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Comparative study of Avicennia marina (Forsk.) Vierh. pollens allergenicity in two regions of Bushehr province in Iran.

Introduction

Respiratory diseases such as allergy are increasing due to the pollens have scattered in the air [16]. Several studies have proved that pollens especially those are anemophilous cause allergy [11]. These allergenic pollens have special proteins in their walls but other substances like glycoproteins or a single peptide located in pollen walls or cytoplasm may cause allergy [12]. Environmental effects such as temperature and air pollution can influence the allergenicity of allergenic pollens [10]. Avicennia marina (Forsk.) Vierh. in Bushehr province has grown in two regions: Bordekhoon (Mond Protected Area) and Assaluyeh (Marine National Park of Nayband).

Assaluyeh (polluted area) is an industrial city and there are many petrochemical factories that pollute area but these factories do not exist in Bordekhoon (control area). Sulfur compounds are one of the important pollutants in petrochemical areas [13]. Several investigations revealed that the abundance of allergic diseases is higher in industrial areas [2, 8]. High levels of pollutants that these factories produce intensify allergic symptoms [2, 10]. The aim of this study was to determine pollen allergenicity of this plant that is no data about it till now and compare it in control area and polluted area in order to investigate the environmental effects on pollens allergenicity.

Materials and Methods

Method for sampling:

During spring and summer 2011 (June-July) the pollens were collected from flowers of Avicennia marina (Forsk.) Vierh. trees grown in Bordekhoon (control area) and Assaluyeh (polluted area) in Bushehr province. Some of the collected pollens were briefly dried and stored at -20[degrees]C for studying pollen allergenicity and some of them were dried for scanning electron microscopy. Storage was the same in two regions.

Method for Scanning Electron Microscopy (SEM):

Dried pollens were coated with gold by physical vapor deposition (Sputter coater SCDOOS, BAL-TEC. CO, Switzerland) for morphological analysis and then were studied with a scanning electron microscope (Philips XL30).

Method for preparing the pollen extract:

For preparation the pollen extract, 0.05 gram of pollens of each samples were defatted and then extracted in 1ml of PBS (phosphate buffered saline) pH = 7.4 (1:20). The mixture was stirred overnight at 4[degrees]C. The suspension was centrifuged at 4600 rpm for 45 min and supernatants were dialyzed against distilled water.

Animals:

Forty female 6-8 week old Balb/c mice were obtained from Pasteure Institute (Karaj, Iran). Animals were maintained at the animal care service of the Tehran University of Medical Sciences. They were divided in four groups (n = 10). First and second groups as control groups received PBS and Aluminum hydroxide (Alum) respectively. Third group was treated by control area pollens extract plus Alum and forth group was treated by polluted area pollens extract plus Alum.

Method for treatment the mice by pollens extracts:

The mice of control groups (first and second groups) received 150 [micro]l PBS and Alum respectively by the intraperitoneal route on days 0, 7, 14, and 21 [3,5]. The mice of third and forth groups were treated intraperitoneally at the same time points by 150 [micro]l pollen extracts of control area and polluted area containing 20 [micro]g protein adsorbed to 2mg of Alum respectively [3,5]. The injections were performed by insulin syringe.

Method for intradermal skin test:

Intradermal skin test was performed on day 24. Animals of first group (negative control) were tested with 15[micro]l of PBS and animals of second group (positive control) were tested with 15ul histamine acid phosphate. Animals of third and forth groups were tested with 15ul of control area and polluted area pollen extracts containing 5[micro]g protein respectively. The wheal diameter (local edema in the point of injection) of intradermal skin test was measured after 30 min from the start. Mice were anaesthetized with ether and blood samples were collected from the heart on day 28. Blood smears were prepared and stained with Gimsa solution to determine eosinophil rate and serum samples were prepared and stored at -20[degrees]C for enzyme-linked immunosorbent assay (ELISA).

Method for Enzyme-Linked Immunosorbent Assay (ELISA):

Total serum IgE (immunoglobulin E) was measured using the mouse IgE measurement kit (Koma Co., Seoul, Korea) according to the manufacturer's instructions. Briefly the precoated wells with anti-mouse IgE monoclonal antibodies were incubated with 100 [micro]l of 1:2 diluted test sera for one hour at room temperature. After washing, each well was incubated with diluted detection antibody (1:6000). The wells were finally filled with substrates and allowed to develop colors for 15 minutes and stopped. The plate was read at 450 nm wavelength by ELISA reader and total serum IgE levels were calculated from the standard curve.

Method for determining Protein contents:

The total protein contents of control area and polluted area pollen extracts were estimated as explained in the Bradford protein assay using bovine serum albumin as standard [6].

Methods for statistical analysis:

SPSS was used to perform statistical analysis. Wheal diameter of intradermal skin tests, rate of eosinophils in blood smears and total serum IgE levels of experimental groups were compared using nonparametric Two-Sample Kolmogorov-Smirnov test.

