Comparison of cytotoxic and genotoxic effects of the synthetic fungicide nimrod and the natural fungicide rhizo – N

The present study was carried out to compare the mutagenic effects of the synthetic fungicide nimrod and the natural fungicide rhizo-N (produced by the bacterium Bacillus subtilis) on mitosis of Allium cepa cells. This study also concerned with the changes in seed protein profile after treatment with both pesticides using sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE). Different concentrations for each fungicide were applied for 3, 6, 24, and 48 h. The obtained results indicate that the two fungicides caused reduction in mitotic activity and induced different types of mitotic abnormalities such as C-metaphase, laggard chromosomes, bridges, stickiness, and disturbed phases as well as micronuclei and multinucleate interphase cells. These changes appeared in varying degree depending on the duration of treatment and concentration applied. The results showed that the synthetic fungicide nimrod have more mutagenic potentialities than the natural fungicide. At electrophoretic level, these fungicides induced alterations in the protein banding patterns of A. cepa seeds as compared with untreated samples.


INTRODUCTION
Chemical pesticides are widely used in Egypt and other countries to minimize the loss of economic crops due to pest invading.Although, pesticides have been useful in pest control and plant disease, there is a considerable risk on human health (Ergonen et al., 2005).The continuous production and use of these pesticides led to many side effects including mutagenic and / or carcinogenic effects in plant, animals, and human (Gopalan, 1999).Evidences accumulated in the past two decades have indicated that a large number of chemical pesticides have mutagenic effects and are capable of inducing chromosomal aberration or DNA damage in cells of different organisms (Singh, 2007;Asya et al., 2012;Mossa and Abbassy, 2012;Sibhghatulla et al., 2012).The danger of these pesticides is not necessary due to direct contact, since it was found that some of these pesticides may accumulate in the food to a toxic level and therefore could be dangerous on the public health (Ergonen et al., 2005).Santovito et al. (2012) reported that human lymphocytes treated in vitro with the fungicide thiran showed a clastogenic effect with respect to residue limits found in some fruits and vegetables in Italy.
Man today is concerned very much with the pollution of his environment.Scientists began trying to use natural compounds to protect plants.Biological control agents are currently being used as alternatives to synthetic pesticides due to their perceived increased level of safety and minimal environmental impacts.Different species of plants, fungi, and bacteria are commercially applied as biological control agents against plant pathogen.The extract of bacterium Bacillus subtilis is used for the control of a range of seedling fungal pathogens including Fusarium spp., Pythium spp.and Rhizoctonia spp (Yoshihero et al., 2003).This bacterium is a common component of soil, being particularly abundant in the rhizosphere of germinating plants.This strain is selected for commercialization because of its effectiveness at controlling fungal infections.
A number of bioassay test system have been developed to monitor the action of pesticides and other environmental hazards on living organisms.Higher plants provide valuable genetic assay systems for screening and monitoring of environmental pollutants (Grant and Owens, 2006;Pesnya and Romanovsky, 2013).Plant genotoxicity assays are relatively inexpensive, fast and give reliable results.Allium cepa test provides a useful tool to estimate the genetic risk derived from an integrated exposure to different chemicals (Mustafa and Arikan, 2008;Yuzbasioglu et al., 2009;Asita and Matebesi, 2010).Chemicals, which cause chromosomal aberration (CA) in plant cells, also produce CA in cultured animal cells that are frequently identical (Santovito et al., 2012).
The present study was planned to compare the mutagenic effect of the biological pesticide (rhizo-N) which contain the bacterium Bacillus subtilis with the synthetic fungicide (nimrod).There is no available published data about the cytotoxic effect of the biological pesticide (rhizo-N).This study aims to investigate the effect of both pesticides on cell division and their capacity to induce chromosomal abnormalities in root tips of A. cepa, in addition, their effect on seed protein electrophoretic profiles.

MATERIALS AND METHODS
Bulbs of A. cepa (Giza 6) were kindly supplied by the Agricultural Research Center, Ministry of Agriculture, and Giza, Egypt.Two pesticides were used in the present study.Both fungicides have been kindly supplied by the General Administration of Pesticides, Ministry of Agriculture, Egypt.The chemical structure of synthetic fungicide (nimrod) is 5-butyl-2-ethyl amino-6 methyl pyrimidin-4dimethylsulfamate and its molecular formula is C13H24N4O3S.The recommended dose is 7 cm 3 /L water.The biological fungicide rhizo-N contains 30 million living bacterium cells of B. subtilis per gram.The recommended dose is 4 gm/L water.

