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Toxicological & Environmental Chemistry

ISSN: 0277-2248 (Print) 1029-0486 (Online) Journal homepage: http://www.tandfonline.com/loi/gtec20

Cytotoxic effect of benzo(a)pyrene ondevelopment and protein pattern of sunflowerpollen grains

Z. Baghali , A. Majd , A. Chehregani , Z. Pourpak , S. Ayerian & M. Vatanchian

To cite this article: Z. Baghali , A. Majd , A. Chehregani , Z. Pourpak , S. Ayerian & M.Vatanchian (2011) Cytotoxic effect of benzo(a)pyrene on development and protein patternof sunflower pollen grains, Toxicological & Environmental Chemistry, 93:4, 665-677, DOI:10.1080/02772248.2011.560851

To link to this article: http://dx.doi.org/10.1080/02772248.2011.560851

Published online: 18 Mar 2011.

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Toxicological & Environmental ChemistryVol. 93, No. 4, April 2011, 665–677

Cytotoxic effect of benzo(a)pyrene on development and protein pattern of

sunflower pollen grains

Z. Baghalia, A. Majda, A. Chehreganib*, Z. Pourpakc, S. Ayeriana and M. Vatanchianb

aLaboratory of Plant Cell Biology, Department of Biology, Tarbiat Moallem University, Tehran,Iran; bLaboratory of Plant Cell Biology, Department of Biology, Bu-Ali Sina University, Hamedan,Iran; cDepartment of Infectious Diseases, Children’s Medical Center, Medical Sciences/Universityof Tehran, Tehran, Iran

(Received 21 November 2010; final version received 2 February 2011)

Benzo(a)pyrene (BaP) is an important part of diesel exhaust particles (DEP). Theoverall objective of this research was to elucidate some microscopic effects of BaPon the formation, development, and structure of pollen grains and its proteins insunflower (Helianthus annuus L). Sunflower plants were grown in experimentalpots and treated with the different concentrations of BaP. Flowers and youngbuds were removed, fixed in FAA70 (formalin:acetic acid:ethanol, 2 : 1 : 17) andsubjected to developmental studies. Our results show that BaP-treatment causesabnormalities during pollen development. Shape of microspore tetrads changedfrom spherical to polygonal or irregular. Pollen grains in normal plants arespherical in both equatorial and polar views; in BaP-treated plants, they changedinto irregular shapes in equatorial view and triangular shape in polar view. Delayin degeneration of the tapetum layer in anther, and formation of giant, irregular,and non-fertile pollen grains are other results of BaP treatment. Gel electropho-retic studies revealed that a band with a molecular mass of 30 kDa disappeared inthe BaP-treated group and two new proteins with molecular masses of 15 and20 kDa were formed. Some pollen proteins may act as allergens, considering thefact that pollen allergy frequency is increased in polluted areas, especially DEP-polluted ones; the possibility arises that BaP, as an important part of DEP, couldbe an effective agent entailing formation of detoxifying proteins which, on theother hand, can also act as allergens.

Keywords: air pollution; benzo(a)pyrene; Helianthus annuus; pollen development;pollen proteins

Introduction

Air pollution by diesel exhaust particles (DEP) is common in both industrial anddevelopmental countries (Helender, Sarolainen, and Ahlholm 1997). DEP appear to playa role in causing respiratory and allergic diseases (Yokota, Ohara, and Kobayashi 2008;Lin et al. 2010; Lubitz et al. 2010). They may affect pollen grains, either indirectly viagrowth stress on the plant or directly through contamination of the anthers of a plant orduring the flight of pollen grains through the air (Chehregani, Mohsenzadeh, and Hosseini2011). DEP constitute an important part of air pollutants (Chehregani and Kouhkan 2008;Yokota, Ohara, and Kobayashi 2008) and consist of a complex mixture of particulate

*Corresponding author. Email: chehregani@basu.ac.ir

ISSN 0277–2248 print/ISSN 1029–0486 online

� 2011 Taylor & Francis

DOI: 10.1080/02772248.2011.560851

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matter, including elemental carbon and polycyclic aromatic hydrocarbons (PAH), aswell as aerosols and volatile organic compounds such as hydrocarbons (Hagemann et al.1982; EPA 1998). PAHs are released due to both natural and man-made processes,e.g., burning of biomass or fossil fuels and they are widespread in the environment(Sverdrup et al. 2007).