Results:

Scanning electron microscopy (SEM) results:

Scanning electron microscopy (SEM) analysis of pollen grains of Avicennia marina (Forsk.) Vierh. showed that they are elliptic in shape (Figs. 2, 5), 24 um in diameter (Figs. 1, 4) and 30 [micro]m in length (Figs. 2, 5), the exine is 3-colporate (Figs. 1, 4) and the ornamentation of exine is reticulate (Figs. 3, 6). On the pollens surfaces of Bordekhoon (control area) the pores on the exine were opened (Figs. 3) but these pores in Assaluyeh (polluted area) pollens were closed (Fig. 6).

[FIGURE 1-3 OMITTED]

[FIGURE 4-6 OMITTED]

Intradermal skin tests results

The results of intradermal skin tests are shown in Table 1. The results indicated that average of wheal diameter after 30 min from the start in mice received PBS and histamine acid phosphate as negative and positive controls were 5.22 mm and 5.30 and in mice treated by control area and polluted area pollen extracts were 16 and 16.33 mm respectively. Statistical analysis showed that pollen extracts of two regions considerably increased skin reactions (P = 0) Compared with control groups. Wheal diameter of intradermal skin test in forth group (treated by polluted area pollens extract) was more than third group (treated by control area pollens extract) but this increasing was not significant (Fig. 7).

[FIGURE 7 OMITTED]

Blood factors assay results:

The results of blood smears in four experimental groups are summarized in Table 2. The results revealed that eosinophil rate in the blood of mice treated by pollen extracts of two regions were more than control ones, but it was significantly higher in group treated by polluted area pollen extract (P = 0.011). Eosinophil rate in mice of forth group (treated by polluted area pollen extract) was more than third group (treated by control area pollen extract) but it was not considerable (Fig. 8).

Groups: 1: Control group that received PBS; 2: Control group that received Alum; 3: The group that treated by Bordekhoon pollen extract plus Alum; 4: The group that treated by Assaluyeh pollen extract plus Alum.

As shown in Table 2 blood smears of groups 3 and 4 showed Eosinophilia in comparison with control ones but it was significantly higher in group 4 (P = 0.011). Rate of eosinophils in group 4 increased compared with group 3 but it was not significant. Mean of total serum IgE level was also increased in group 3 and 4 in comparison with control ones but interestingly it was considerably higher in group 4 (P = 0.003). Total serum IgE level in group 4 increased considerably compared with group 3 (P = 0.05).

[FIGURE 8 OMITTED]

Evaluation of total serum IgE (Table 2) showed that the amount of IgE increased in sera of mice treated by pollens extracts of two regions compared with control ones. Statistical analysis showed that this increasing was significant in forth group (P = 0.003) in comparison with control group. Total serum IgE level in forth group (treated by polluted area pollen extract) was considerably more than third group (treated by control area pollen extract) (P = 0.05) (Fig. 9).

Bradford protein assay results:

Bradford protein assay showed non significance increasing in total protein contents in polluted area (575ug/ml) compared with control area (525 [micro]g/ml) pollens extracts.

[FIGURE 9 OMITTED]

Discussion:

One of the causes of allergy is plant pollination [11]. The abundance of allergic symptoms depends on several factors such as relative humidity, sunlight intensity and wind speed [11]. Assaluyeh is an industrial city with petrochemical factories. Sulfur compounds are one of the most important pollutants in petrochemical regions [13]. Air pollutants stick to the pollens surfaces, and also interact with the pollen allergens and can act as adjuvant in the immune system therefore enhance allergenic properties of pollens [2,15]. Petrochemical factories don't exist in Bordekhoon (control area) thus the differences between the pollens of two regions may be result of air pollution that is abundant in Assaluyeh (polluted area).

Scanning electron microscopy analysis (Figs. 3, 6) showed that in control area and polluted area the pores on the surface of Avicennia marina (Forsk.) Vierh. pollens were different. In control area these pores were opened but in polluted area these pores were closed. Rezanejad (2007) and Shahali et al. (2008) also observed that air pollution can affect and change the surfaces of pollen grains [14, 17].

Our results showed that allergic reactions (wheal diameter of Intradermal skin test, eosinophil rate in blood smears and total serum IgE level) increased in mice treated by pollen extracts of two regions in comparison with control groups, interestingly these reactions (specially total serum IgE level) in the group treated by polluted area pollens extract were more than the group treated by control area pollens extract. Therefore Avicennia marina Pollens have allergenic properties but after air pollution exposure these allergenic characteristics (specially total serum IgE level) increase. Thus activities of petrochemical factories and air pollution, affected allergenic properties of this plant. These findings are in accordance with the observations of Armentia et al. (2002), Barta et al. (2007) and Jianan et al. (2007) [2, 4, 10] and also Chehregani et al. (2004), Shahali et al. (2008), and Arbabian et al (2011) in Iran that observed air pollution increases allergenicity of pollens [7, 17, 1]. However studies of Helander et al (1997) did not show any significant difference between the pollens allergenicity of polluted and nonpolluted area [9].

