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Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis
Food Chemistry 192 (2016) 370–373
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis Nurcan Cetinkaya a,⇑, Demet Ercin b, Sümer Özvatan b, Yakup Erel b a b
Ondokuz Mayis University, Faculty of Veterinary Medicine, 55139 Kurupelit, Samsun, Turkey Turkish Atomic Energy Authority, Sarayköy Nuclear Research and Training Center, Saray Mah., Atom Cad., No: 27, 06983 Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 7 April 2014 Received in revised form 16 December 2014 Accepted 7 July 2015 Available online 8 July 2015 Keywords: Irradiation DNA Comet Assay Quarantine control method Citrus fruits Image analysis
a b s t r a c t The experiments were conducted for quantification of applied dose for quarantine control in irradiated citrus fruits. Citrus fruits exposed to doses of 0.1 to 1.5 kGy and analyzed by DNA Comet Assay. Observed comets were evaluated by image analysis. The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Irradiated citrus fruits showed the separated tails from the head of the comet by increasing applied doses from 0.1 to 1.5 kGy. The mean tail length and mean tail moment% levels of irradiated citrus fruits at all doses are significantly different (p < 0.01) from control even for the lowest dose at 0.1 kGy. Thus, DNA Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Irradiation has been considered by ICGFI. (1994), Marcotte (1998) and Hallman (1999) and now a days, it has been used as an alternative to phytosanitary and quarantine treatments for international trade of fresh fruits and vegetables (Bustos-Griffin, Hallman, & Griffin, 2012; Hallman, 2011; Hallman, 2012) so it can be replaced with chemical fumigants such as methyl bromide that is going to be phased out by the year 2015 in developing countries. In 2009, International Plant Protection Commission (IPPC) approved the generic radiation dose of 150 Gy for tephritid fruit flies (IPPC, 2009). Irradiation proved to be extremely beneficial in terms of prolonging the fruit and vegetable shelf life by 3–5 times (Arvanitoyannis, Stratakos, & Tsarouhas, 2009). Recently, irradiation has been used as generic phytosanitary treatment for exported fresh fruits in Asian countries such as India, Pakistan, Thailand and Vietnam (Follett & Weinert, 2012). Ionizing radiation damages the DNA of insects and pathogenic microorganisms in foods (Delincee & Soika, 2002). The DNA Comet Assay is one of ten approved standard detection methods in many food items such as meat, fish, grains, and fruits (CEN. EN 13784, 2001) and has also been utilized to identify irradiated grapefruits (Delincée, 1998) but not for quantification of applied doses. Analytical detection of radiation treatment of food is an ⇑ Corresponding author. E-mail address: [email protected] (N. Cetinkaya). http://dx.doi.org/10.1016/j.foodchem.2015.07.027 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
important means to implement such control, once the food items have left the irradiation facility (Khan, Khan, & Delincée, 2003). Quarantine inspectors only control the irradiation certificate travel with irradiated produces. That’s why the irradiation facilities are specifically designed and established for low dose quarantine method application to fresh fruits. However, multipurpose irradiation facilities should also be used for commercial fresh fruit irradiation. Beside of irradiation certificate, there is a need for a justification method to control of generic low dose irradiated fresh fruits to be certain about good radiation processing not changing of sensory properties of fruits. In present study, DNA Comet Assay was used together with image analysis for quantification of applied low dose to grapefruit, lemon, mandarin and orange in order to propose of DNA Comet Assay as potential quarantine control method for inspectors.
2. Materıals and methods 2.1. Irradiation of samples Seed samples for each dose from grapefruit, lemon, mandarin and orange were exposed at different dose levels of 0.1, 0.3, 0.5, 1.0 and 1.5 kGy in gamma cell (Gammacell Co60, dose rate 1.31 kGy/h) at Turkish Atomic Energy Authority, Saraykoy Nuclear Research and Training Center. Harwell Gammachrome YR Batch 62 dosimeters were used for the measurement of radiation dose.
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Dose kGy
Grapefruit
Lemon
Mandarin
Orange
0.0
0.1
0.3
0.5
1.0
1.5
Fig. 1. DNA Comets after microgel electrophoresis of cells from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses.
