Rapid and Reliable Method for Determining Irradiation Histories of

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Rapid and Reliable Method for Determining Irradiation Histories of Ground Beef and Prawns by Measuring 5,6-Dihydrothymidine Naoki Fukui,†,‡ Satoshi Takatori,*,† Yoko Kitagawa,† Takuya Fujiwara,† Etsuko Ishikawa,‡ Takatomo Fujiyama,‡ Keiji Kajimura,† Masakazu Furuta,‡ and Hirotaka Obana†,§ †

Division of Hygienic Chemistry, Osaka Institute of Public Health, Nakamichi 1-3-69, Higashinari-ku, Osaka, Japan Laboratory of Quantum-Beam Chemistry and Biology, Radiation Research Center, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka, Japan

‡

ABSTRACT: A rapid and reliable method for determining irradiation histories of ground beef and prawns was developed on the basis of a method for determining the irradiation history of beef liver by liquid chromatography−tandem mass spectrometry (LCMS/MS) of 5,6-dihydrothymidine (DHdThd). Improvements in the method included the following: (1) 50% ethanol precipitation in the DNA extraction step was conducted before the RNase step, (2) snake venom phosphodiesterase I was used for DNA digestion to boost liberation of DHdThd, and (3) a matrix-matched calibration curve was used for determining DHdThd by LC-MS/MS analysis. This method successfully determined irradiation histories of ground beef and prawns. Furthermore, a close correlation between the formation of DHdThd and 2-alkylcyclobutanones, which are an established index of irradiation histories, was observed in ground beef. DHdThd in DNA could be a promising candidate for a new index of irradiation histories of various foods. KEYWORDS: food irradiation, DNA, 5,6-dihydrothymidine, ground beef, prawns, LC-MS/MS

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INTRODUCTION Irradiating food is an effective method for controlling foodborne pathogens or killing insects that contaminate food such as spices, frozen seafood, and meat, whose quality can be degraded by heating or fumigation.1−3 Thus, irradiation of certain foods is approved in more than 57 countries.4 Accompanying the approval of irradiation, methods to verify irradiation histories of foods are essential for assessing regulatory compliance and facilitating international trade. A variety of methods for determining irradiation histories of foods have been developed and evaluated.5,6 The European Committee for Standardization established 10 official standard methods for determining the irradiation histories of foods by 2004, and nine methods have already been authorized as general Codex methods. These methods are based on physical, chemical, or biological changes in food components following irradiation. The applicability of these methods depends on the components or contamination of foods. The following are relevant examples. Method EN 17857 would be suitable for fat-rich foods. This method detects radiolytic products of fat, 2-alkylcyclobutanones (ACBs), including 2dodecylcyclobutanone (DCB) and 2-tetradecylcyclobutanone (TCB). Methods EN 17888 and EN 137519 would be applicable to foods with silicate mineral contamination. These methods detect thermoluminescence (TL) and photostimulated luminescence (PSL), respectively. Both TL and PSL are radiospecific phenomena resulting from thermal or photostimulated release of energy that is stored in minerals, by means of irradiation, as detectable luminescence. Thus, these methods are applicable to foods containing minerals, such as herbs, spices, prawns, shellfish, and potatoes. Methods EN 1786,10 EN 1787,11 and EN 1370812 detect electron spin resonance of stable radicals formed in hydroxyapatite in bone, cellulose, or crystalline sugar as a result of © XXXX American Chemical Society

irradiation. They are applicable to foods, such as meat/fish with bones, nuts, and dried fruit, containing components that produce stable radicals. These methods are unsuitable for foods such as boneless, low-fat meat, including liver and lean meat, that contain either insufficient levels or none of the components (i.e., fat, minerals, or bone) requisite for these methods. Physical and chemical changes in DNA caused by irradiation are also useful markers for determining irradiation histories of various foods. Currently, a DNA comet assay and detection of modified DNA bases using an enzyme-linked immunosorbent assay (ELISA) have been established.13,14 The DNA comet assay (EN 13784) for detecting irradiation-induced DNA strand breaks is applicable to various foods from which cells with intact DNA can be isolated.15 To obtain dose−response curves, which are essential for determining irradiation histories of foods, complex image analyses of electrophoretic data are required.16 Because enzymatic degradation of DNA during food storage can lead to ambiguous results,13 EN 13784 is regarded as a screening method. Another official method is required to confirm positive results obtained by this method. In contrast, an ELISA measuring modified DNA bases can be used to produce dose−response curves without complex analysis. Kikuchi et al.17 successfully determined irradiation histories of meat by detecting 8-oxo-2′deoxyguanosine (8-oxo-dGuo) in DNA using chemiluminescence ELISA. Tyreman et al.18 also successfully determined irradiation histories of prawns by detecting 5,6-dihydrothymidine (DHdThd) residues in DNA using ELISA. The compounds 8-oxo-dGuo, 5,6-dihydroxy-5,6-dihydro-2′-deoxythymidine, 5Received: Revised: Accepted: Published: A

July 15, 2017 September 27, 2017 September 27, 2017 September 27, 2017 DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Irradiation Doses and Storage Periods of Ground Beef and P. monodon Prawn Samples Prior to DNA or Fat Extraction ground beef samples Ia

Ib

II

III

irradiation doses (kGy) storage period before DNA extraction (days) storage period before fat extraction (days)

0.47, 0.97, 1.4, 5.2, 6.8, 9.8 3 7 IVa

IVb

V

VI

irradiation doses (kGy) storage period before DNA extraction (days)

0.49, 0.89, 1.1, 3.9, 6.5, 9.4 4

0.49, 0.89, 1.1, 3.9, 6.5, 9.4 102

0.58, 0.98, 1.7, 4.0, 7.9, 10.7 4

0.49, 0.82, 1.6, 3.7, 7.8, 9.6 5

a

0.47, 0.97, 1.4, 5.2, 6.8, 9.8 0.30, 0.69, 1.7, 4.1, 6.4, 9.7 56 151 a 2 P. monodon prawn samples