Conclusion:

The present study showed that Avicennia marina (Forsk.) Vierh., pollens have allergenic properties and their allergenicity increased in Assaluyeh (polluted area) in comparison with Bordekhoon (control area). It seems that air pollution in Assaluyeh was the cause of increasing in pollen allergenicity of this plant.

Acknowledgements

This work was supported by Tehran University of Medical Sciences grants.

References

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[2.] Armentia, A., M. Lombardero, A. Callejo, D. Barber, F.G. Martin Gill, J.M. Martin Santos and M.L. Arranz, 2002. IS Lolium pollen from an urban environment more allergic than rural pollen? Allergologia Et Immunopathologia, 30(4): 218-224.

[3.] Barral, P., M. Villalba, R. Rodrigues and E. Batanero, 2006. The role of major olive pollen allergen Ole e 1, Ole e 9, and Ole e 10 on mice sensitization. Annals of Allergy Asthma and Immunology, 96: 466-471.

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[5.] Batanero, E.P., Barral, M. Villaba and R. Rodrigues, 2002. Sensitization of mice with olive pollen allergen Ole e1 induces a Th2 response. International Archives of Allergy and Immunology, 127: 269-275.

[6.] Bradford M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein -dye binding. Analytical Biochemistry, 72: 248-254.

[7.] Chehregani A., A. Majd, M. Moin and M. Gholami, 2004. Increasing allergy potency of Zinnia pollen grains in polluted areas. Ecotoxicology and Environmental Safety, 58: 267-272.

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[10.] Jianan, X., O. Zhiyum, Z. Hua, W. Xiaoke and M. Hong, 2007. Allergenic pollen plants and their influential factors in urban areas. Acta Ecologica Sinica, 27(9): 3820-3827.

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[12.] Mondal, A.K., S. Parui and S. Mandal, 1998. Protein profile of the allergenic pollen of Ipomea fistula L.--A comparative study. Annals of Agricultural and Environmental Medicine, 5: 131-134.

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[14.] Rezanejad, F., 2007. The effects of air pollution on microsporogenesis, pollen development and soluble pollen proteins in Spartium junceum L. (Fabaceae). Turk Journal of Botany, 31: 183-191.

[15.] Rezanejad, F. and A. Chehregani, 2008. Allergenicity and identification of specific IgE binding proteins in pollen of Spartium junceum L. (Fabaceae) and Lagerstroemia indica L. (Lytraceae): The effect of air pollution on their allergenicity. Iranian Journal of Science and Technology, 32: 129-134.

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(1) Mandana Salehi, (2) Ahmad Majd, (3) Gholamali Kardar, (1) Parissa Jonoubi, (3) Ahad Zare, (1) Leila Karami, (3) Zahra Pourpak

(1) Department of Biology, Tarbiat Moallem University, Tehran, Iran.

(2) Department of Biology, Faculty of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran.

(3) Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran.

Mandana Salehi, Ahmad Majd, Gholamali Kardar, Parissa Jonoubi, Ahad Zare, Leila Karami, Zahra Pourpak; Comparative study of Avicennia marina (Forsk.) Vierh. pollens allergenicity in two regions of Bushehr province in Iran

Corresponding Author

Ahmad Majd, Department of Biology, Faculty of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran.

E-mail: Ahmad_Majd2005@yahoo.com
Table 1: Wheal diameters of intradermal skin test
(mm) in the mice treated by pollen extracts of two
regions and control ones.

Groups (n = 10)       Wheal diameters of
                  intradermal skin test (mm)

1                      5.22[+ or -]0.97
2                      5.30[+ or -]0.94
3                       16[+ or -]1.06
4                       16.33[+ or -]1

Table 2: Rate of eosinophils (%) and Total serum IgE level
(ng/ml) in the mice treated by pollen extracts of two
regions and control ones.

Groups           Rate of          Total serum IgE
(n = 10)     eosinophils (%)       level (ng/ml)

1            1.44[+ or -]0.52    51.88[+ or -]11.70
2             2[+ or -]0.81      65.40[+ or -]24.90
3            2.5[+ or -]0.75     81.49[+ or -]38.42
4            2.72[+ or -]0.78   142.16[+ or -]116.58
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Article Details
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Title Annotation:Original Article
Author:Salehi, Mandana; Majd, Ahmad; Kardar, Gholamali; Jonoubi, Parissa; Zare, Ahad; Karami, Leila; Pourpa
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:May 1, 2012
Words:2616
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