Cytological studies
Young, healthy and uniformed A. cepa bulbs were allowed to germinate in tap water.When the roots reached 2-3 cm long, the bulbs were transferred to new bottles which contained the test substances.The roots were treated for 3, 6, 24 and 48 h for each fungicide.Three bulbs were taken for each treatment and 3 meristematic roots were taken from each bulb (9 roots for each treatment).The root tips were then, fixed in Carnoy solution (3:1) absolute ethyl alcohol: acetic acid glacial for 24 h.Each series of experiments included a simultaneous control treated with tap water.After fixation, the roots were stored in 70% ethanol at 20-22°C until cytological analysis.Permanent preparations were made according to Darlington and La-Cour (1976) using Feulgens quash technique.90 fields were completely analyzed microscopically for each concentration.The frequencies of the different mitotic phases and the mitotic abnormalities after each treatment were statistically analyzed using (t-test).
Where S, Standard deviation.
Where, SE, Standard error.

Protein banding patterns electrophoresis
Total soluble proteins were extracted from M2 seeds of A. cepa treated with the pesticides under study.Characterization of protein profiles was carried out using one dimentional sodium dodecylesulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli, 1970.Run was performed in 12% acrylamide slab gel at 15 mA till the tracing bromophenol blue dye reached the gel bottom.Gels were stained with Comassie blue R-250.The banding profile was analyzed.

Cytological studies
Results obtained from the present study showed that, both pesticides examined induced change in the frequency of mitotic phases that is accompanied by a decrease in mitotic index (MI) and appearance of different mitotic abnormalities.In general, there is an inverse correlation between the frequency of prophase and that of metaphase (Table 1).The results indicated that all treatment with the synthetic fungicide nimrod induced marked reduction in mitotic activity.At long treatments for 48 h there was an obvious decrease in MI as compared with those treated for 3, 6 and 24 h.Such reduction was clearly dose and time dependent and showed statistical significant effect in roots treated with most concentrations (Table 1 and Figure 1A).On the other hand, the biological fungicide rhizo-N showed reduction in mitotic index in roots treated with different concentrations for 3, 6, 24 and 48 h as compared with the control.This reduction increased gradually with the increase of the concentrations applied; but there was no clear decrease in mitotic index values with increasing time of treatment.The statistical analysis of the data reveals that all treatments for 3 and 6 h with the biocide rhizo-N had no significant effect on MI.
Only, the highest concentrations applied for 24 and 48 h and the concentration 1.25 gm/L applied for 48 h induced a significant or highly significant decrease in MI value (Table 1 and Figure 2A).This inhibition of mitotic index may be due to the interference of fungicides with normal process of division by reducing the number of the cell entering mitosis.In the roots treated for 48 h with the highest concentration of nimrod, the percentage of prophase decreased to the extent that they recorded 1.98% as compared with the control value which was recorded 49.74 (Table 1).It can be concluded that the synthetic fungicide nimrod is more effective in inducing reduction in mitotic activity accompanied with high percentage of chromosomal abnormalities as compared with the natural fungicide rhizo -N (Tables 2 and 3 and Figures 1B and 2B).Treatment of root tips cells of A. cepa with the two fungicides produced different types of chromosomal abnormalities such as Cmetaphase, disturbed, laggard chromosomes, chromosome bridge, stickiness, micronuclei and multinucleate (Tables 2 and 3 and Figures 3 and  4).The frequency of total abnormalities produced by nimrod increased as the concentration and the period of treatment was increased (Table 2).On the other hand, the percentages of total abnormalities produced by natural fungicide are slightly increased with the increasing concentration and duration of treatments (Table 3).The scored values of abnormalities induced by the natural fungicide were generally low as compared with that of the chemical fungicide (Figures 1B and 2B).