Benzo(a)pyrene (BaP) is a typical five-ring PAH that is a by-product of incompletecombustion or burning of organic (carbon-containing) items, e.g., cigarettes, gasoline, andwood (Sverdrup et al. 2007). BaP is also found in ambient (outdoor) air, indoor air, and insome water sources and has been used as an indicator of environmental PAH exposure(Ono-Ogasawara and Smith 2004). BaP, like other PAHs, shows the ability to accumulatein plants, animals, and soils and because of this accumulation, its concentration increase asorganisms take higher places in the food chain.

BaP is metabolized (chemically modified in the body) in humans and animals to form anumber of metabolites that may elicit toxicity (Sverdrup et al. 2007). BaP and BaPmetabolites can bind to DNA forming a structure called BaP–DNA adducts. BaP isclassified as having a mutagenic mode of action for inducing tumor formation, and isthought to require metabolic activation to become carcinogenic (Alarcorn et al. 2006;Kang et al. 2010).

The toxicity of BaP to several standard test organisms including the seed emergenceand early life stage growth of three terrestrial plants (Trifolium pretense L., Lolium perenneL., and Brassica alba (L.) Boiss.) was checked. The results showed that BaP did not affectseed emergence for any of the plants, but the growth of B. alba was significantly reduced atthe highest concentration tested (375mgkg�1; Sverdrup et al. 2007). The effect ofincreasing concentration of BaP (0.1, 1, 10, 50, 100mgL�1) on the growth of root andhypocotyls of lettuce was studied. The results showed that inhibition of growth wasdemonstrated in the plants treated by BaP in 100mgL�1 concentration (Kummerov,Slovak, and Holoubek 1995). The genotoxicity of BaP on Trifolium repens L. was studiedand changes in DNA content and sequences were observed (Aina, Plain, and Citterio2006). Plant germination and growth are strongly inhibited by the presence of volatile,water soluble and low-molecular weight hydrocarbons but high-molecular weight PAHslike BaP did not show any phytotoxicity (Henner et al. 1999).

Although several researches have shown detrimental effects of BaP, we were not able tofind any report on effects of BaP during developmental stages of pollen grains and theirprotein banding pattern. In this study, we have studied the effect of BaP on pollenin Helianthus annuus L.

Materials and methods

Plant material and treatments

Sunflower plants (H. annuus L. cv. Azargol from Asteraceae) were planted in differentgroups at the research farm of Bu-Ali Sina University. Forty pots (30 cm diameter and40 cm high) were filled with 3 kg of agricultural soil containing about 30% sand, 50%loam, and 20% humus. The plants were grown from seeds. The pots were divided intofour groups each including 10 pots, and each pot contained two seedlings. They werekept under identical conditions (27� 4�C, about 70% humidity and 15 h day light) in agreenhouse.

The experimental groups were classified as following: (1) 0.002 gL�1, (2) 0.02 gL�1,and (3) 0.04 gL�1 BaP in phosphate buffer saline (PBS), and (4) the control group that was

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treated by PBS. Two weeks before the flowering and continuing for the next 3 weeks,during flowering and pollen development, the plants of each pot were treated by above-mentioned BaP solutions. For preparation of BaP solutions, 1.2 g BaP (Sigma–Aldrich,UK) was mixed with 3L of PBS and kept in an ultrasonic apparatus for 20min. Otherconcentrations were made using the dilution of this solution. Shoots of experimental plants(leaves and developing buds) were sprayed with different concentrations of BaP in PBSwhile controls were sprayed with PBS. Each plant was sprayed with about 20mL of theabove-mentioned solutions in each day.

Microscopic studies

Flowers and young buds were removed from the experimental and control plants atanthesis, fixed in formalin:acetic acid:ethanol, 2 : 1 : 17 (FAA70), stored in 70% ethanol,dehydrated in a graded alcohol series ending with xylene 100%, embedded in paraffin, andsliced longitudinally at 7–10 mm thickness with a microtome (Leitz 1512, Germany).Staining was carried out with periodic acid Schiff’s reagent (PAS) according to theprotocol of Yeung (1984) and contrasted with modified Meyer’s hematoxylin (Chehregani,Mohsenzadeh, and Hosseini 2011). The prepared sections were fixed after dehydrationthrough increasingly graded alcohol series, and then studied under a light microscope(Zeiss Axiostar, Germany) with magnifications ranging from 400 to 1000. For eachdevelopmental stage of anther and pollen grains, the best sections were used forphotography with a Canon digital camera (G11). The developmental stages ofexperimental and control samples were compared for any difference. At each develop-mental stage, at least 20 flowers were studied and differences between experimental andcontrol plants were determined microscopically.