2.2. DNA Comet Assay Standard DNA Comet Assay was used in this study (CEN, 2001. EN 13784). Seed samples were crushed and mixed with 5 mL of cold phosphate buffered saline (PBS, pH 7.4), which was stirred for 5 min at about 500 rpm, filtered through 200 lm and 100 lm nylon sieve clothes, and then let for sedimentation for about 30 min 100 lL of cell suspension was mixed with 1000 lL of 0.8% low melting point agarose (LMA) and 100lL of this mixture was spread onto the pre-coated slide glass using a cover glass and left cooling for 45 min on ice bucket. The casted slides were immersed in lysis buffer (2.5% SDS in 45 mM Tris–borate, 1 mM EDTA, pH 8.4)
for 20 min then slides were kept 5 min in electrophoresis buffer solution. The electrophoresis was performed in TBE buffer (Tris borate electrophoresis buffer, pH 8.4) at 2 V/cm for 2 min The slides were put into distilled water for 5 min and dried at 45 °C approximately 1 h. Propidium iodide staining was employed to visualize DNA. 2.3. Evaluation of comets by image analysis Observed comets from DNA Comet Assay were evaluated by image analysis for linking the comet parameters to estimate applied doses.
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The slides were evaluated with a standard transmission microscope (Olympus BX 51 model) at 20X and evaluated by digital color video camera (Pixera) with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Comet measurements and estimated parameters are defined as following: 1. Tail Area: Area of the comet tail in pixels (sum of pixels in the head). 2. Head DNA: Amount of DNA in the comet head (sum of intensities of pixels in the head). 3. Tail DNA: Amount of DNA in the comet tail (sum of intensities of pixels in the tail). 4. Tail DNA%: Percent of DNA in the comet tail. 5. Tail Length: Length of the comet tail measured from right border of head area to end of tail (in pixels). 6. Comet Length: Length of the entire comet from left border of head area to end of tail (in pixels). 7. Tail Moment: Tail DNA% x TailLength ([percent of DNA in the tail] x [tail length]).
Fig. 2. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for grapefruit.
There are semi (New Manual) and fully automatic (AutoComet) comet measurements of BAB Imaging System. The comet was put in frame and comet head was identified with mouse in the semiautomatic measurement which was used for the evaluation of comets in the present study. The quantitative comet parameters such as tail length, tail moment and tail DNA% were available immediately after 3 s. 3. Statistical analysis
Fig. 3. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for lemon.
Data were summarized with descriptive statistics for means, and the standard errors of the means were analyzed with analysis of variance (ANOVA), using the Least Square Method of the GLM procedureof the SAS (SAS Statistical Software, 2009). The differences between the mean values were analyzed and all results were summarized as mean ± standard error. Ordinary linear regression and Pearson correlation analyses were performed with the use of variables, mainly tail DNA%, tail length%, and tail moment% of comets observed from grapefruit, lemon, mandarin and orange seeds at the irradiation doses between 0.1–1.5 kGy. 4. Results Photographs of DNA comets after microgel electrophoresis of cells from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses are shown in Fig. 1. The mean values of tail DNA%, tail length%, and tail moment of comets observed from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses are presented in Table 1. Tail length% and tail moment which are estimated by image analysis of observed comets versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for grapefruit, lemon, mandarin and orange are presented in Figs. 2–5 respectively. 5. Discussion Control seed samples of grapefruit, lemon, mandarin and orange showed no comets because there was no DNA damage at 0.0 kGy as seen in Fig. 1. However irradiated citrus fruits seed samples at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy showed comets representing damaged appearance with increased doses (Fig. 1). Moreover, the tail length increased depending on the irradiation dose. The tail
Fig. 4. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for mandarin.
