Tradescantia pallida as a biomonitoring tool to assess the influence of vehicle exhaustion and benzene derivatives

1 Research Laboratory of Toxicological Genetics (LAPGENIC), Federal University of Piauí (UFPI), Teresina (Piauí)64.049-550, Brazil. 2 Northeast Biotechnology Network (RENORBIO), Postgraduate Program in Pharmaceutical Science, Federal University of Piauí, 64.049-550, Teresina, Brazil. 3 Center for Education College São Camilo (CEPROSC), Teresina, Brazil. 4 Department of Pharmacy, Southern University Bangladesh, Mehedibag (Chittagong)-4000, Bangladesh..

interference from vehicular exhaust, considered the main cause of air pollution.Volatile organic compounds, particulate materials, nitrogen oxides, carbon and sulfur, and benzene derivatives, such as polycyclic aromatic hydrocarbons (PAHs) cause damage to the environment, affecting the flora, fauna, and people, for making improper air, inconvenient, harmful and detrimental to health in general (Pereira et al., 2013;Inomata et al., 2015).
The chemical modification air quality standard can be measured through the use of bioindicators, plant or animal beings, characterized by the use in biomonitoring of air quality (Billet et al., 2015).As a result of, the plants are inexpensive, easy to use and cultivation, have shown as the most effective means of an assessment and response quality of the environment, presenting practical use mainly related to air pollution control (Rodríguez et al., 2015).
The variation in biological monitoring techniques is sufficient to encompass the use of various species, believed to be active biological monitors (Güez et al., 2012;Ávila et al., 2013).The use of superior plants such as angiosperms, in biomonitoring studies has grown sharply (Santos, 2015).The Tradescantia pallida, is popularly known as tetrapoeraba-roxa.It is an ornamental plant widely distributed in Brazil, as it has a great adaptability to climate variations.It is a small angiosperm in the Commelinaceae family for presenting genetic characteristics favorable to studies, such as large chromosomes, it is often used as a test system in biomonitoring (Carvalho, 2005;Crispim et al., 2012).
One of the parameters for biomonitoring is the comparative analysis of the structural pattern of leaves with anatomical features arising from the exposure to a particular agent.These features can be macro or microscopic, as the decrease in leaf size and stomata, organelles responsible for gas exchange (Mott et al., 2014).
Other trials from Tradescantia are cytogenetic tests, related to the analysis of the cellular genome.Two of these tests are widely accepted on the observation of somatic mutations or changes in the structure of chromosomes: The test with analysis of mutations in stem hairs (Trad-SHM) and bioassay of MN in pollen grains of stem cells (Trad-MCN) (Carvalho, 2005;Savóia et al., 2009;Andrade-Vieira et al., 2011).
Thus, this study aimed to evaluate the toxic effects by increasing frequency of micronuclei (MN) in T. pallida exposed to few zones in the city of Teresina, Piauí, Brazil.

MATERIALS AND METHODS
T. pallida was identified by the Herbarium Graziela Barroso -TPB (Federal University of Piauí) with the voucher number 28339.Then 50 seedlings of T. pallida were cultivated in small pots with fertile soil and regular irrigation.We distributed the plants in 4 points in the city of Teresina -Piauí, during the period of August to October de Carvalho et al. 281 in 2012.It is because, August to October is the dry season with more dust, temperature, and vehicle exhausts in this state of Brazil.The municipality is located on the right bank of the Parnaíba River and occupies a total area of 1,809 km 2 , with geographical coordinates: 05º05'12" south latitude and 42º48'42" west longitude (Branco, 2003).
After the cultivation, 10 seedlings were sent for display in each of their previously cited environments, and they were weekly watered with deionized water.Plant structures used in laboratory analysis were young inflorescences (Figure 2), where pollen grains appear more abundantly on tetrad stage.The inflorescences withdrawals were carried in aqueous solution (for maintenance adequate humidity and integrity) to the laboratory and analyzed in the range of periods: 15, 30 and 60 days after implantation.This procedure related to frequency of MN found in pollen grains of stem cells with the time of exposure to clastogenic effects of pollutants being studied.
Once young inflorescences were removed, they were fixed in methanol: acetic acid (3:1) for 24 h and stored in ethanol (70%).The flower bud clumps were dissected out and macerated in glass slide for staining acetic carmine (2%) and further heating to about 60°C for impregnating the dye.In an optical microscope at magnification of 400 X, 3000 tetrads were viewed for each exposure period.The occurrence frequency of MN was obtained from the ratio between the number of MN in 300 tetrads for each exposure place at all points, including the NC, in each time period described above, and follows the method described by Andrade-Vieira et al. (2011).