Polluted and normal pollen grains were studied by scanning electron microscopy. Aftercoating with gold, samples were analyzed using an SEM model SEM-EDS, EX 300(Link, UK). The purified pollen grains were fixed by glutaraldehyde, post-fixed withosmium, dehydrated, and dried prior to gold coating.

Pollen fertility

Pollen fertility was checked by staining a minimum of 1000 pollen grains from each groupusing a mixture of equal volumes of a solution of 1% acetocarmine (in distilled water) and50% glycerine (1 : 1) for 24 h. Well-stained and perfect pollen grains were considered asfertile, while unstained and empty pollen grains were counted as infertile (Sheidai andInamdar 1992).

Electrophoresis

The total protein contents of normal and BaP-treated pollen extracts were determined andcompared according to Bradford protein assay (Bradford 1976) using bovine serumalbumin as standard. For protein extraction, dried pollen grains (0.2 g) were homogenizedwith 1.2mol L�1 Tris–HCl buffer, pH 8, for 1 h. The mixture was put in an ultrasonicapparatus for 20min and then centrifuged at 12,000 g for 20min. Fifty microliters of theextract were mixed with an equal volume of a sample buffer (0.125mol L�1 Tris–HCl, pH6.8, 2% sodium dodecyl sulfate (SDS), 10% sucrose, 0.5% mercaptoethanol), denaturizedby boiling for 5min in a water bath, and cooled; then, 0.1% bromophenol blue was added

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to the solution as a dye. For separation of protein components, 20 mL of this mixture wasloaded on 12.5% gel slabs, prepared as described by Laemmli (1970). Electrophoresis wascarried out in Tris–glycine buffer (pH 8.3) at 4�C and 100V for 2 h in a Bio-Rad VerticalSlab Gel Apparatus using a low-molecular weight protein mixture as standard marker(Sigma, St. Louis, MO). Gels were stained with Coomassie brilliant blue for about 16 h atroom temperature. The dye solution was then replaced by a de-staining solution, whichwas changed regularly until a clear background was observed. The gels were scanned withHP Scan Jet 3000 (South Korea) and the best photos were used for comparing the bands ofcontrol and experimental groups.

Statistical analysis

In order to detect a possible significant difference between the experimental groups andcontrol ones, analysis of variance (ANOVA) followed by the least significant differencetest (LSD) that was performed between studied groups (Chehregani et al. 2006). Each datawas represented as the means� SD of 20 samples for experimental groups as well as 20samples for control one.

Results

Microscopic studies of this research revealed that each anther of H. annuus L., in normalplants, consists of four pollen sacs. These four pollen sacs display synchronousdevelopment, are initiated very early during flower development, and occupy themajority of each flower. Pollen development followed the same way as other dicotyle-donous plants. Primary sporogenous cells were developed directly as microsporocytes(Figure 1a). Meiosis, in each microsporocyte, results in a microspore tetrad via prophase I(Figure 1b), metaphase I (Figure 1c), anaphase I (Figure 1d), telophase I (Figure 1e),prophase II, metaphase II, anaphase II, and telophase II (Figure 1f). No cell wall isdetected between the two newly formed nuclei at telophase I (Figure 1e). The cytokinesis isof the simultaneous type. The tetrads are mostly tetrahedral (Figure 1g) and rarelytetragonal (Figure 1h). Sometimes callose is well discerned around the tetrad andbetween each monad (Figure 1h). Microspores in the two neighboring sporangia aresynchronized in development. The microspore, when released from tetrad, has notvacuolated; yet, it has a dense cytoplasm and is somewhat irregular in shape, with aprominent and centrally placed nucleus (Figure 1i). As the central vacuole develops, thenucleus takes up a peripheral position (Figure 1j), i.e., a large vacuole squashes thecytoplasm together with the nucleus toward the microspore margin. Mature pollen grainsare spherical shaped, tricoplated type and contained long needle (echinated) as pollensculpturing (Figure 1k and l).