0.35, 0.79, 1.4, 4.7, 6.7, 8.6 153 4

Not done. and III. These GB samples were from different animals (I, domestic 1; II, imported from Australia; III, domestic 2). Each GB sample was formed into sticks and wrapped with a poly(vinylidene chloride) film designed for food. The size of each stick was approximately 12 × 100 mm (diameter × length) with a weight of ∼10 g. The four sticks were placed in a polyethylene bag (Ziploc, Asahi Kasei Home Products Co., Tokyo, Japan) and were stored at −20 °C prior to γ-ray irradiation. Penaeus monodon (PM) is one of the most popular prawns in Japan. The raw PM samples (imported from India) were obtained from a retail market in Osaka on three different days and were provided decapitated and with shells. The PM samples were designated as PM IV, V, and VI. The weight of each individual PM was around 10−12 g. Each PM sample consisted of two individuals placed lengthwise in a 50 mL polypropylene (PP) tube (Thermo Fisher Scientific, Inc., MA) and was stored at −20 °C prior to γ-ray irradiation. Chemicals. The chemicals used for examining DHdThd were as described previously except for the following two enzymes for DNA digestion. Alkaline phosphatase (ALP) and snake venom phosphodiesterase I (SVPD) were obtained from Roche Diagnostics GmbH (Mannheim, Germany) and Sigma−Aldrich Co. (St. Louis, MO), respectively. The standard for DHdThd used in this study comprised 15% 5R isomer [(5R)-DHdThd] and 85% 5S isomer [(5S)-DHdThd]. Total DHdThd is expressed as the sum of these isomers. In this study, the ACBs are defined as the sum of DCB and TCB. DCB, TCB, 2cyclohexylcyclohexanone, which was used as an internal standard (IS), and other chemicals used for the determination of ACBs were obtained as described before.30 γ-Irradiation and Storage of Samples. γ-Irradiation of samples was conducted at the radioisotope centers of Osaka Prefecture University using 60Co (2.9 kGy/h). GB and PM samples were irradiated under frozen and aerobic conditions. Planned irradiation doses were 0.5 1, 2, 5, 8, and 11 kGy. The sample temperature during irradiation was maintained by use of a sodium chloride−ice mixture as a refrigerant (−20 and −18 °C at the initial and end stages of 11 kGy irradiation, respectively). The number of irradiation cycles at each dose for GB (I− III) and PM (IV−VI) samples was one. The actual irradiation doses were determined by use of radiochromic film dosimetry patches on the surfaces of the plastic bags or tubes. After irradiation, the samples were stored at −20 °C until analysis. The storage periods of GB and PM samples from irradiation until analysis are shown in Table 1. DNA Extraction and Purification. To extract the DNA from PM samples, the shells were removed and then the bodies were cut into 2−3 mm sized pieces. Aliquots (6.0 g) of the prepared GB or PM sample were put into a high-durability 50 mL PP tube (Hitachi Ko-ki, Tokyo, Japan) with 12 mL of lysis solution, containing water, 0.5 M ethylenediaminetetraacetic acid (EDTA), 1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 8.0), and 10% sodium dodecyl sulfate (SDS) at a ratio of 14/4/1/1 (v/v/v/v), and then the sample was homogenized with a TissueRuptor homogenizer (Qiagen, Venlo, Netherland). After addition of 80 μL of proteinase K solution (20 mg/mL), samples were incubated at 55 °C overnight for 16 h. Subsequently, equal volumes of Tris/EDTA (TE)-saturated phenol were added and the solution was shaken vigorously for 10 min. After centrifugation (13000g, 10 min, 4 °C), supernatants were transferred to fresh 50 mL PP tubes and were mixed with equal volumes of chloroform

formyl-2′-deoxyuridine, and 5-hydroxymethyl-2′-deoxyuridine can be formed by oxidative radical reactions in the absence of irradiation.19 Thus, 8-oxo-dGuo could be an artifact formed during the process of DNA extraction and subsequent procedures.20 Unlike these modified nucleosides, DHdThd is a radiospecific product of 2′-deoxythymidine (dThd).21,22 From this perspective, DHdThd could be used as a potential marker of irradiation histories of foods; however, a high-quality antibody for the detection of DHdThd should be prepared to perform ELISAs in laboratories.23 Currently, a liquid chromatograph equipped with a tandem mass spectrometer (LC-MS/MS) is the most powerful tool for analyzing modified nucleosides formed by radical reactions.24 Apart from ELISA methods, we have developed a method for determining irradiation histories of beef liver, which involves detection of DHdThd with LC-MS/ MS.25 This method involves three steps: (1) DNA extraction from γ-irradiated samples, (2) digestion of DNA to nucleosides, and (3) determination of the ratio of DHdThd to dThd in the test solution by LC-MS/MS. The method could successfully determine irradiation histories of beef liver, although the following three issues need to be resolved: (1) the DNA extraction step can be laborious, (2) undigested DNA fragments containing DHdThd may persist after the DNA digestion step, and (3) ion suppression of DHdThd in LC-MS/MS analysis can occur. In the present study, we developed a rapid and reliable method by resolving these issues and examined its applicability in detecting irradiation histories of ground beef (GB) and prawns. The reasons for selecting GB and prawns are as follows: (1) they are the major livestock and seafood products irradiated for pathogen control;26 (2) appropriate examination methods for determining their irradiation history are different (i.e., EN 1785 would be applicable for GB with sufficient fat content, and EN 1788 would be applicable for prawns that contained silicate mineral contaminants). In the United States, irradiation of GB using up to 7.0 kGy is approved.27 In the European Union, Belgium, Czech Republic, and France approved an irradiation limit of up to 5 kGy for frozen shrimp (peeled or decapitated);28 in fact, frozen peeled or decapitated shrimp were irradiated at a dose of 4.0−4.3 kGy in Belgium in 2015.29 Furthermore, we validated the method by comparing the amounts of DHdThd/ dThd determined by this method and the concentrations of ACBs in fat determined by other methods that we have formerly developed30 for the same irradiated GB samples.

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MATERIALS AND METHODS

Ground Beef and Prawn Samples. Raw GB (mince made from beef without additives) was obtained from three different retail markets in Osaka on different days. These samples were designated as GB I, II, B

DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry and shaken for 10 min. After centrifugation (13000g, 10 min, 4 °C), supernatants were collected in fresh 50 mL PP tubes. To the supernatants, one-ninth volume of 5 M NaCl was added with 2-fold volume of cold ethanol; the samples were mixed gently and then allowed to stand at −80 °C for 20 min to precipitate crude DNA. To further purify DNA, additional precipitation steps were performed with 50% ethanol as follows. The crude DNA was dissolved in 3 mL of water and transferred to a fresh 15 mL PP tube, and one-ninth volume of 5 M NaCl was added with an equal volume of cold ethanol; the samples were gently mixed and then allowed to stand at −80 °C for 10 min. The precipitated DNA was collected by centrifugation (12000g, 10 min, 4 °C). The collected DNA was washed with 70% ethanol, lyophilized, and dissolved in 2 mL of TE buffer. The DNA was incubated with 20 μg/mL RNase A at 37 °C for 1 h. Equal volumes of TE-saturated phenol/ chloroform/isoamyl alcohol solution were then added, and the samples were shaken for 10 min. After centrifugation (12000g, 10 min, 4 °C), supernatants were transferred to fresh 15 mL PP tubes. After removal of phenol and chloroform from the supernatant, the DNA was precipitated by mixing with a 2-fold volume of ethanol as described above. After being washed with 70% ethanol and lyophilized, DNA was dissolved in 0.25 mL of water, and the UV absorbance was measured at 230, 260, 280, and 340 nm. DNA Digestion and Preparation of Test Solution. DNA digestion to nucleosides and subsequent test solution preparation was conducted as reported by Madugundu et al.31 After denaturation of 40 μL of DNA (5 mg/mL) in a 1.5 mL PP tube, 5.0 μL of sodium acetate buffer (0.5 M, pH 5.5), 5.0 μL of nuclease P1 (NP1; 5 units), and 5.0 μL of water were added to the tube, and this was incubated for 1 h at 37 °C. Then 10 μL of Tris-HCl (0.5 M, pH 8.0), 5.0 μL of ALP (1 unit/μL), and 5.0 μL of SVPD (0.001 unit/μL) were added to the tube, which was further incubated at 37 °C for 1 h. After termination of the reaction by addition of 10 μL of phosphoric acid (0.19 M), 40 μL of chloroform was added, and the solution was shaken vigorously for 30 s and then centrifuged (6800g, 10 min, ambient). Instead of solid-phase extraction (SPE) and concentration of the fraction of total DHdThd, the supernatant was processed by centrifugation through an ultrafiltration membrane (UM; Centricut supermini, Kurabo Industries Ltd., Osaka, Japan) (5000g, 10 min, ambient) to obtain a test solution for LC-MS/ MS analysis. For determination of dThd, the test solution was diluted 1.0 × 104-fold with water. These test solutions were analyzed by LC-MS/ MS. The concentration ratio of total DHdThd to dThd in the test solution was calculated as an indicator of irradiation. Liquid Chromatographic−Tandem Mass Spectrometric Analysis. The LC-MS/MS system consisted of the 4000 QTRAP mass spectrometer (AB Sciex; Framingham, MA) equipped with an electrospray ionization interface and the Nexera ultraperformance liquid chromatography (UPLC) instrument comprising the CBM-20A controller, two LC-30AD UPLC pumps, SIL-30AC autosampler, and CTO-30A column oven (Shimadzu Co., Kyoto, Japan). The analytical column was the UPLC Acquity HSS T3 (2.1 × 100 mm, 1.8-μm particle; Waters) with a guard column (Acquity HSS T3, 2.1 × 5 mm, 1.8-μm particle; Waters). The conditions for LC-MS/MS analysis are shown in Table 2. Recovery Tests of Total 5,6-Dihydrothymidine and 2′Deoxythymidine. Recoveries of total DHdThd and dThd in the DNA digestion process were examined by fortifying total DHdThd and 5-ethyl-2′-deoxyuridine (EtdUrd; a homologue of dThd) in the DNA digestion mixtures of nonirradiated GB or PM samples, respectively. For total DHdThd, 1.0 and 5.0 ng of total DHdThd were added to the DNA digestion mixtures; these corresponded to concentrations of test solutions of 11.8 and 58.8 ng/mL, respectively. For EtdUrd, 1.5 × 104 ng of EtdUrd was added to the DNA digestion mixtures, because dThd was found at approximately 1.8 × 105 ng/mL in the test solutions. In recovery tests, total DHdThd and EtdUrd were determined by use of calibration curves prepared with total DHdThd and EtdUrd standard solutions, respectively, serially diluted with water. Calibration Curves of Total 5,6-Dihydrothymidine and 2′Deoxythymidine. To compensate for the recovery loss derived both from the process of preparing test solutions, including DNA digestion and following purification, and from ion suppression caused by

Table 2. Liquid Chromatography−Tandem Mass Spectrometry Conditions column temperature mobile phase A mobile phase B flow rate injection volume gradient profile ionization mode capillary temperature and voltage analysis mode transition

50 °C 0.5 mM ammonium acetate in aqueous solution 0.5 mM ammonium acetate in methanol solution 0.2 mL/min 5 μL 5% B (hold, 7 min); 70% B (linear, 5 min); 95% B (stepwise and then hold, 4 min); 5% B (stepwise and then hold, 14 min) electrospray ionization, positive mode 500 °C, +5.5 kV multiple reaction monitoring precursor ion (m/z) product iona (m/z)

total DHdThd dThd EtdUrd

245 243 257

117; 99 127; 117 141; 117

CEb (eV) 19; 21 15; 17 15; 17

a

Monitoring and qualifier ions. bCollision energy (CE) for monitoring and qualifier ions.

impurities derived from samples in the test solutions (i.e., matrix) in LC-MS/MS analysis, matrix-matched calibration curves were used. Standard solutions for the matrix-matched calibration curves were prepared by fortification of total DHdThd standard solutions in DNA digestion mixtures of nonirradiated samples and were processed as samples, except for the addition of 5.0 μL of water before the first incubation (Table 3). In brief, 40 μL of DNA solution (5 mg/mL) of the

Table 3. Components of DNA Digestion Mixtures for Test Solution and Matrix-Matched Standard Solution mixing volume (μL) component

test solution

First Incubationa DNA solution (5 mg/mL) 40 sodium acetate buffer (0.5 M, pH 5.0 5.5) nuclease P1 (1 unit/μL) 5.0 water 5.0 standard solution of total DHdThd Second Incubationa Tris-HCl (0.5 M, pH 8.0) 10 ALP (1 unit/μL) 5.0 SVPD (0.001 unit/μL) 5.0 Termination of Reaction phosphoric acid (0.19 M) 10 final volume (μL) 85

matrix-matched standard solution 40 5.0 5.0 5.0 10 5.0 5.0 10 85

a First incubation, processed with nuclease P1 for 1 h at 37 °C; second incubation, processed with alkaline phosphatase (ALP) and snake venom phosphodiesterase I (SVPD) for 1 h at 37 °C.

corresponding nonirradiated sample, 5.0 μL of sodium acetate buffer (0.5 M, pH 5.5), 5.0 μL of nuclease P1 (1 unit/μL), and 5.0 μL of standard solution of total DHdThd (instead of 5.0 μL of water) were mixed, and this was then processed as a sample (known as the first incubation). After the first incubation, 10 μL of Tris-HCl (0.5 M, pH 8.0), 5.0 μL of ALP (1 unit/μL), and 5.0 μL of SVPD (0.001 unit/μL) were added to the mixture, which was then processed as a sample (known as the second incubation). After the second incubation, 10 μL of phosphoric acid (0.19 M) was added to the mixture (final volume 85 μL). The mixture was further processed with chloroform and centrifuged, and the resulting aqueous layer was filtered with UM. C

DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry The resulting filtrate was injected as the matrix-matched standard solution. The matrix-matched calibration curves of total DHdThd for the GB samples comprised seven points (0.588, 1.18, 2.35, 5.88, 11.8, 29.4, and 58.8 ng/mL), and those of PM samples comprised 10 points (0.588, 1.18, 2.35, 5.88, 11.8, 29.4, 58.8, 118, 176, and 235 ng/mL). The calibration curves of dThd for both GB and PM samples comprised six points (2.0, 5.0, 10, 20, 50, and 100 ng/mL). Determination of 2-Alkylcyclobutanones Formed in Irradiated Ground Beef Samples. The procedures used for determination of ACBs in this study were previously reported30 with a minor change; to optimize defatting conditions, the solvent used to dissolve the aliquot of fat (0.2 g) was changed from a combination of 2.5 mL of acetone and 0.5 mL of acetonitrile to 2 mL of acetone and 1 mL of acetonitrile.