RESULTS
The results allowed the increase in the frequencies of MN in T. pallida, from exposure to gaseous pollutants originating from the vehicle combustion and benzene derivatives action in periods of 15, 30 and 60 days of exposure as shown in Figures 3 to 5.
Figure 6 shows all points with the NC, total period of 60 days, allowing an analytical correlation between the genotoxic influence of air pollutants, absorption time and accumulation of these air pollutants by plants and environments more significance for induction of MN.
The median frequency of MN in T. pallida obtained by tetrads counts 3000 per point in each period demonstrated great variance as compared to the NC as shown in Table 1.Exposure times were compared to determine the presence or absence of relevant statistics on the test points as shown in Table 2.

DISCUSSION
The plants are more sensitive to pollution than animals and man.Thus, they favor subsidies and an easier study of the effects of pollutants in the environment (Klumpp et al., 2006;Maioli et al., 2008).Chemical and physical agents exert influence vastly on the rate of change of biochemical activities in plants, thus increasing the frequency of changes in DNA molecules has been widely studied (Carvalho, 2005).
Frequency of MN showed elevated in all points when compared to the NC group.According to Figure 1, although the increase in the percentage of this type of chromosomal damage was not significant in points 1 and 3 within 15 days after implantation of T. pallida, points 2 and 4 had high statistical significance.
Low MN rates found in sections 1 and 3, respectively petrol station and parking, are justified by the reduced exposure to vehicle exhaust, the main cause of air pollution.Since the high frequency of MN in points 2 and 4, respectively an avenue of the great movement of vehicles and a garage, it is justified by the greater presence of toxic compounds due to the intense vehicular exhaust and direct manipulation of benzene derivatives in these environments.
The amount of carrier flow and handling content of compounds derived from benzene led to a wide variability in the results between the test points.The tests, as well  literature showed a direct and proportional relation between the significant number of chromosomal damage observed in T. pallida and elevation in the presence of these toxic gases, according to Figures 2 to 4 (Gábelová et al., 2004).
It was evident that local with exceeding the flow of vehicles has higher clastogenic effects and aneugenic indices correlated to Table 1.The longer period of exposure to polluting compounds induced an increase in the percentage of genetic damage of T. pallida as is  shown in Table 2.In the 15 days analysis only point 4 was significant in relation to the other, as in the analysis of 30 days was observed that point 2 became significant to the point 4. In consonance, the analysis to 60 days of exposition shown that all the points has become significant according to the increasing on MN.
Points 2 and 4 showed high relevance, the fact can be proven by the index and type of pollutants in these places is more intense.What determines the influence of the amount and the constant movement of vehicles in point 2  is the constant manipulation of benzene derivatives at point 4, that influence the expressively increase in MN frequency.
The frequency of MN found at points with higher vehicular traffic is according to records of Carreras et al. (2006) and Klumpp et al. (2006) who observed a direct relationship between mutagenic index and expressively increase of population concentration in urban areas.Metropolitan areas tend to present more atmospheres contaminated by pollutants than areas with less flow of vehicles and people, leading to a higher incidence of genetic damage in plants used as bio-indicators.
The data cited by Carreras et al. (2006) from a study with the same focus, using 3 test points in the city of Cordoba, Argentina, showed values between 2.4 ± 2.08 in distant area from the center and 4.2 ± 2.6 in region central city, revealing lower rates in inducing genetic damage in T. pallida compared with our study.The results showed mean values of 5.6 ± 0.7 and 7.1 ± 1.0, equating to the average percentage of this study, since the highest percentage found in sections that had only the pollutant factor of vehicle exhaust it was 7.5 ± 2.27, as shown in Table 1.
In a study, Costa and Droste (2012) suggested that rainfalls may decrease the genotoxic impacts in T. pallida.For this reason, we targeted dry season in this study to avoid the rain effects on this plant.Our study confirms the genotoxic effects in T. pallida at the points exposed in the city of Teresina.
It is noteworthy that during the analysis of T. pallida other structural changes in the cells of pollen grains were identified.These findings understood increased formation of non-viable pollen (characterized by the absence or reduction in the cytoplasm and a weak staining), which involves the interference of the fertility of the plant organism.According to Costa and Droste (2012), reduced pollen viability and increased in the frequency of MN, it is resulting from the influence of the vehicle pollution and extent of drought, factors that correlate to the points tested in research and high temperature in the city, Teresina.
In urban areas, air pollution is demonstrated by the photochemical smog resulting from the interaction between nitrogen oxides, hydrocarbons such as benzene and sunlight to form oxidation products, which can cause harm to humans and plants.As cars emit carbon monoxide, sulfur dioxide, aromatic compounds and hydrocarbons, contribute directly to the formation of photochemical smog and other pollution (Proncove, 2006;Semace, 2011).
The growing demand for new technologies makes the benzene is one of the industrial substances produced in higher volume.It comes mainly in production and oil refining, one of the constituents of gasoline.The exhaust of automotive vehicles accounts for about 5% of total hydrocarbons emitted into the environment.It is understood that the primary means of benzene poisoning in the workplace, either by direct contact with the substance or its derivatives (Abiquim, 1999;Costa and Costa, 2002).Thus, the high frequency of MN in T. pallida is related in point 4 with the studies of Alves et al. ( 2011), which found a positive association between high frequency of MN in pollen mother cells T. pallida exposed at different times of the large amount of polycyclic aromatic hydrocarbons -HPA's.Among the molecules considered mutagenic and / or carcinogenic, the HPA's have a prominent place in environmental and biological studies (Conpet, 2006;Jung et al., 2009;Brito et al., 2013;Silva et al., 2015).
When exposed to environmental pollutants, T. pallida suffers in short, physiological and structural changes resulting from the genotoxic potential.Thus, many injuries can be caused by these compounds, serving as an excellent indication, for example, its clastogenic effects, chromosomal breakage, and aneugenic, partial or total loss of chromosomes, detected by exposing the plant to the environment (Ma and Grant, 1982;Savóia, 2008;Sisenando, 2011;Crispim et al., 2014).
According to Thewes et al. (2011), chromosomal variations found in this experiment are explained by environmental changes that affect the errors in the process of formation and development of cellular structures of these vegetables (Rodríguez, 2015) Identification of the potential harm in the genome of T. pallida, through exposure of polluted air in situ may be an important indicator for genotoxic events in mammals, since these eukartotic cells are continuously entering into different divisional phases and constantly mutate (Carvalho, 2005).