In plants treated by different concentrations of BaP, some abnormalities were observedduring pollen development. Pollen sacs formed were smaller in size than normal ones.Microspore tetrads formed were spherical shape in all the normal plants (Figure 1g and h),but they were polygonal and irregularly formed in BaP-treated samples (Figure 2a). Thisdifference was more obvious in the group treated with 0.04 gL�1 of BaP (84% of tetrads,Table 1). The shape of pollen grains was spherical in both equatorial and polar views innormal plants (Figure 1j and k), but abnormal shapes including serrate margined andovate were observed in plants treated by Bap (Figures 2b, g, and 3). In the groups treatedby 0.002, 0.02 and 0.04 gL�1 BaP, 4%, 32%, and 68% of pollen grains showed abnormal

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Figure 1. Microsporogenesis and pollen development in normal sunflower plants. (a) Longitudinalsection of anther showing anther wall, tapetum layer (ta) and pollen mother cells (PMC); (b–f)Showing various stages of meiosis in microsporocytes; (b) Prophase I; (c) Metaphase I (arrow); (d)Anaphase I (arrow); (e) Telophase I; (f) Telophase II (arrow); (g) Tetragonal microspore tetrads; (h)Tetrahedral microspore tetrads. The shape of microspore tetrads is spherical; (i) Microspores justreleased from tetrads. The shape of microspores is spherical to ovate; (j) Some microsporesvacuolated slightly at maturation process (arrow); (k) Mature pollen grains with spherical shape andprominent nucleus; arrow indicates the position of apertures. As results showed, tapetal cells (Ta)were digested and disappeared in this phase. The pollens were not vacuolated obviously; (l) Scanningelectron micrograph of mature pollen grain with long needle as sculpturing and tricoplate (arrow).The shape of pollen is spherical. Scale bars are 30 mm in the Figure 1(a)–(k), and 10 mm in Figure 1(l).Abbreviations: En, endothecium; Ta, tapetum layer; PMC, pollen mother cells.

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shapes, respectively. Gigantism of some pollen grains was one of the most obviouseffects of BaP treatment (Figure 2c and d). The pollen grains were 2–3 times larger in BaP-treated plants than normal ones (Figures 2c, d, and g and 4, Table 1). Accumulation ofblack particles was evident in the cytoplasm of pollen grains in BaP-treated samples

Figure 2. Microsporogenesis and pollen development in BaP-treated plants. (a) Microspore tetradsin BaP-treated plants. The shape of microspore tetrads (Te) changed from spherical to polygonal, asa result of BaP treatment; (b) Released microspores (young pollens) (m) in BaP-treated plants. Theshape of microspores and pollens changed to irregular and triangle form; (c) Formation of a largevacuole in young pollens (") is a result of BaP treatment; (d) In BaP-treated plants, most of thepollens were vacuolated dramatically and have a larger size than normal pollens; (e) In BaP-treatedplants, starch particles (black particles) were accumulated in pollen grains ("); (f) Remaining maturepollen grains in BaP-treated plants. Pollens are vacuolated considerably and tapetum cells (Ta) werenot digested ("); (g) Scanning electron micrographs of DEP-treated pollen. Pollen is ovate shapedand colporate ("). Scale bars are 30mm in the Figure 2(a)–(f) and 10mm in Figure 2(g).Abbreviations are similar to Figure 1.

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(Figures 2e and 4). These particles were observed in the more than 50% of pollen grainstreated by 0.04 gL�1 of BaP solution (Figures 2e and 4), but rarely in the control plants. Innormal plants, tapetum cells were degraded very soon, in the microspore stage, forming aplasmodium useful in nourishment of developing pollen grains (Figure 1j), but in BaP-treated plants the degeneration of tapetum cells were took place later and was visible at thetime of pollen grains maturation (Figure 2f). Some large vesicles and vacuoles alsoappeared in the cytoplasm of pollen grains in BaP-treated plants (Figure 2f), but only fewsmall vacuoles were visible in the control plants. Otherwise, the cytoplasm of pollen grainswas not vacuolated obviously in normal plants (Figure 1k). In conclusion, statisticalanalysis showed that the formation of abnormal pollen grains including irregular, giant,and vacuolated ones increased in BaP-treated plants significantly compared with controlplants (Figures 3, 4 and Table 1). All aforementioned effects are visible in the all BaP-treated groups but they are more significant in the group treated by 0.04 gL�1 of BaP-solution.