end was wider and thicker than the head of comet at 1.5 kGy dose (Fig 1). Quantification of comets was carried out by image analysis to evaluate the head and tail of comet intensity related with applied doses. Evaluation of DNA comets by image analysis to control the freshness of quail meat has previously reported by Erel, Yazici, Ozvatan, Ercin, and Cetinkaya (2009). Tail length changed linearly with increasing applied doses in seed samples of grapefruit (R2 = 0.97), lemon (R2 = 0.88), mandarin (R2 = 0.89) and orange (R2 = 0.87) as seen in Figs 2–5. Tail moment also changed linearly with increasing applied doses in seed samples of grapefruit (R2 = 0.94), lemon (R2 = 0.86), mandarin (R2 = 0.92) and orange (R2 = 0.97) as seen in Figs 2–5. The percentage calculation of tail moment covers both of tail DNA% and tail length% (Table 1). The mean tail length and moment% levels of irradiated samples of citrus fruits at all doses are significantly different (p < 0.01) from control even for the lowest dose at 0, 1 kGy (Table 1). With
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minimum absorbed dose would be 0.058 or 0.069 kGy. When irradiation applied on a commercial scale, many irradiated fruits in each load can be expected to receive three times the minimum required dose for treatment efficacy (Hallman & Martinez, 2001). However there is no published report available to quantify as minimum as 0.058 absorbed irradiation dose. Present study indicates that quantification of applied irradiation doses for citrus fruits is possible by DNA comet analysis when combined with image analysis and it may be used as quarantine control method for Mediterranean fruit fly. 6. Conclusion
Fig. 5. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for orange.
Table 1 Mean values of tail DNA%, tail length%, and tail moment of comets observed from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses. Irradiation dose1 (kGy)
Tail DNA%a Mean ± SE
Tail lengthb Mean ± SE
Tail momentc Mean ± SE
Grapefruit 0.0 0.1 0.3 0.5 1.0 1.5
46.75 ± 5.30 48.39 ± 4.21 72.33 ± 5.71 80.84 ± 3.92 85.05 ± 4.23 86.69 ± 5.10
22.00 ± 4.75 48.73 ± 5.01 90.36 ± 6.18 130.96 ± 7.12 138.07 ± 5.85 168.53 ± 4.98
10.29 ± 3.01 23.58 ± 4.03 65.35 ± 4.03 105.87 ± 6.03 117.43 ± 5.78 146.09 ± 4.39
Lemon 0.0 0.1 0.3 0.5 1.0 1.5
55.53 ± 4.06 59.38 ± 2.97 74.20 ± 3.92 78.39 ± 5.01 86.04 ± 5.23 81.66 ± 4.05
25.38 ± 3.56 35.53 ± 3.89 108.63 ± 4.87 139.09 ± 3.56 219.29 ± 5.67 310.66 ± 5.43
14.09 ± 3.06 21.10 ± 2.90 80.61 ± 5.07 109.03 ± 4.81 188.68 ± 5.35 253.68 ± 4.03
Mandarin 0.0 0.1 0.3 0.5 1.0 1.5
34.17 ± 4.34 66.58 ± 3.74 72.47 ± 5.01 66.34 ± 3.84 72.40 ± 4.53 79.41 ± 2.98
17.56 ± 4.32 77.16 ± 3.43 119.80 ± 5.21 120.81 ± 6.12 145.18 ± 5.51 169.54 ± 5.28
6.00 ± 1.04 51.37 ± 3.96 86.81 ± 6.07 80.14 ± 4.90 105.11 ± 5.38 134.64 ± 6.05
Orange 0.0 0.1 0.3 0.5 1.0 1.5
77.52 ± 4.87 68.68 ± 5.12 81.83 ± 4.23 80.65 ± 3.96 86.26 ± 3.88 89.73 ± 4.33
52.79 ± 5.12 84.26 ± 5.01 111.68 ± 3.98 148.22 ± 4.13 150.25 ± 6.01 185.79 ± 5.95
40.92 ± 3.50 57.88 ± 3.92 90.83 ± 4.07 119.54 ± 4.19 129.61 ± 6.09 166.70 ± 5.05
a
Tail DNA% = percent of DNA in the comet tail. Tail length = length of the comet tail measured from right border of head area to end of tail (micron = pixels). c Tail moment = tail DNA% tail length ((DNA% in the tail) (tail length)). 1 Differences are significant between the mean values of tail length% and tail moment% at 0.1–1.5 kGy applied doses in columns (p < 0.01). b
increasing radiation dose more DNA fragmentation occurs, and these fragments migrate further during the electrophoresis. Thus, irradiated cells will show an increased extension of the DNA from the nucleus towards the anode, whereas unirradiated cells will appear nearly circular or with only slight tails (Delincee, 2002; Marín-Huachaca, Delincée, Mancini-Filho, & Villavicencio, 2005). Analytical detection of irradiated food is an important means to implement such control, once the food items have left the irradiation facility (Khan et al., 2003). Follett suggests that 150 Gy is an effective dose to control Mediterranean fruit fly (Follett, 2004). Depending on the level of quarantine security required, the