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RESULTS AND DISCUSSION

DNA Extraction and Purification. By conducting DNA precipitation with 50% ethanol prior to RNase digestion, a large proportion of the impurities were removed early in the process of DNA extraction. After this improvement, the phenol−chloroform (including phenol−chloroform−isoamyl alcohol) extraction could be reduced from six times to twice. After incubation for proteolysis, the time required to process eight samples was almost halved; this was reduced from approximately 16 to 8 h. The yield and quality of DNA obtained by the improved procedure would be sufficient for further analysis. In the averages of four independent experiments, approximately 2.4 and 3.8 × 10−3 g of DNA were obtained from each 6.0 g of GB and PM samples, respectively. The average OD260/280 and OD260/230 values of DNA extracted from the GB samples were approximately 1.8 and 2.4, respectively. The average OD260/280 and OD260/230 values of DNA extracted from the PM samples were approximately 2.1 and 2.2, respectively. We also tried to apply a DNA extraction method involving a chaotropic ion technique that was applicable to beef liver to GB and PM samples, and we found that the efficiencies of DNA extraction were poor because of interference by fat in GB samples and impurities in PM samples. Further improvements would be required to apply this method to GB and PM samples. Thus, the method of using phenol−chloroform extraction may be the first choice when attempting to extract DNA from animal food products. DNA Digestion and Preparation of Test Solution. DNA digestion step was improved by a procedure using SVPD. The DNA digestion conditions described in the former report were optimized by using a combination of NP1 and ALP.25 The efficiency of DNA digestion to nucleoside monophosphates by NP1 declines at the DHdThd moiety, especially its 5S isomer.32,33 This implies that undigested DNA fragments containing the DHdThd moiety may remain under the previous digestion conditions. To overcome this problem, SVPD was recruited for the DNA digestion step because the digestion efficiency of SVPD is not affected by the DHdThd moiety.32 The effects of SVPD were confirmed by an examination using salmon sperm DNA (SS DNA) irradiated at 6.5 kGy. Figure 1 shows the effects of SVPD on liberation of total DHdThd from undigested DNA. The amounts of total DHdThd and dThd liberated from 200 μg of DNA into the test solution increased for longer incubation times and reached a plateau at 30 min incubation. In the presence of SVPD, the amount of total DHdThd in the test solution at the plateau level was approximately 2.3 times that in the absence of SVPD. Thus, the amount of DNA required to obtain sufficient sensitivity in the LC-MS/MS analysis could be halved. In the presence of SVPD, the amount of dThd reached a plateau within 5 min of incubation; however, the plateau level

Figure 1. Time course of liberation of 5,6-dihydrothymidine (total DHdThd) and 2′-deoxythymidine (dThd) from salmon sperm DNA (SS DNA; 200 μg) irradiated at 6.5 kGy into the test solutions. SS DNA was processed with nuclease P1, followed by incubation with alkaline phosphatase (ALP) and snake venom phosphodiesterase I (SVPD) or with ALP alone. Liberated total DHdThd and dThd were determined by LC-MS/MS by use of standard solutions without addition of matrix. Conditions in the presence of SVPD: (●) total DHdThd; (■) dThd. Conditions in the absence of SVPD: (○) total DHdThd; (□) dThd. Points represent the averages of three independent experiments. Each bar represents the standard deviation (n = 3).

was almost same as that in the absence of SVPD. Thus, the DNA digestion was almost completed even in the absence of SVPD; however, the presence of SVPD was essential for complete liberation of the DHdThd moiety. Accompanying the increase in total DHdThd liberation, a change in the isomer ratio of DHdThd in the test solution was also observed by LC-MS/MS analysis. Under these LC-MS/MS conditions, the peak responses, recovery of DNA digestion step, and ion suppression of (5S)- and (5R)-DHdThd were almost equal (as described below). Thus, the peak area ratio of these isomers reflects that in the test solution. In the absence of SVPD, the ratio (5S)/(5R)-DHdThd was 30:70, whereas this changed to 55:45 in the presence of SVPD (Figure 2). These observations indicate that the (5S)-DHdThd moiety was more abundant than the (5R)-DHdThd moiety in the remaining DNA fragment. This finding is also supported by the report that the (5S)-DHdThd moiety was more resistant to NP1 digestion than the (5R)DHdThd moiety. 32,33 Because the DNA digestion was conducted in the absence of SVPD in our previous report, the ratio (5S)/(5R)-DHdThd might have been underestimated. By changing the purification process of the test solution from SPE to a combination of liquid−liquid extraction with chloroform and UM filtration, the time for preparing the test solutions for eight samples after enzymatic digestion was reduced from approximately 4 to 0.5 h. Thus, the total time required for preparing 8 samples was shortened from approximately 4 days to one overnight incubation and 2 working days. Recovery Tests and Calibration Curves of Total 5,6Dihydrothymidine and 2′-Deoxythymidine. Recovery of total DHdThd in the process of test solution preparation and LC-MS/MS analysis was estimated by fortification of total DHdThd in the DNA digestion mixtures of nonirradiated GB or PM samples. The average recoveries (n = 3) of 1.0 and 5.0 ng of total DHdThd fortified in DNA digestion mixtures of GB samples were 71% [relative standard deviation (RSD) = 5.5%] and 70% (RSD = 4.8%), respectively. The average recoveries (n = D

DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Typical multiple reaction monitoring chromatograms, monitoring (A−C) 5,6-dihydrothymidine (DHdThd; m/z 245 → 117) and (D−F) 2′deoxythymidine (dThd; m/z 243 → 127). The peaks S and R in panel A correspond to (5S)- and (5R)-DHdThd, respectively. The retention times of (5S)-DHdThd, (5R)-DHdThd, and dThd were 4.8, 5.5, and 7.4 min, respectively. For analysis of total DHdThd, test solutions were applied without dilution. For analysis of dThd, test solutions were diluted 1.0 × 104-fold with water. (A, D) Standard solutions of (A) total DHdThd (100 ng/mL) and (D) dThd (50 ng/mL). (B, E) 6.5 kGy-irradiated salmon sperm DNA (SS DNA) processed in the absence of snake venom phosphodiesterase I [SVPD (−)]. (C, F) 6.5 kGy-irradiated SS DNA processed in the presence of snake venom phosphodiesterase I [SVPD (+)].