The results of pollen fertility analysis using aceto-carmine staining showed that thefertility of pollen grains decreased in BaP-treated plants (Figure 3). Normal plantsproduced 97% fertile pollen grains but the fertility values were evaluated as 85%, 50%,and 35% in the groups treated by 0.002, 0.02, and 0.04 gL�1 of BaP-solution, respectively(Figure 2). The decrease in pollen fertility was significant in the groups treated with 0.02and 0.04 gL�1 of BaP (p� 0.01).

The total protein banding pattern was also considerably different among normal andBaP-treated plants (Figure 5). Results showed that protein banding pattern was shiftedobviously in the group treated by 0.04 gL�1 of BaP-solution. In this group, a band withmolecular mass of 30 kDa disappeared and two new bands with molecular masses 15 and20 kDa were formed (Figure 5).

Table 1. Comparison of properties of pollen grains between normal and BaP-treated pollen grains.

Specimens

Pollen characters Normal plants BaP-treated plants

Tetrad shape Spherical Polygonal and irregularPolar shape Circular Triangle and irregularEquatorial shape Spherical and regular Different and irregularPollen size variation Uniform Normal and giant pollen

Polar length (m) Small pollen (34%) Giant pollen (56%)Minimum 26.5 23.5 32.5Mean 27.8 24.2 36.5Maximum 28.5 25.8 38.7

Equatorial length (m) Small pollen Giant pollenMinimum 25 23.1 31Mean 25.5 24 32.2Maximum 26 25.2 34.4

Polar/equatorial diameter Small pollen Giant pollenMinimum 1.06 1.02 1.05Mean 1.09 1.01 1.13Maximum 1.07 1.02 1.12

Notes: Each data represented the means of 15–25 anthers. Differences between normal and DEP-treated groups are significant (p� 0.01).

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Discussion

Several reports have indicated that air pollutants, including DEP and its constituent

chemicals, might affect the development of pollen grains (Chehregani and Kouhkan 2008;

Lubitz et al. 2010; Chehregani, Mohsenzadeh, and Hosseini 2011). The results of this studyshowed that the developmental stages and the proteins banding profile were affected by

BaP as a major part of DEP.Our microscopic studies showed that in control sunflower plants, the developmental

process of pollen grains took place in an ordinary way, like other dicotyledonous plants.

However, the plants treated by different concentrations of BaP-solutions showed some

abnormalities during pollen developmental process (Figure 2). The shape of tetradschanged from spherical to polygonal or irregular, which is probably correlated with BaP

solutions (Figure 2a). It seems that BaP treatments cause changes in cell division pattern

and the direction of cell plate formation. Prior reports showed that some environmentalpollutants cause the same results in plants that were under treatment of SO2 (Majd and

Chehregani 1992), air pollutants (Majd et al. 2008), acid rain (Chehregani et al. 2006), and

DEP (Chehregani, Mohsenzadeh, and Hosseini 2011).The interior layer of the anther contains large cells rich in nutrients, the so-called

tapetum layer, and serves in providing nutrients for the developing microspores and pollen

Figure 3. Effect of BaP on the pollen fertility and abnormality. Results showed that non-fertile andempty pollen grains increased in BaP-treated plants. Irregular and abnormal pollen grains alsoincreased in BaP-treated plants. Increase of abnormal pollen grains and decrease of pollen fertilityare significant (p� 0.01). Each datum represented the means� 20 samples (at least 1000 pollengrains).

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grains (Pacini, Franchi, and Hess 1985). The tapetum layer is digested during maturation

of pollen grains, so that most of its cells disappear in mature anthers (Figure 1k).

Microscopic observations show that degeneration of the tapetum layer occurs later in the

plants treated by BaP-solutions (Figure 2f) than in the control plants. These phenomena

result in deficiency of nourishment for the developing pollen grains and cause them to

form abnormal pollen grains (Chehregani et al. 2006; Chehregani, Mohsenzadeh, and

Hosseini 2011). A similar finding was reported about the effect of DEP (Chehregani,

Mohsenzadeh, and Hosseini 2011).The shape of pollen grains is spherical in both equatorial and polar views in normal

plants (Figure 1j–l), but pollen grains with different shapes including irregular ones were

observed in the BaP-treated plants (Figures 2b and 3). It seems that delay in degeneration

of tapetal cells and their consequent inability in providing nutrients for developing pollen

grains are the main reasons of this phenomenon. According to some prior reports, other

environmental pollutants have also similar effects (Chehregani et al. 2004). Some reports

showed that the presence of other environmental pollutants, such as cadmium, reduces cell

wall plastic extensibility and impairs normal cell elongation in growing pollen tubes,

causing morphological and structural alterations. This can be explained by the interaction

of pollutants with the anionic contents of secretory vesicles and the fact that pollen cell

walls contain large quantities of pectins and callose, but less cellulose (Sawidis and Reiss

1995; Sawidis 2008). This is a new finding about the effect of BaP on pollen grain and we

have reported similar findings in DEP-treated plants (Chehregani, Mohsenzadeh, and

Hosseini 2011).