The image analysis results of citrus fruits comets indicate that DNA Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis. Acknowledgement The authors gratefully thank to the Turkish Atomic Energy Authority (TAEK) for the financial support and also to Nizamettin Yazici for his help on image analysis of comets. References Arvanitoyannis, I. S., Stratakos, A. Ch., & Tsarouhas, P. (2009). Irradiation applications in vegetables and fruits: a review. Critical Reviews in Food Science and Nutrition, 49(5), 427–462. Bustos-Griffin, E., Hallman, G. J., & Griffin, R. L. (2012). Current and potential trade in horticultural products irradiated for phytosanitary purposes. Radiation Physics and Chemistry, 81, 1203–1207. CEN. (2001). EN 13784 Foodstuffs – DNA Comet Assay fort he detection of irradiated foodstuffs - screening method. Brussles: European Committee for Standardization. Delincée, H. (1998). Detection of food treated with ionizing radiation. Trends in Food Science and Technology, 9, 73–82. Delincee, H. (2002). Rapid detection of irradiated frozen hamburgers. Radiation Physics and Chemistry, 63, 443–446. Delincee, H., & Soika, C. (2002). Improvement of the ESR detection of irradiated food containing cellulose employing a simple extraction method. Radiation Physics and Chemistry, 63, 437–441. Erel, Y., Yazici, N., Ozvatan, S., Ercin, D., & Cetinkaya, N. (2009). Detection of irradiated quail meat by using DNA Comet Assay and evaluation of comets by image analysis. Radiation Physics and Chemistry, 78, 776–781. Follett, P. A. (2004). Irradiation to control insects in fruits and vegetables for export from Hawaii. Radiation Physics and Chemistry, 71, 161–164. Follett, P. A., & Weinert, E. D. (2012). Phytosanitary irradiation of fresh tropical commodities in Hawaii: Generic treatments, commercial adoption, and current issues. Radiation Physics and Chemistry, 81, 1064–1067. Hallman, G. J. (1999). Ionizing radiation quarantine treatment against tephritid fruit flies. Postharvest Biology and Technology, 16, 93–106. Hallman, G. J. (2011). Phytosanitary applications of irradiation. Comprehensive. Reviews of Food Science and Food Safety, 10, 143–151. Hallman, G. J. (2012). Generic phytosanitary irradiation treatments. Radiation Physics and Chemistry, 81, 861–866. Hallman, G. J., & Martinez, L. R. (2001). Ionizing irradiation quarantine treatment against Mexican fruit fly (Diptera: Tephritidae) in citrus fruits. Postharvest Biology and Technology, 23(19), 71–77. ICGFI. (1994). Irradiation as a Quarantine Treatment of Fresh Fruits and Vegetables. A report of the Working Group Convened by ICGFI, U.S. Department of Agriculture, Washington, D.C., March 22 to 25. International Plant Protection Convention (IPPC). (2009). International Standards for Phytosanitary Measures (ISPM) No.28, Phytosanitary treatments for regulated pests FAO, Rome. Khan, A. A., Khan, H. M., & Delincée, H. (2003). DNA Comet Assay – a validity assessment for the identification of radiation treatment of meats and seafood. European Food Research and Technology, 216, 88–92. Marín-Huachaca, N., Delincée, H., Mancini-Filho, J., & Villavicencio, A. L. C. H. (2005). Use of the DNA Comet Assay to detect beef meat treated by ionizing radiation. Meat Science, 71, 446–450. Marcotte, M. (1998). Irradiation as a disinfestation method – update on methyl bromide phase out regulatory action and emerging opportunities. Radiation Physics and Chemistry, 52, 85–90. SAS Statistical Software. (2009). SAS Compus drive, Carry, NC 27513, USA.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis Nurcan Cetinkaya a,⇑, Demet Ercin b, Sümer Özvatan b, Yakup Erel b a b
Ondokuz Mayis University, Faculty of Veterinary Medicine, 55139 Kurupelit, Samsun, Turkey Turkish Atomic Energy Authority, Sarayköy Nuclear Research and Training Center, Saray Mah., Atom Cad., No: 27, 06983 Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 7 April 2014 Received in revised form 16 December 2014 Accepted 7 July 2015 Available online 8 July 2015 Keywords: Irradiation DNA Comet Assay Quarantine control method Citrus fruits Image analysis