3) of 1.0 and 5.0 ng of total DHdThd fortified in DNA digestion mixtures of PM samples were 66% (RSD = 0.55%) and 63% (RSD = 4.1%), respectively. The recoveries of (5S)- and (5R)DHdThd would be almost the same, since the (5S)/(5R)DHdThd ratio in test solutions was the same as that in the

standard of total DHdThd. Moreover, ion suppression levels of the test solutions were estimated by fortification of total DHdThd at a concentration of 100 ng/mL in the test solutions of nonirradiated GB and PM samples. The average peak area (n = 3) of total DHdThd in the test solutions of GB and PM samples E

DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 4. Calibration Curve Profiles of Total 5,6-Dihydrothymidine average (RSD, %) matrix matcheda

slope

yesb noc

1.04 × 104 (1.68) 1.47 × 104 (2.84)

yese noc

1.05 × 104 (3.65) 1.68 × 104 (7.56)

intercept Ground Beef Matrix 1.64 × 103 (95.7) 1.71 × 102 (895) P. monodon Prawn Matrix 1.35 × 103 (117) −3.56 × 103 (106)

r2

range (ng/mL)

1.00 1.00

0.588−58.8 0.500−50.0d

1.00 1.00

0.588−235 0.500−200d

a

Yes indicates matrix-matched calibration curves; no denotes calibration curves without the addition of GB or PM matrix. RSD, relative standard deviation; r2, coefficient of determination. bAverages and standard deviations were calculated from matrix-matched calibration curves independently prepared from DNA digestion mixtures of nonirradiated GB Ia, Ib, II, and III samples, respectively (total, n = 4). cDifferences would reflect the change of response on LC-MS/MS analysis during this study. dCalibration curves of total DHdThd without the addition of GB or PM matrix comprised seven points (0.500, 1.00, 2.00, 5.00, 10.0, 20.0, and 50.0 ng/mL) and nine points (0.500, 1.00, 2.00, 5.00, 10.0, 20.0, 50.0, 100, and 200 ng/mL), respectively. eAverages and standard deviations were calculated from matrix-matched calibration curves independently prepared from DNA digestion mixtures of nonirradiated PM IVa, IVb, V, and VI samples, respectively (total, n = 4).

nonirradiated GB and PM samples. No significant recovery loss of EtdUrd was observed in either GB or PM samples. Thus, the recovery loss of dThd throughout the DNA digestion and analysis by LC-MS/MS must be negligible. 5,6-Dihydrothymidine Moiety Formed in Irradiated Ground Beef and P. monodon Prawn Samples. Total DHdThd was radiospecifically and dose-dependently formed in GB and PM samples. The product ion spectra of these samples were identical to those of the standards (data not shown). Typical multiple reaction monitoring chromatograms are shown in Figure 3. In the GB samples, the (5S)/(5R)-DHdThd ratio was consistently approximately 80:20 in the irradiation range 0.30−9.8 kGy. In the PM samples, the (5S)/(5R)-DHdThd ratio was consistently approximately 90:10 in the irradiation range 0.49−10.7 kGy. The ratios found in the GB and PM samples were similar; however, these isomer ratios were different than those quoted in our former report on irradiated beef liver, in which the (5S)/(5R)-DHdThd ratio was approximately 40:60. In the former report, DNA digestion was conducted with a combination of NP1 and ALP, and (5S)-DHdThd remained more abundant than (5R)-DHdThd in undigested DNA fragments, so that the (5S)/(5R)-DHdThd ratio was underestimated. The actual (5S)/(5R)-DHdThd ratios observed in the GB and PM samples (80:20 and 90:10) were different from that of salmon sperm DNA (55:45). The DHdThd moiety is formed via an intermediate, a 5,6-dihydrothymidine-5-yl radical moiety.35,36 The isomer ratio of DHdThd depends on the diversion rate from the intermediate to each isomer in the hydrogen reaction at the 5-position. The hydrogen reaction of the 5,6-dihydrothymidine-5-yl radical moiety at the 5-position may be stereochemically affected by nucleosome structures. In the DNA of intact cells, which is coiled around histones, the diversion rate of (5S)-DHdThd would be higher than that of (5R)-DHdThd. Dose−response curves of total DHdThd/dThd of GB and PM samples are shown in Figure 4. Because (5S)-DHdThd is more abundant than (5R)-DHdThd in both standard and test solutions, the limits of quantitation (LOQ) of total DHdThd in GB and PM matrix-matched standard solutions were defined as the concentrations at which the (5S)-DHdThd peak exhibited signal-to-noise ratio (S/N) > 10 and the (5R)-DHdThd peak exhibited S/N > 3 in this study. The LOQs of total DHdThd in both GB and PM matrix-matched standards were 0.588 ng/mL. Similarly, the limit of detection (LOD) of total DHdThd in GB and PM matrix-matched standard solutions was defined as the

decreased to 73% (RSD = 0.53%) and 76% (RSD = 1.4%), respectively, compared with that of the pure standard solution of 100 ng/mL total DHdThd. Thus, a large part of the recovery loss may be explained by ion suppression. Fortification of isotope-labeled DHdThd in the DNA digestion solution as a surrogate would be the most reliable method for determining recovery loss; however, the isotopelabeled DHdThd should be synthesized from isotope-labeled dThd.34 Instead of using the surrogate, a matrix-matched calibration curve was used for determination of total DHdThd in this study. The matrix-matched calibration curve can be expected to compensate for the loss of total DHdThd in the process of test solution preparation and the loss due to ion suppression, because standard solutions for the matrix-matched calibration curves were prepared by fortification of total DHdThd in the DNA digestion mixtures of nonirradiated samples. Profiles of matrix-matched calibration curves for GB and PM samples are shown in Table 4. These matrix-matched calibration curves were independently prepared from DNA digestion mixtures of nonirradiated samples (GB Ia, Ib, II, and III and PM IVa, IVb, V, and VI); however, the RSD values of the slope for GB and PM samples were 1.68% and 3.65%, respectively. This suggests that the effect of different matrices (in this case, GB and PM) in causing total DHdThd ion suppression would be limited. 2′-Deoxyguanosine (dGuo), a common component of the DNA digestion mixtures, may exert the most potent ion suppression on total DHdThd because the peak of dGuo overlapped with those of total DHdThd in the LCMS/MS analysis (retention time of dGuo, 5.1 min). Thus, the matrix-matched calibration curves for DNA of nonirradiated GB or PM samples could be sufficiently stable for application to examine the irradiation history of other GB or PM samples, respectively. Furthermore, the average percentages (n = 4) of the slopes of matrix-matched calibration curves of GB and PM samples compared to those of matrix-free calibration curves were 71% (RSD = 2.5%) and 63% (RSD = 3.7%), respectively. Because the average percentages of the slopes were similar to the recovery of total DHdThd fortified in the DNA digestion mixtures, the matrix-matched calibration curves could compensate for the recovery loss throughout DNA digestion and analysis by LC-MS/MS. This observation may support the reliability of the matrix-matched calibration curves. The recovery of dThd in the process of test solution preparation and LC-MS/MS analysis was also estimated by fortification of EtdUrd in the DNA digestion mixture of F

