleus (MN) assay.In situ assay included spraying Pisum plants at seedling and flowering stage with two concentrations (500 and 1000 ppm) of pesticides.Cytogenetic analysis proved the efficiency of conducting Micronucleus assay in F1 cells relative to the meristematic region of root tips.Results of MN assay also exhibited Allium a more sensitive test compared to Pisum.Spraying the flower buds of Pisum plants with the applied concentrations of the two pesticides resulted in a significant increase of total abnormal PMC's.The types of meiotic abnormalities induced were: stickiness, lagging, bridges, micronucleus and disturbed chromosomes.Results of biomarkers assay revealed a pronounced mutagenic effect on the hereditary material of Pisum M1 seeds confirmed by reduction in the DNA and RNA contents and alteration in protein synthesis.In addition, the protein banding patterns of M1 seeds exhibited both qualitative and quantitative changes.Overall, it can be concluded that the two pesticides have potential mutagenic effects on genetic material at different levels.So, it will be necessary to test the mutagenic potential of bentazone and lannate on a more intensive basis especially on non-target systems before it is recommended for wider use in agricultural field.
it was thought of interest, in this work to investigate the cytological and biochemical effects of two different pesticides namely, the herbicide bentazone and the insecticide lannate, using the following approaches: 1) Micronucleus assay in root tips of both Allium cepa and Pisum sativum, 2) Monitoring of meiotic irregularities in Pisum plants and 3) biochemical analysis of the yielded M1 seeds of the treated Pisum plants which include assessment of: storage protein banding patterns using SDS-PAGE , protein and nucleic acids content.
Fig. (2): Linear dose-response curves for micronucleus frequency in F1 and M cells of the treated meristem of Allium and Pisum.

Fig. ( 3
Fig. (3): Percentage of the different types of abnormalities in P. sativum PMC's sprayed with different concentrations of lannate and bentazone.

Table ( 1
): T/C values at different concentrations of lannate and bentazone estimated from micronuclei frequency in F1 and M cells of both Allium and Pisum.

Table ( 2
): Percentages of abnormal pollen mother cells (PMC's) in the different meiotic stages in Pisum plants sprayed with different concentrations of lannate and bentazone.