a b s t r a c t The experiments were conducted for quantification of applied dose for quarantine control in irradiated citrus fruits. Citrus fruits exposed to doses of 0.1 to 1.5 kGy and analyzed by DNA Comet Assay. Observed comets were evaluated by image analysis. The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Irradiated citrus fruits showed the separated tails from the head of the comet by increasing applied doses from 0.1 to 1.5 kGy. The mean tail length and mean tail moment% levels of irradiated citrus fruits at all doses are significantly different (p < 0.01) from control even for the lowest dose at 0.1 kGy. Thus, DNA Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Irradiation has been considered by ICGFI. (1994), Marcotte (1998) and Hallman (1999) and now a days, it has been used as an alternative to phytosanitary and quarantine treatments for international trade of fresh fruits and vegetables (Bustos-Griffin, Hallman, & Griffin, 2012; Hallman, 2011; Hallman, 2012) so it can be replaced with chemical fumigants such as methyl bromide that is going to be phased out by the year 2015 in developing countries. In 2009, International Plant Protection Commission (IPPC) approved the generic radiation dose of 150 Gy for tephritid fruit flies (IPPC, 2009). Irradiation proved to be extremely beneficial in terms of prolonging the fruit and vegetable shelf life by 3–5 times (Arvanitoyannis, Stratakos, & Tsarouhas, 2009). Recently, irradiation has been used as generic phytosanitary treatment for exported fresh fruits in Asian countries such as India, Pakistan, Thailand and Vietnam (Follett & Weinert, 2012). Ionizing radiation damages the DNA of insects and pathogenic microorganisms in foods (Delincee & Soika, 2002). The DNA Comet Assay is one of ten approved standard detection methods in many food items such as meat, fish, grains, and fruits (CEN. EN 13784, 2001) and has also been utilized to identify irradiated grapefruits (Delincée, 1998) but not for quantification of applied doses. Analytical detection of radiation treatment of food is an ⇑ Corresponding author. E-mail address: [email protected] (N. Cetinkaya). http://dx.doi.org/10.1016/j.foodchem.2015.07.027 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
important means to implement such control, once the food items have left the irradiation facility (Khan, Khan, & Delincée, 2003). Quarantine inspectors only control the irradiation certificate travel with irradiated produces. That’s why the irradiation facilities are specifically designed and established for low dose quarantine method application to fresh fruits. However, multipurpose irradiation facilities should also be used for commercial fresh fruit irradiation. Beside of irradiation certificate, there is a need for a justification method to control of generic low dose irradiated fresh fruits to be certain about good radiation processing not changing of sensory properties of fruits. In present study, DNA Comet Assay was used together with image analysis for quantification of applied low dose to grapefruit, lemon, mandarin and orange in order to propose of DNA Comet Assay as potential quarantine control method for inspectors.
2. Materıals and methods 2.1. Irradiation of samples Seed samples for each dose from grapefruit, lemon, mandarin and orange were exposed at different dose levels of 0.1, 0.3, 0.5, 1.0 and 1.5 kGy in gamma cell (Gammacell Co60, dose rate 1.31 kGy/h) at Turkish Atomic Energy Authority, Saraykoy Nuclear Research and Training Center. Harwell Gammachrome YR Batch 62 dosimeters were used for the measurement of radiation dose.
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N. Cetinkaya et al. / Food Chemistry 192 (2016) 370–373
Dose kGy
Grapefruit
Lemon
Mandarin
Orange
0.0
0.1
0.3
0.5
1.0
1.5
Fig. 1. DNA Comets after microgel electrophoresis of cells from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses.