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Figure 4. Dose−response curves of the concentration ratio of total 5,6dihydrothymidine to 2′-deoxythymidine (total DHdThd/dThd) in test solution obtained from irradiated ground beef samples (●) GB Ia, (▲) GB Ib, (■) GB II, and (◆) GB III and from P. monodon samples (○) PM IVa, (△) PM IVb, (□) PM V, and (◇) PM VI. Each symbol represents the average of three independent experiments after DNA digestion and LC-MS/MS analysis. Each bar represents the standard deviation (n = 3). (Inset) Magnified graph area for a low irradiation dose (0−1.2 kGy). Total DHdThd/dThd ratio at the lowest irradiation point (0.30 kGy) of GB-II (■) was calculated from the concentration of total DHdThd extrapolated from its matrix-matched calibration curves.

concentration at which the (5S)-DHdThd peak exhibited S/N > 3. The LODs of total DHdThd in both GB and PM matrixmatched standards were 0.235 ng/mL. The critical judgment point of irradiation-positive was set as detection of total DHdThd over the LOQ in the test solution in this study. Thus, the threshold line for the total DHdThd/dThd ratio for GB and PM samples, calculated from the typical concentration of dThd (1.8 × 105 ng/mL) in the test solution, was found to be 0.033 × 10−4. In both GB and PM samples judged irradiationpositive (total DHdThd/dThd ratio > 0.033 × 10−4), no overlap of the total DHdThd/dThd ratio between adjacent dosages was observed. Therefore, by comparing the total DHdThd/dThd ratio, approximately 0.5−1 kGy difference between irradiation doses in the lower range (planned 0.5, 1, and 2 kGy irradiation) and approximately 2−3 kGy difference in the upper range (planned 5, 8, and 11 kGy irradiation) may be distinguishable. The boundary for judging the signal as positive was set as detection of total DHdThd at a concentration between LOD and LOQ (0.235−0.588 ng/mL). In the case where (5S)-DHdThd is less than LOD, the sample should be judged to be negative. In the case when the signal is near the boundary, detection of both (5S)and (5R)-DHdThd peaks at a peak area ratio of approximately 8:2−9:1 may be judged to be positive. In the case of detecting only (5S)-DHdThd, a reanalysis conducted by increasing the DNA concentration in the digestion mixture with careful monitoring of DNA digestion efficiency, or analysis of the sample on another LC-MS/MS system with higher sensitivity, could be helpful in making a correct judgment. Formation of total DHdThd in the GB sample (II) irradiated at 0.30 kGy and the PM samples (IVa and VI) irradiated at 0.49 kGy could be detected (Figure 4, inset). The concentrations of total DHdThd

Figure 3. Typical multiple reaction monitoring chromatograms, monitoring (A−E) 5,6-dihydrothymidine (DHdThd) and (F−J) 2′deoxythymidine (dThd). The peaks S and R in panel A correspond to (5S)- and (5R)-DHdThd, respectively. Transitions and retention times were the same as in Figure 2. For analysis of total DHdThd, test solutions were applied without dilution. For analysis of dThd, test solutions were diluted 1.0 × 104-fold with water. (A, F) Standard solutions of (A) total DHdThd (20 ng/mL) and (F) dThd (20 ng/mL); (B, G) nonirradiated ground beef (GB) Ia; (C, H) GB Ia irradiated at 5.2 kGy; (D, I) nonirradiated P. monodon (PM) IVa; (E, J) PM IVa irradiated at 3.9 kGy. G

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Correlation between Total 5,6-Dihydrothymidine/2′Deoxythymidine and 2-Alkylcyclobutanones in Irradiated Ground Beef. ACBs, the sum of DCB and TCB, were radiospecifically and dose-dependently formed in the GB samples. Typical chromatograms are shown in Figure 5. In the

in these test solutions of GB II, PM IVa, and PM VI were 0.30, 2.9, and 1.5 ng/mL, respectively. The concentration of total DHdThd in GB II was estimated by extrapolation; the peak of (5S)-DHdThd exhibited S/N > 10, but the peak of (5R)DHdThd exhibited S/N < 3; the peak area ratio (5S)/(5R)DHdThd was speculated to be approximately 9:1. Thus, the detection limit (DL) of irradiation of the method would be around 0.3−0.5 kGy. The DL of the method would be comparable to those of both the ELISA method detecting the DHdThd moiety in DNA for irradiated prawns (0.5 kGy)18 and the ACB-detecting methods for irradiated GB (0.7 kGy).37 The actual irradiation dose for pathogen control in meat, including GB and prawns, is at least 1 kGy. Thus, the sensitivity of the method would be satisfactory for determining the irradiation history of meat and prawns irradiated for that purpose. The dose−response curves were concave; initially, the formation rate of total DHdThd was slow and this increased gradually at higher irradiation doses. DHdThd is efficiently produced from dThd under anaerobic conditions.22 The gradual degradation of oxygen in samples, as a result of consumption by other reactions, may promote the formation of DHdThd with higher irradiation doses, as discussed in a former report.25 Dose−response curves of total DHdThd/dThd of the GB samples (GB Ia, II, and III) were similar. These samples were derived from individual animals and the storage periods were also different (Table 1). Furthermore, the dose−response curves of GB Ia and Ib almost overlapped. The storage times of GB Ia and Ib before DNA extraction were 3 and 56 days, respectively (Table 1). Thus, total DHdThd/dThd in the DNA of GB samples would be stable under the storage conditions for at least 53 days. Dose− response curves of total DHdThd/dThd of PM samples (PM IVa, V, and VI) were also similar. These samples were derived from prawns from different arrival days. Dose−response curves of PM IVa and IVb also almost overlapped. The storage times of PM IVa and IVb for DNA extraction were 4 and 102 days, respectively (Table 1). Thus, total DHdThd/dThd in the DNA of PM samples would be stable under the storage conditions for at least 98 days. Dose−response curves of total DHdThd/dThd, reflecting the production efficiency of DHdThd, were similar in the same types of samples as described above. However, dose−response curves may be different in different foods. The slopes of dose−response curves of the PM samples were approximately 3-fold higher than those of the GB samples. This indicates differences in the efficiencies of radical reactions that form the DHdThd moiety in GB and PM samples. One possible difference between GB and PM samples may be the effect of water content on production efficiency of the DHdThd moiety, because the radical species taking part in DHdThd formation, such as hydrogen atoms and anions, originate from the irradiation of H2O. The typical nutritional content of GB (beef, ground, 90% lean and 10% fat, raw) is 69.5% water, 20.0% protein, 10.0% fat, and others;38 that of prawn (shrimp, mixed species, raw) is 83.0% water, 13.6% protein, 1.0% fat, and others.39 The water content in prawn is higher than in GB, and the contents of other components (protein, fat, and others) in prawn are lower than in GB. Water would promote the production of radical species related to formation of the DHdThd moiety and protein and fat may protect DNA from radical reactions. This method would be applicable to a variety of foods from which appropriate DNA could be extracted for analysis. However, the DLs for dried foods, including spices and herbs, may be higher than those for foods with high water contents.