Conclusion
Air pollution is an emerging problem in society; it's leading to major changes in ecosystems thus a significant number of diseases in humans, animals and plants in general.The T. pallida is an important form to detect air pollution.TRAD-MCN bioassay is a relatively simple technique, easy reproducibility and high efficiency in the evaluation of chromosomal damage arising from the interference of the pollutants.The results showed the genotoxic potential of the four tested environments when compared to the negative control.Thus, increased frequency of micronuclei was correlated with the local exposure time and, as the largest potential to cause chromosomal aberrations occurred in most vehicle flow and manipulation of benzene derivatives environments.These data, although relevant, are early.Necessitating the need for investigations in different seasons and chemical analyzes of atmospheric air of the places to be studied.

Figure 1 .
Figure 1.Tradescantia pallida exposure places in the city of Teresina: fuel refueling point (point 1), access way, avenue Frei Serafim (point 2), vehicle parking lot of a Higher Education Institution (point 3), and workshop manipulation of coating materials and solvents (point 4).

Figure 3 .
Figure 3. Frequency the micronuclei (MN) in (mean ± SD) of young T. pallida inflorescences after 15 days of seedlings exposure to local testing and control test.ANOVA followed by Dunnett's multiple comparison test; frequency of MN at significance of p<0.001*** compared to the NC.

Figure 4 .
Figure 4. Frequency the (MN) in 300 tetrads (mean ± SD) of young T. pallida inflorescences after 30 days of seedlings exposure to local testing and control test.ANOVA followed by Dunnett's multiple comparison test; frequency of MN at significance of p<0.001** and p<0.001*** compared to the NC.

Figure 5 .
Figure 5. Frequency the (MN) in 300 tetrads (mean ± SD) of young T. pallida inflorescences after 60 days of seedlings exposure to local testing and control test.ANOVA followed by Dunnett's multiple comparison test; frequency of MN at significance of p<0.01** and p<0.001*** compared to the NC.

Figure 6 .
Figure 6.Frequency of MN in 300 tetrads of young (mean ± SD).T. pallida inflorescences in all periods of exposure, 15, 30 and 60 days.Two-way ANOVA followed by Bonferroni post-hoc test; comparison between the exposure days and points at p<0.05*, p<0.01** and p<0.001*** compared to the NC.

Table 1 .
Micronuclei (MN)  formation in tetrads of young T. pallida inflorescences in all periods of exposure, 15, 30 and 60 days.and Dunnett's multiple comparison test.Mean ± standard deviation (SD) with a significance of 15-days p<0.001*** in points 2 and 4; for 30 days of p<0.01** in point 1 and p<0.001*** in points 2 and 4; and 60-days p<0.05* in point 3, p<0.01** in point 1 and p<0.001*** in points 2 and 4 in the test group, compared to the NC.

Table 2 .
Influence of exposure time to compare the percentage of micronuclei (MN) in T. pallida between test groups, assuming the significant value of each.