Biomedical subjects

Masatake Toyoda

Publications and source records attributed to Masatake Toyoda.

32 records · Page 2Linked to original sources

PubMed2002

Levels and tissue-dependent distribution of dioxin in Japanese domestic leafy vegetables--from the 1999 national investigation.

In 1999, Japanese domestic leafy vegetables were successively investigated for levels of dioxins, including 17 dibenzo-p-dioxins/dibenzofurans (PCDD/Fs), four non-ortho co-planar PCBs (co-PCBs) and eight mono-ortho co-PCBs, all of which had been assigned toxic equivalency factors (TEFs) by WHO in 1997. The mean levels of dioxins in the edible portions were 0.07 (0.09) pg TEQ/g in spinach, 0.13 (0.14) pgTEQ/g in garland chrysanthemum, 0.01 (0.04) pg TEQ/g in mitsuba (marsh parsley) and 0.01 (0.03) pg TEQ/g in chingentsuai (Brassica Campestris var. chinesis), when non-detects were set to zero (and set to half the limit of detection). In order to understand the dioxin pollution of leafy vegetables in detail, a further investigation of dioxin levels in the tissues of spinach was conducted. As a result, the dioxin levels in the leaves were found to be higher than those in the stem and red collar, but they were much lower than those found in the primary and secondary roots, which are considerably affected by the soil, which is recognized as a sink of airborne dioxins. The dioxin levels in edible portions (leaves, stem and red collar) were obviously lower than those in non-edible portions (primary and secondary roots). In addition, from the finding that several lower-chlorinated PCDD/Fs and co-PCBs, namely 2,3,7,8-TCDD/F, 1,2,3,7,8-PeCDD, 1,2,3,7,8/2,3,4,7,8-PeCDFs, 1,2,3,7,8,9-HxCDD, 1,2,3,4,7,8-/1,2,3,6,7,8-HxCDFs, 3,3',4,4'-TCB, 2,3,3',4,4'-/2,3',4,4',5-PeCBs, and 2,3',4,4',5,5'-HxCB, were more highly represented in the dioxins in the leaves than in those in the secondary roots, it was suggested that in leafy vegetables the deposition of gaseous, presumably moderately volatile dioxins in leaf wax is another pollution pathway in addition to the adhesion of dioxin-contaminated particles including soil.

Dioxins↗

PubMed2002

Preliminary screening of the inhibitory effect of food extracts on activation of the aryl hydrocarbon receptor induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

A preliminary screening for the inhibitory effects on the activation of the aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by applying AhR-based bioassays for dioxins, the Ah-Immunoassay and CALUX assay, was attempted. Thirty-nine food extracts including vegetables, fruits, herbs, and teas were initially screened in vitro. We first examined the application of both bioassay methods using green tea extracts and (-)-epigallocatechin gallate, reported antagonists of the AhR, since the results could reveal an inhibitory effect versus the control in both assays. Food extracts were then tested. Among the herbs, extracts of sage, among the vegetables, green leafy ones such as spinach, and among the fruit, citrus showed inhibitory effects on AhR activation by TCDD, although some tested samples did not show parallel behavior in both assays. Sage had a remarkable inhibitory effect (79% in the CALUX assay and 83% in the Ah-Immunoassay compared with control) and its effects were dose dependent. The results suggest that these assays might be applicable to the preliminary screening of antagonist activity against the AhR. Moreover, based on these results, the potential benefit of factors that function as dietary ligands of the AhR and are present in several foodstuffs is indicated.

Animals↗

PubMed2002

[Simultaneous determination of dichlorvos and trichlorfon in agricultural products by GC-FPD].