Figure 5. Typical selected ion-monitoring chromatograms (m/z 98) of gas chromatography−mass spectrometry (GC-MS) analysis monitoring 2-dodecylcyclobutanone (DCB) and 2-tetradecylcyclobutanone (TCB) in nonirradiated and 5.2 kGy-irradiated ground beef sample GB Ia. (A) Standard solution (400 ng/mL); (B) nonirradiated GB Ia; (C) 5.2 kGyirradiated GB Ia. The retention times of internal standard (IS: 2cyclohexylcyclohexanone), DCB, and TCB were 8.1, 11.5, and 13.8 min, respectively. The IS concentration in the test solution was 100 ng/mL.

GB Ia sample irradiated at 5.2 kGy, 509 ± 100 ng of DCB and 588 ± 74 ng of TCB were detected in 1.0 g of extracted fat. In the irradiation range 0.3−9.8 kGy, the concentration ratio of DCB to TCB was approximately 1:1. This was similar to that quoted in a former report by Stewart et al.;40 however, the concentrations of DCB and TCB per 1.0 g of fat reported in this study were lower than those reported by Stewart et al. This is probably a result of a lower irradiation temperature used in this study than that used in the study by Stewart et al.40 (irradiated at 4 ± 1 °C). In addition, the difference in the methods of fat extraction may affect the concentrations of ACBs per gram of fat because the components of extracted fat, including the contamination rate of impurities, may vary with different methods of fat extraction. In method EN 1785, a Soxhlet apparatus is adopted for extraction of fat from a sample; however, this process would be time-consuming (the time for reflux with n-hexane is 6 h).7 Thus, various extraction procedures have been studied to promote the efficiency of fat extraction, such as supercritical fluid extraction (SFE) and accelerated solvent extraction methods.37,40 Here, we extracted fat from the GB samples dispersed in diatomaceous earth particles by shaking with n-hexane under ambient conditions, H

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Journal of Agricultural and Food Chemistry whereas Stewart et al.40 adopted SFE with carbon dioxide as the extraction fluid. Total DHdThd/dThd in the irradiated GB samples was proportionally correlated to the concentration of ACBs in fat (Figure 6). Dose−response curves of total DHdThd/dThd were

the method was not applicable to foods with low fat content. Indeed, ACBs can be formed in prawns as a result of irradiation as with GB; however, the amount of ACBs in irradiated prawns would be much lower than that in GB at the same irradiation dose. Chen et al.42 have successfully achieved the determination of ACBs in irradiated prawns by intensive purification and concentration of the test solution for GC-MS analysis; the test solution (0.2 mL) was prepared from 15 g of prawns. A comparison of total DHdThd/dThd and TL response in PM samples is not described in this study because TL measurement is not available at our laboratory. The comparison will be performed, by use of an outsourcing service for TL analysis to validate the proposed method with the TL method, in the near future to promote the reliability of the proposed method. In conclusion, these results suggest that the total DHdThd/ dThd ratio in DNA would be a promising candidate for a new reliable index to measure irradiation histories of various foods. The ACB method depends on fat content; this method would require special procedures to allow it to be applied to low-fat foods such as prawns. The TL method also depends on isolation of minerals from foods, and the results can vary according to differences in the isolated minerals.43 This method is not applicable to GB, even though prawns when the minerals are removed with sand vein from them. The sand vein is removable in the process of prawns. The developed method only depends on isolation of a sufficient amount and quality of DNA and is expected to be applicable to foods with sufficient DNA content. Dose−response curves of GB and PM were different; however, the dose−response curves for different GB or PM samples were similar. This method not only would be useful for determining irradiation histories but also could be used to estimate irradiation doses by referencing dose−response curves for the same types of foods. Furthermore, a close proportional correlation between total DHdThd/dThd and ACBs was observed in the same irradiated GB samples. This demonstrates the potential for crosschecking results between two independent methods, which improves mutual reliability of the methods and validates the data.

Figure 6. Correlation between total DHdThd/dThd and 2alkylcyclobutanones (ACBs; nanograms per gram of fat) observed in irradiated ground beef samples. Here, total DHdThd/dThd corresponds to the concentration ratio of total 5,6-dihydrothymidine to 2′deoxythymidine in the test solution obtained from irradiated GB samples: (●) GB Ia, (■) GB II, and (◆) GB III. Correlation curves obtained by least-squares methods are shown as broken lines. The functions (y, total DHdThd/dThd; x, ACBs ng/g of fat) and correlation coefficient (r) of the line of best fit are as follows: for GB Ia, y = 0.863 × 10−7 x − 0.771 × 10−5, and r2 = 0.978; for GB II, y = 1.11 × 10−7 x − 1.11 × 10−5, and r2 = 0.976; and GB III, y = 2.44 × 10−7 x − 0.725 × 10−5, and r2 = 0.923. Each point represent the average of three independent experiments including DNA digestion and analysis for total DHdThd/ dThd and cleanup and analysis of ACBs. Each bar represents the standard deviation (n = 3). (Inset) Magnified graph area for low ACB concentrations, (0−0.4) × 103 ng/g of fat.