2.2. DNA Comet Assay Standard DNA Comet Assay was used in this study (CEN, 2001. EN 13784). Seed samples were crushed and mixed with 5 mL of cold phosphate buffered saline (PBS, pH 7.4), which was stirred for 5 min at about 500 rpm, filtered through 200 lm and 100 lm nylon sieve clothes, and then let for sedimentation for about 30 min 100 lL of cell suspension was mixed with 1000 lL of 0.8% low melting point agarose (LMA) and 100lL of this mixture was spread onto the pre-coated slide glass using a cover glass and left cooling for 45 min on ice bucket. The casted slides were immersed in lysis buffer (2.5% SDS in 45 mM Tris–borate, 1 mM EDTA, pH 8.4)
for 20 min then slides were kept 5 min in electrophoresis buffer solution. The electrophoresis was performed in TBE buffer (Tris borate electrophoresis buffer, pH 8.4) at 2 V/cm for 2 min The slides were put into distilled water for 5 min and dried at 45 °C approximately 1 h. Propidium iodide staining was employed to visualize DNA. 2.3. Evaluation of comets by image analysis Observed comets from DNA Comet Assay were evaluated by image analysis for linking the comet parameters to estimate applied doses.
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The slides were evaluated with a standard transmission microscope (Olympus BX 51 model) at 20X and evaluated by digital color video camera (Pixera) with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Comet measurements and estimated parameters are defined as following: 1. Tail Area: Area of the comet tail in pixels (sum of pixels in the head). 2. Head DNA: Amount of DNA in the comet head (sum of intensities of pixels in the head). 3. Tail DNA: Amount of DNA in the comet tail (sum of intensities of pixels in the tail). 4. Tail DNA%: Percent of DNA in the comet tail. 5. Tail Length: Length of the comet tail measured from right border of head area to end of tail (in pixels). 6. Comet Length: Length of the entire comet from left border of head area to end of tail (in pixels). 7. Tail Moment: Tail DNA% x TailLength ([percent of DNA in the tail] x [tail length]).
Fig. 2. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for grapefruit.
There are semi (New Manual) and fully automatic (AutoComet) comet measurements of BAB Imaging System. The comet was put in frame and comet head was identified with mouse in the semiautomatic measurement which was used for the evaluation of comets in the present study. The quantitative comet parameters such as tail length, tail moment and tail DNA% were available immediately after 3 s. 3. Statistical analysis
Fig. 3. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for lemon.
Data were summarized with descriptive statistics for means, and the standard errors of the means were analyzed with analysis of variance (ANOVA), using the Least Square Method of the GLM procedureof the SAS (SAS Statistical Software, 2009). The differences between the mean values were analyzed and all results were summarized as mean ± standard error. Ordinary linear regression and Pearson correlation analyses were performed with the use of variables, mainly tail DNA%, tail length%, and tail moment% of comets observed from grapefruit, lemon, mandarin and orange seeds at the irradiation doses between 0.1–1.5 kGy. 4. Results Photographs of DNA comets after microgel electrophoresis of cells from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses are shown in Fig. 1. The mean values of tail DNA%, tail length%, and tail moment of comets observed from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses are presented in Table 1. Tail length% and tail moment which are estimated by image analysis of observed comets versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for grapefruit, lemon, mandarin and orange are presented in Figs. 2–5 respectively. 5. Discussion Control seed samples of grapefruit, lemon, mandarin and orange showed no comets because there was no DNA damage at 0.0 kGy as seen in Fig. 1. However irradiated citrus fruits seed samples at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy showed comets representing damaged appearance with increased doses (Fig. 1). Moreover, the tail length increased depending on the irradiation dose. The tail
Fig. 4. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for mandarin.
end was wider and thicker than the head of comet at 1.5 kGy dose (Fig 1). Quantification of comets was carried out by image analysis to evaluate the head and tail of comet intensity related with applied doses. Evaluation of DNA comets by image analysis to control the freshness of quail meat has previously reported by Erel, Yazici, Ozvatan, Ercin, and Cetinkaya (2009). Tail length changed linearly with increasing applied doses in seed samples of grapefruit (R2 = 0.97), lemon (R2 = 0.88), mandarin (R2 = 0.89) and orange (R2 = 0.87) as seen in Figs 2–5. Tail moment also changed linearly with increasing applied doses in seed samples of grapefruit (R2 = 0.94), lemon (R2 = 0.86), mandarin (R2 = 0.92) and orange (R2 = 0.97) as seen in Figs 2–5. The percentage calculation of tail moment covers both of tail DNA% and tail length% (Table 1). The mean tail length and moment% levels of irradiated samples of citrus fruits at all doses are significantly different (p < 0.01) from control even for the lowest dose at 0, 1 kGy (Table 1). With
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minimum absorbed dose would be 0.058 or 0.069 kGy. When irradiation applied on a commercial scale, many irradiated fruits in each load can be expected to receive three times the minimum required dose for treatment efficacy (Hallman & Martinez, 2001). However there is no published report available to quantify as minimum as 0.058 absorbed irradiation dose. Present study indicates that quantification of applied irradiation doses for citrus fruits is possible by DNA comet analysis when combined with image analysis and it may be used as quarantine control method for Mediterranean fruit fly. 6. Conclusion
Fig. 5. Tail length and tail moment versus irradiation doses at 0.1, 0.3, 0.5, 1.0 and 1.5 kGy for orange.