We studied a gas chromatographic method for the determination of dichlorvos (DDVP) and trichlorfon (DEP) in agricultural products. DDVP and DEP were extracted from agricultural products with acetone and re-extracted with ethyl acetate instead of dichloromethane. DDVP and DEP were eluted in one fraction on a silica gel column using n-hexane-acetone (1:1). DEP is a thermally labile compound, so it was derived to a more thermally stable compound by acylation with N-methylbis(trifluoroacetamide) and pyridine in acetone at 60 degrees C for 2 hours. DDVP and the DEP-TFA derivative were determined simultaneously by GC-FPD. The recoveries of DDVP and DEP from agricultural products spiked at levels of 0.1 microgram/g were 72.6-117.7% and 86.2-106.6%, respectively. The detection limits were 0.03 microgram/g in powdered tea and < or = 0.01 microgram/g in other samples. An interlaboratory study by 6 laboratories was conducted to validate this proposed method for 6 crops. Repeatability ranged from 3.1 to 7.8% for DDVP and from 3.4 to 8.3% for DEP, and reproducibility, from 6.9 to 15.5% for DDVP, and from 7.9 to 21.8% for DEP. Precision values were well within statistically predicted levels.

Chromatography, Gas↗

PubMed2002

[A detection method of recombinant DNA from genetically modified potato (NewLeaf Plus potato) and detection of NewLeaf Plus potato in snack].

A detection method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) potato (NewLeaf Plus potato; NL-P), which has not been authorized as safe in foods in Japan. The potato sucrose synthase gene was used as an internal control. The DNA from NL-P specifically provided an amplified band using PCR with a primer pair recognizing PLRV-rep gene. In addition, to prevent false-positive results in processed potato foods infected with PLRV, we designed a primer pair recognizing sequences derived from two organisms to detect specifically NL-P in processed potato. The PCR product obtained using the designed primer pair was specific for NL-P. The DNA introduced into NL-P could be detected from potato powder samples containing 0.05% NL-P. The proposed method was applied to the detection of NL-P in 25 processed potato foods. NL-P was detected in 3 snack products.

DNA, Plant↗

PubMed2002

Effect of subchronic feeding of genetically modified corn (CBH351) on immune system in BN rats and B10A mice.

Subchronic animal feeding studies to examine the effect on the immune system of genetically modified corn CBH351, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi, were conducted in female BN rats and B10A mice. The studies were designed to compare the effect of a line of genetically modified corn CBH351 (GM corn) with that of isoline corn (non-GM corn). Heat-treated corn meal was incorporated into the diets of the rats and mice at a concentration of 50%. The study duration was 13 weeks. Growth, food intake, and organ weights of the thymus, spleen, and liver were compared between animals fed the non-GM and GM lines. The histological findings in thymus, spleen, mesenteric lymph nodes, Peyer's patches, small intestines, liver, kidney, and bone marrow, and the presence of Cry9C-specific IgE, IgG, IgG1 and IgA antibodies in serum were also compared. The results showed no significant differences in growth, feeding value, or the histological findings in immunity-related organs between the animals fed the GM and non-GM lines. Production of Cry9 C-specific IgE and IgA was not detected in the serum of either group. Production of Cry9C-specific IgG and IgG1 was slightly increased in the 50% GM groups of BN rats. No Cry9C-specific IgG or IgG1 was detected in the serum of BN rats fed the diet containing 5% GM-corn In conclusion, no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study.

Animals↗

PubMed2002

[Determination of ethychlozate and its degradation product in fruits by HPLC and LC/MS].

A method was developed for the analysis of ethychlozate (CIE) and its decomposition compound, 5-chloro-3(1H)-indazolylacetic acid (CIA) in fruits by HPLC and LC/MS. The sample was homogenized with 1 mol/L HC1, and CIE and CIA were extracted with 5 mol/L HCl and acetone. They were extracted from the acetone extract with diethylether-n-hexane (2:1). CIE was hydrolyzed to CIA with methanol-4 mol/L KOH (1:1). The solution was made acidic, and CIA was extracted with diethylether-n-hexane (2:1). The extract was cleaned up on a silica gel column. CIA was determined by HPLC-UV and LC/MS (Scan or SIR). Four fruits were spiked with CIE or CIA at 0.5 microgram/g and analyzed by the proposed method with HPLC. The average recoveries were 77.2-83.2% for CIE and 71.2-89.2% for CIA. The concentrations determined by LC/MS were 10-25% higher than the values by HPLC. The limit of detection (LOD) of CIA standard solution by HPLC corresponds to 0.015 microgram/g of CIE in the sample. In the same way, the LOD of CIA by LC/MS (SIR) corresponds to 0.009 microgram/g of CIE in the sample.