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AUTHOR INFORMATION

Corresponding Author

similar for GB Ia, II, and III; however, the correlation curve of GB III was different from those of GB Ia and II. The formation rate of ACBs in GB III seemed to decrease at high irradiation doses. The fat content of GB samples, estimated from the weight of extracted fat from 5.0 g of GB sample, was as follows: Ia, 9.6%; II, 8.6%; III, 6.7% (w/w). Gadgil et al.41 reported that the formation rates of ACBs in GB containing 15% fat and 25% fat were almost the same in the irradiation range 1.0−4.5 kGy. However, from our observation, the formation rate of ACBs may decline at high irradiation doses in samples with relatively low fat content. On the other hand, total DHdThd/dThd would be more stable with respect to differences between individual animals than ACBs. From the discussion on the difference in total DHdThd/dThd between GB and PM samples, total DHdThd/dThd may vary according to the water content of samples rather than the differences between individual animals, such as fat content, strain, and age. Typical GB would contain sufficient fat for the determination of ACBs. However, the extraction of sufficient fat from liver or lean beef could be laborious. Thus, in the case of examination of various beef products for irradiation history, the method proposed in this study may be more convenient than methods that involved the use of ACBs. A comparison of total DHdThd/dThd and concentration of ACBs in PM samples was not conducted in this study, because

*Phone +81 6972 1321; fax +81 6972 2393; e-mail sts-tkt@iph. osaka.jp. ORCID

Satoshi Takatori: 0000-0001-6258-2593 Present Address

§ (H.O.) San-Ei Gen F.F.I., Inc., 1-1-11 Sanwa-cho, Toyonaka, Osaka, Japan.

Funding

This work was partially supported by JSPS KAKENHI Grants 25460833 and 16H07502. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ACBs, 2-alkylcyclobutanones; ALP, alkaline phosphatase; CEN, European Committee for Standardization; DCB, 2-dodecylcyclobutanone; EtdUrd, 5-ethyl-2′-deoxyuridine; dGuo, 2′-deoxyguanosine; DHdThd, 5,6-dihydrothymidine; dThd, 2′-deoxythymidine; ELISA, enzyme-linked immunosorbent assay; GB, ground beef; LC-MS/MS, liquid chromatography−tandem mass spectrometry; LOD, limit of detection; LOQ, limit of quantitation; NP1, nuclease P1; 8-oxo-dGuo, 8-oxo-2′-deoxyI

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(17) Kikuchi, M.; Gunawardane, C. R.; Alam, M. K.; Dzomir, A. Z. M.; Pitipanaarachchi, R. C.; Funayama, T.; Hamada, N.; Sakashita, T.; Wada, S.; Satoh, K.; Narumi, I.; Kobayashi, Y. Chemiluminescence ELISA for the detection of Oxidative DNA base damage using Anti-8-hydroxy-2deoxyguanosine Antibody: Application to the detection of irradiated foods. Radioisotopes 2007, 56, 509−517. (18) Tyreman, A. L.; Bonwick, G. A.; Smith, C. J.; Coleman, R. C.; Beaumont, P. C.; Williams, J. H. H. Detection of irradiated food by immunoassay − development and optimization of an ELISA for dihydrothymidine in irradiated prawns. Int. J. Food Sci. Technol. 2004, 39, 533−540. (19) Cadet, J.; Davies, K. J. A.; Medeiros, M. H.; Di Mascio, P.; Wagner, J. R. Formation and repair of oxidatively generated damage in cellular DNA. Free Radical Biol. Med. 2017, 107, 13−34. (20) Ravanat, J. L.; Douki, T.; Duez, P.; Gremaud, E.; Herbert, K.; Hofer, T.; Lasserre, L.; Saint-Pierre, C.; Favier, A.; Cadet, J. Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up. Carcinogenesis 2002, 23, 1911− 1918. (21) Sharpatyi, V. A.; Cadet, J.; Teoule, R. Final products obtained from the gamma radiolysis of frozen aqueous solutions of thymidine. Int. J. Radiat. Biol. Relat. Stud. Phys., Chem. Med. 1978, 33, 419−23. (22) Furlong, E. A.; Jorgensen, T. J.; Henner, W. D. Production of dihydrothymidine stereoisomers in DNA by gamma-irradiation. Biochemistry 1986, 25, 4344−9. (23) Hubbard, K.; Ide, H.; Erlanger, B. F.; Wallace, S. S. Characterization of antibodies to dihydrothymine, a radiolysis product of DNA. Biochemistry 1989, 28, 4382−7. (24) Cadet, J.; Douki, T.; Frelon, S.; Sauvaigo, S.; Pouget, J. P.; Ravanat, J. L. Assessment of oxidative base damage to isolated and cellular DNA by HPLC-MS/MS measurement. Free Radical Biol. Med. 2002, 33, 441−9. (25) Fukui, N.; Takatori, S.; Kitagawa, Y.; Okihashi, M.; Ishikawa, E.; Fujiyama, T.; Kajimura, K.; Furuta, M.; Obana, H. Determination of irradiation histories of raw beef livers using liquid chromatographytandem mass spectrometry of 5,6-dihydrothymidine. Food Chem. 2017, 216, 186−93. (26) Pillai, S. D.; Shayanfar, S. Electron Beam Technology and Other Irradiation Technology Applications in the Food Industry. Top Curr. Chem. (J) 2017, 375, No. 6. (27) U.S. Food and Drug Administration. Ionizing radiation for the treatment of food. Code of Federal Regulations, Title 21, Section 179.26, 2016; http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/ CFRSearch.cfm?fr=179.26 (accessed September 21, 2017). (28) European Commission. List of Member States’ authorisations of food and food ingredients which may be treated with ionising radiation. Off. J. Eur. Union C283, Vol. 52, 2009/C 283/02, 2009; http://eur-lex. europa.eu/legal-content/EN/TXT/PDF/?uri= OJ:C:2009:283:FULL&from=EN (accessed September 21, 2017). (29) Report from the Commission to the European Parliament from the Council: On Food and Food Ingredients Treated with Ionizing Radiation for the Year 2015; COM (2016) 738 final; European Commission, Brussels, Belgium, 2016; http://eur-lex.europa.eu/legal-content/EN/TXT/ PDF/?uri=CELEX:52016DC0738&from=EN (accessed September 21, 2017). (30) Kitagawa, Y.; Okihashi, M.; Takatori, S.; Kajimura, K.; Obana, H.; Furuta, M.; Nishiyama, T. A Rapid and Simple Method for the Determination of 2-Alkylcyclobutanones in Irradiated Meat and Processed Foods. Food Analytical Methods 2014, 7, 1066−72. (31) Madugundu, G. S.; Cadet, J.; Wagner, J. R. Hydroxyl-radicalinduced oxidation of 5-methylcytosine in isolated and cellular DNA. Nucleic Acids Res. 2014, 42, 7450−60. (32) Weinfeld, M.; Soderlind, K. J.; Buchko, G. W. Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA. Nucleic Acids Res. 1993, 21, 621−6. (33) Falcone, J. M.; Box, H. C. Selective hydrolysis of damaged DNA by nuclease P1. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1997, 1337, 267−75.

guanosine; PM, Penaeus monodon; PP, polypropylene; PSL, photostimulated luminescence; S/N, signal-to-noise ratio; SPE, solid-phase extraction; SS DNA, salmon sperm DNA; SVPD, snake venom phosphodiesterase I; TCB, 2-tetradecylcyclobutanone; TL, thermoluminescence

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DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.7b03266 J. Agric. Food Chem. XXXX, XXX, XXX−XXX