SDS-PAGE studies
The SDS-PAGE protein profiles of M 2 seeds of A. cepa plants treated with the nimrod and rizho-N fungicides are illustrated in Figure 5 and Table 4.The total number of the protein bands recorded was 18 bands.The protein patterns showed variations among the investigated samples includ-ing the appearance of new bands, disappearance of some bands and changes in band intensity (Table 4).The synthetic fungicide nimrod induced 5 new bands and 4 bands disappeared.The treatment with the natural fungicide rizho-N induced only 1 new band with molecular weight of KDa (Table 4 and Figure 5).

DISCUSSION
Many investigators attributed the inhibition of mitotic activity to blocking of mitotic cycle and accumulation of cells at G 1 or G 2 periods due to the inhibition of nuclear protein synthesis required for the progress of cell cycle (Polit et al., 2003;Cvikrova et al., 2003).Binarova et al. (1998) showed that treatment of Vicia faba root tip cells with specific inhibitors to cyclin -dependent kinases leads to abnormal spindle formation; arrest cells at the G 1 /S and G 2 /M regulatory points; decrease cyclin A and B levels as well as cdc2 kinase indicating the role of this enzyme.This view is in agreement with the result obtained from the present study since the two fungicides led to appearance of c-metaphase and disturbed mitotic phases produced from abnormal spindle formation in addition the disappearance of some  (Asita and Makhalemele, 2009;Asita and Matebesi, 2010;Asya et al., 2012;Sibhghatulla et al, 2012;Sarah et al ., 2013).The inhibition of mitotic division may be also attributed to induction of large number of mitotic abnormalities.Mendhulkar (1993) attributed plant inhibition to disturbance in natural growth regulators and mitotic chromosomal irregularities as additional factors.
The most common type of aberration observed after treatments with the two pesticides nimrod and rhizo-N was C-metaphase.Such type of abnormalities causes inhibition of spindle fiber formation by their action on microtubules, which play the major role in the formation of spindle fibers.On the other hand, the induction of disturbed mitotic configurations by these fungicides may be due to partial inhibition on spindle fiber that affects the orientation of these chromosomes at the equatorial plate.Lagging chromosomes appeared at metaphase, anaphase, and telophase stages.The induction of laggard could be attributed to disturbance in the mechanism of chromosomes movement.
Induction of chromosomal and chromatin bridges at anaphase and telophase stages were also observed.Bridges may be result from chro-mosome stickiness or from breakage followed by reunion.In the present investigation, occurrence of bridges may be due to stickiness rather than chromosome breakage and reunion.Stickiness appeared in different mitotic phases after treatment roots with higher concentrations of both fungicides.Such stickiness led to the appearance of chromatin masses where the general appearance of chromosomes is lost.Micronuclei and multinucleated cells were observed in the interphase cells.Micronuclei are true mutagenic aspects, which may lead to a loss of genetic material (Fernandes et al., 2007).The formation of multinucleated cells may be result from a preceding multipolar mitosis.Several investigators studying the effect of different pesticides or The natural fungicide rhizo-N induced reduction in mitotic activity with the increase of concentrations, but there is no clear increase in reduction of mitotic index percentage as duration of treatment increased.On the other hand, the scored values of the induced abnormalities after treatment with the rhizo-N were low as compared with that scored after treatments with the chemical fungicides nimrod.This means that the chemical fungicides are more effective in inducing mitotic abnormalities than that of the biological fungicide.The genotoxicity of the synthetic fungicide nimrod, as indicated by their capacity to produce chromosomal aberrations, was confirmed by their effect on cell cycle phases as well as protein banding pattern and they are more dangerous than the biological fungicides rhizo-N.The result of the present investigation, point out the importance of taking proper measure in order to avoid contamination with these pollutants.The toxic effect of the synthetic fungicides has created a demand for new environmentally safe fungicides.

Table 1 .
Number and percentage of different mitotic phases and mitotic index after treatment Allium cepa roots with rhizo-N and nimrod fungicides.

Table 2 .
Frequency of micronuclei, abnormal mitotic phases and total abnormal mitosis after treatment Allium cepa roots with nimrod fungicide.
*Significant from control at 0.05 level (t-test).**Significant from control at 0.01 level (t-test).

Table 3 .
Frequency of micronuclei abnormal mitotic phases and total abnormal mitosis after treatment Allium cepa roots with rhizo-N fungicide.