Chromatography, High Pressure Liquid↗

PubMed2002

[A detection method for recombinant DNA from genetically modified potato (NewLeaf Y potato)].

A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.

DNA Primers↗

PubMed2002

Comparison of sulfuric acid treatment and multi-layer silica gel column chromatography in cleanup methods for determination of PCDDs, PCDFs and dioxin-like PCBs in foods.

Two typical cleanup methods, sulfuric acid treatment and multi-layer silica gel column chromatography, for the determination of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dioxin-like PCBs) in seventeen food samples were examined and compared. Vegetables, fruits, cereals, fish, meat and dairy foods were extracted by conventional methods (shaking with acetone/n-hexane or with n-hexane after alkaline treatment). The extracts were cleaned up by sulfuric acid treatment or multi-layer silica gel column chromatography, followed by several column chromatographic steps. Of the samples treated, the vegetable, fruit and cereal samples could be directly applied to the multi-layer silica gel column after extraction. However, the samples containing fats and oils such as fish, meat and dairy foods needed to be treated several times with concentrated sulfuric acid before multi-layer column chromatography, because these samples plugged the column with oily residues. Both cleanup methods gave similar values of isomeric concentrations and showed similar efficiency of purification, and the recoveries ranged from 40 to 120%. These results are considered to provide useful data for the efficient analysis of dioxins in foods which have wide-ranging compositions.

Benzofurans↗

PubMed2002

[Comparison of soyasaponin and isoflavone contents between genetically modified (GM) and non-GM soybeans].

Soyasaponins and isoflavones are main secondary metabolites in soybeans. In this report we compared the content of secondary metabolites between genetically modified (GM) and non-GM soybeans. Six cultivars/lines of GM and six cultivars/lines of non-GM soybeans were extracted with methanol. Each extract was partitioned with aqueous methanol and hexane and the aqueous methanol fraction was partially purified by HP-20 and LH-20 column chromatography to afford crude soyasaponin and isoflavone fractions. The main A-type soyasaponin, acetylsoyasaponin A1 (AcA1), and the main B-type soyasaponins, soyasaponins I and II (I and II), in the crude fractions were identified by LC/MS analyses with authentic samples. The main isoflavones, daidzin, genistin, daidzein and genistein (DI, GI, DE and GE), in the crude fractions were identified by LC photo-diode array analyses with authentic samples. The contents of AcA1, I and II in the crude soyasaponin fractions and those of DI, GI, DE and GE in the crude isoflavone fractions were analyzed by reversed-phase HPLC. The average contents (mg/100 g) of AcA1, I, II and total of B-type soyasaponins in GM soybeans were 36.4 +/- 24.2, 51.2 +/- 11.8, 26.4 +/- 7.6 and 77.7 +/- 18.5, respectively, and those in non-GM ones were 22.3 +/- 14.7, 46.3 +/- 17.8, 19.8 +/- 9.1 and 65.9 +/- 26.9, respectively. The average contents (mg/100 g) of DI, GI, DE, GE and total isoflavones in GM soybeans were 93.1 +/- 15.5, 121.8 +/- 19.4, 0.073 +/- 0.178, 0.320 +/- 0.082 and 215.3 +/- 33.3, respectively, and those in non-GM ones were 78.8 +/- 34.6, 106.7 +/- 28.3, 2.206 +/- 4.468, 0.822 +/- 0.754 and 188.5 +/- 26.7, respectively. There were no statistically significant differences in soyasaponin and isoflavone contents between GM and non-GM soybeans. Therefore, it was estimated that the GM soybeans are equivalent to the non-GM ones in terms of the composition of the main secondary metabolites.