Table 1 Mean values of tail DNA%, tail length%, and tail moment of comets observed from grapefruit, lemon, mandarin and orange seeds irradiated at 0, 0.1, 0.3, 0.5, 1.0 and 1.5 kGy doses. Irradiation dose1 (kGy)
Tail DNA%a Mean ± SE
Tail lengthb Mean ± SE
Tail momentc Mean ± SE
Grapefruit 0.0 0.1 0.3 0.5 1.0 1.5
46.75 ± 5.30 48.39 ± 4.21 72.33 ± 5.71 80.84 ± 3.92 85.05 ± 4.23 86.69 ± 5.10
22.00 ± 4.75 48.73 ± 5.01 90.36 ± 6.18 130.96 ± 7.12 138.07 ± 5.85 168.53 ± 4.98
10.29 ± 3.01 23.58 ± 4.03 65.35 ± 4.03 105.87 ± 6.03 117.43 ± 5.78 146.09 ± 4.39
Lemon 0.0 0.1 0.3 0.5 1.0 1.5
55.53 ± 4.06 59.38 ± 2.97 74.20 ± 3.92 78.39 ± 5.01 86.04 ± 5.23 81.66 ± 4.05
25.38 ± 3.56 35.53 ± 3.89 108.63 ± 4.87 139.09 ± 3.56 219.29 ± 5.67 310.66 ± 5.43
14.09 ± 3.06 21.10 ± 2.90 80.61 ± 5.07 109.03 ± 4.81 188.68 ± 5.35 253.68 ± 4.03
Mandarin 0.0 0.1 0.3 0.5 1.0 1.5
34.17 ± 4.34 66.58 ± 3.74 72.47 ± 5.01 66.34 ± 3.84 72.40 ± 4.53 79.41 ± 2.98
17.56 ± 4.32 77.16 ± 3.43 119.80 ± 5.21 120.81 ± 6.12 145.18 ± 5.51 169.54 ± 5.28
6.00 ± 1.04 51.37 ± 3.96 86.81 ± 6.07 80.14 ± 4.90 105.11 ± 5.38 134.64 ± 6.05
Orange 0.0 0.1 0.3 0.5 1.0 1.5
77.52 ± 4.87 68.68 ± 5.12 81.83 ± 4.23 80.65 ± 3.96 86.26 ± 3.88 89.73 ± 4.33
52.79 ± 5.12 84.26 ± 5.01 111.68 ± 3.98 148.22 ± 4.13 150.25 ± 6.01 185.79 ± 5.95
40.92 ± 3.50 57.88 ± 3.92 90.83 ± 4.07 119.54 ± 4.19 129.61 ± 6.09 166.70 ± 5.05
a
Tail DNA% = percent of DNA in the comet tail. Tail length = length of the comet tail measured from right border of head area to end of tail (micron = pixels). c Tail moment = tail DNA% tail length ((DNA% in the tail) (tail length)). 1 Differences are significant between the mean values of tail length% and tail moment% at 0.1–1.5 kGy applied doses in columns (p < 0.01). b
increasing radiation dose more DNA fragmentation occurs, and these fragments migrate further during the electrophoresis. Thus, irradiated cells will show an increased extension of the DNA from the nucleus towards the anode, whereas unirradiated cells will appear nearly circular or with only slight tails (Delincee, 2002; Marín-Huachaca, Delincée, Mancini-Filho, & Villavicencio, 2005). Analytical detection of irradiated food is an important means to implement such control, once the food items have left the irradiation facility (Khan et al., 2003). Follett suggests that 150 Gy is an effective dose to control Mediterranean fruit fly (Follett, 2004). Depending on the level of quarantine security required, the
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