Figure 4. Effect of BaP on the accumulation of dark starch particles in pollen grains. Resultsindicated the significant increases of the particles in BaP-treated plants (p� 0.01). 0.002, 0.02, and0.04, the groups treated by 0.002, 0.02, and 0.04 gL�1 BaP solutions, respectively. Each datumrepresented the means� 20 samples (at least 1000 pollen grains).

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Our results and observations showed that in BaP-treated plants, some pollen grains

were enlarged several times more than normal ones (Figure 2c and g, and Table 1). Similar

findings were reported in the plants treated with water-soluble fraction of DEP

(Chehregani, Mohsenzadeh, and Hosseini 2011). The pollen growth is inhibited by the

formation of exine in normal plants, but when exine is not formed regularly, for example,

due to deficiency of nourishing material of tapetal cells, the inhibition of growth may fail

and the pollen grains continue growing. Although no previous report exists confirming this

theory, it has shown that other environmental pollutants, such as heavy metals, can

stimulate pollen germination and tube growth (Sawidis and Reiss 1995; Sawidis 2008).

Low metal concentrations can also stimulate enzymatic activities, whereas high concen-

trations of metals inhibit these processes (Sawidis 2008). Accumulation of starch particles

in the pollen grains is the other result of BaP treatment (Figures 2e and 4). It seems that

this is a common result of plant growth under the influence of several environmental

pollutants (Chehregani, Mohsenzadeh, and Hosseini 2011).

Figure 5. Protein banding profile of normal and BaP-treated pollen grains. Results showed in BaP-treated plants (0.04 gL�1) that a band with molecular mass of 30 kDa (*) disappeared and two newbands with molecular masses of 15 and 20 kDa (**) are formed. Other BaP-treated groups are thesame as the normal ones. M, molecular markers; 1, the group without any treatment; 2, controlgroup treated with PBS; 3, 4, and 5, the groups treated by 0.002, 0.02, and 0.04 gL�1 BaP-solutions,respectively.

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The results of the comparison of protein banding among BaP-treated and normal

plants (Figure 5) showed that there are major dissidences in the group treated with

0.04 gL�1 of BaP-solution with the control plants. In plants treated with 0.04 gL�1 of

BaP-solution, a band with molecular mass of 30 kDa disappeared and two new bands with

molecular mass of 15 and 20 kDa are formed (Figure 5). This finding is in accordance with

some prior reports on the other air pollutants (Chehregani and Kouhkan 2008;

Chehregani, Mohsenzadeh, and Hosseini 2011) but contrary to some others (Chehregani

et al. 2004). This means that BaP treatment can affect gene expression in pollen grains and

cause them to form new proteins, probably the detoxifying proteins reported previously inresponse to heavy metal toxicity (Yousefi et al. 2009). Since pollen proteins are acting as

allergens, and based on the fact that the frequency of pollen allergy is increasing in areas

with polluted air, we can conclude that BaP could induce to form new proteins (or new

allergens; Shahali et al. 2007; Chehregani and Kouhkan 2008; Majd et al. 2008). Since our

previously reported data about DEP (Chehregani, Mohsenzadeh, and Hosseini 2011) are

similar to the finding of this research, it seems that the effect of DEP is due to its BaP

constitute.DEP, as an important environmental pollutant, has different morphological,

biochemical, and cytological effects on plants (Behrendt and Becker 2001; Chehregani

and Kouhkan 2008; Chehregani, Mohsenzadeh, and Hosseini 2011). The results of this

study indicated that BaP, as a most important part of DEP, induced several abnormalities

in pollen development and protein banding pattern, which can in turn affect fertility, gene

expression, and survival of plants. Although there are several reports about the effects of

BaP on different organisms, this is the first investigation on the influence of BaP in pollendevelopment.

Acknowledgments

This research was done using financial support provided by research council of Bu-Ali SinaUniversity and also Tarbiat Moallem University partly.

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