Food, Genetically Modified↗

PubMed2002

[Performance study of analytical method for ethoxyquin in fruit].

A performance study of an analytical method of ethoxyquin (ET) in apples and pears was conducted. In the proposed method, the sample was homogenized with thiourea and ET was extracted with acetone instead of hexane used in the official analytical method for ET, in order to improve the extraction efficiency. Furthermore, dibutyl hydroxytoluene was added in the test solution to prevent the decomposition of ET. For evaluation of the method. ET spiked into apples and pears at the level of 0.2 microgram/g was determined in replicate in six laboratories. Mean recoveries from apple, pear and Japanese pear were 85.3, 83.0 and 83.9%, respectively. Repeatability relative standard deviation values were 3.8-4.7% and reproducibility relative standard deviation values were 7.8-11.3%. The detection limits were 0.001-0.025 microgram/g in the six laboratories; this value may reflect instrument performance. ET spiked into apples and pears at the level of 0.2 microgram/g was detected by both LC/MS and GC/MS.

Antioxidants↗

PubMed2002

Increased digestibility of two products in genetically modified food (CP4-EPSPS and Cry1Ab) after preheating.

We performed experiments on in vitro digestion of newly expressed proteins by SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) to assess the allergenicity of food components derived from biotechnological modification. For newly expressed proteins, we chose CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4) and Cry1Ab derived from Bacillus thuringiensis subsp. kurstaki strain HD-1. The former is expressed in GM-soybeans and the latter is expressed in GM-corns. Firstly, we examined the digestibility of purified CP4-EPSPS and Cry1Ab by SGF. Both proteins were rapidly digested within 60 sec. After preheating, the digestibility by SGF was slightly increased. Secondly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SGF. The digestion time of both proteins by SGF was almost the same as that of the purified proteins. Thirdly, the digestibility of CP4-EPSPS and Cry1Ab by SIF was examined. The digestion time of these proteins was 240 min or more. However, digestibility of these proteins by SIF was dramatically increased by preheating, and the digestion time was less than 5 sec. Fourthly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SIF. Digestion time of both proteins by SIF was almost the same as that of the purified proteins. From these results, we concluded that the digestibility of both CP4-EPSPS and Cry1Ab by SGF and SIF was increased by preheating. Therefore, we suggest that the allergenicity of both proteins should be extremely low because of the easy digestibility of these proteins by SGF and also by SIF with preheating.

Allergens↗

PubMed2002

[Detection of unexpected recombinant DNA in maize grain].

PCR detection of genetically modified (GM) line-specific recombinant DNA was carried out on Bt 11, Event 176 and Non-GM maize grain harvested in 1999. Of 100 grains of Bt 11 line, Event 176 specific DNA was detected in 11 grains. Of 30 grains of Event 176 line, Mon 810 or Bt 11 specific DNA was detected in 5 grains. In addition, Bt 11 or Event 176 specific DNA was detected in 4 of 30 Non-GM grains. These data suggest that maize grains (not seed) often contain DNAs of different lines from what they were expected to contain. Furthermore, quantitative PCR was performed to estimate the genotype of the Event 176 grains described above. The results showed that the genotype of all the grains containing different recombinant DNA is heterozygous. Therefore, it was considered that the grains containing the unintended recombinant DNA were not accidental contaminants, but that airborne pollination had introduced the recombinant DNA into the grains.

DNA, Recombinant↗

PubMedDate not supplied

Novel reference molecules for quantitation of genetically modified maize and soybean.

New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.

Base Sequence↗

PubMedDate not supplied

Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules.

Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.

DNA, Plant↗