[Plasticizers Used in Polyvinyl Chloride Toys (2019-2020)].

We determined the fifteen types of plasticizers, including four kinds of phthalic acid esters (PAEs) used in 220 polyvinyl chloride (PVC) toys on Japanese market from 2019 to 2020. Three kinds of previously undetected types of PAEs were also detected, but not identified in this study. Di (2-ethylhexyl) terephthalate (DEHTP) was the highest detection rate in 209 soft PVC toys out of 220 toys, with 71.2% for designated toys and 88.9% for not-designated toys, respectively, showing a gradual increase from the previous reports in 2009 and 2014. On the other hand, the usages of o-acetyl tributyl citrate and adipic acid esters decreased, but the six types of PAEs prohibited to use for the designated toys in Japan were not detected in them, the usage of diisobutyl phthalate were increased. In contrast, four types of PAEs were detected in not-designated toys. Among them, the detection ratio of di (2-ethyhexyl) phthalate decreased to about 1/10. The content levels of plasticizers in per each sample were continued to keep low level from the report five years ago. These results showed that the main plasticizer used in PVC toys is DEHTP, and that the usage of other plasticizers was decreased.

[Inter-laboratory Study on the Modified Methods for Analyzing Bisphenol A Content for Migration Tests from Polycarbonate Food Apparatuses, Containers, and Packaging].

An inter-laboratory study involving 24 laboratories was conducted to validate the modified analytical method for the migration solution of heptane for the determination of bisphenol A migrating from polycarbonate food processing materials. In this study, two concentrations of samples were blindly coded. Each laboratory determined the analyte (bisphenol A, phenol and p-tert-butylphenol) concentration in each sample according to the established protocol. The obtained values were analyzed statistically using internationally accepted guidelines. Horwitz ratios were calculated based on the reproducibility relative standard deviation (RSDR), which was estimated from the inter-laboratory study, and predicted RSDR, which was calculated using the Horwitz/Thompson equation. Horwitz ratios of the two samples ranged from 0.15 to 0.37 for the three compounds, meeting the performance criteria of less than 2 set by the Codex Alimentarius for analytical method approval. These results showed that this modified analytical method shows good performance as an analytical method for the migration solution of heptane.

[Modified Methods for Analyzing Bisphenol A Content for Migration Tests from Polycarbonate Food Apparatuses, Containers, and Packaging].

A validation study was performed on the modified analytical method for the migration solution of heptane, 20% ethanol and 4% acetic acid for the determination of bisphenol A migrating from polycarbonate food apparatuses, containers, and packaging. The analytes for the method were bisphenol A, phenol and p-tert-butylphenol. The repeatability, within-laboratory reproducibility and trueness of the method was estimated in the range of 0.2-1.8%, 0.4-2.6% and 95-102% respectively. These results showed that the method is useful as an analytical method for the migration solution of heptane, 20% ethanol and 4% acetic acid. Furthermore, the applicability of the determination methods with a fluorescence detector was verified. As a result of the validation study, the repeatability, within-laboratory reproducibility and trueness of the method was estimated in the range of 0.1-2.9%, 0.2-3.1% and 94-101% respectively. It was confirmed that the measurement with a fluorescence detector is also available.

Dietary Exposure of the Japanese General Population to Elements: Total Diet Study 2013-2018.

Some countries have conducted a total diet study (TDS) focused on the estimation of specific trace elements. Although some results of a Japanese TDS examining trace elements were published, there have been no reports of a nationwide TDS across Japan over a multi-year period to estimate the level of exposure to multiple elements. In the present study, a TDS using a market basket approach was performed to estimate the dietary exposure levels of the general population of Japan to 15 elements, including aluminum (Al), total arsenic (tAs), boron (B), barium (Ba), cadmium (Cd), cobalt (Co), chromium (Cr), total mercury (THg), molybdenum (Mo), nickel (Ni), lead (Pb), antimony (Sb), selenium (Se), tin (Sn), and uranium (U). Samples prepared in eight regions across Japan over a 6-year period were analyzed using validated methods. The robust mean exposure estimates for Al, tAs, B, Ba, Cd, Co, Cr, THg, Mo, Ni, Pb, Sb, Se, Sn, and U were 48, 4.2, 29, 8.6, 0.35, 0.17, 0.49, 0.14, 4.2, 2.8, 0.15, 0.022, 1.8, 0.10, and 0.021 µg/kg body weight/day, respectively. Although the variability in exposure estimates varied greatly from element to element, the relative standard deviations calculated from the robust means and robust standard deviations were ≤ 50% for all elements except Sn. Compared against the health-based guidance values, none of the robust and precise estimates obtained for the target elements would be associated with urgent health risk concern. In addition, the estimated exposure levels were generally in agreement with previously reported estimates, indicating that health risks associated with exposure to these elements have not changed markedly nationwide in Japan in recent years.

[Inter-laboratory Study on the Modified Method Assessment for the Determining Methanol Content in Kitchen Detergents].

We modified a method for determining methanol content in detergents used in kitchens. Furthermore, an inter-laboratory study was conducted in 10 laboratories to validate the modified method. In this study, two concentrations of samples were blindly coded. Each laboratory determined the methanol content in each sample according to a protocol. The determined values were statistically analyzed according to an international harmonized guideline. HorRat values were calculated based on the reproducibility relative standard deviation (RSDR) which was estimated from the interlaboratory study, and predicted RSDR calculated from the Horwitz/Thompson equation. The HorRat values of the two samples were 0.8 and 1.8, meeting the performance criteria of less than 2 set by the Codex Alimentarius for analytical method approval. These results confirm that this modified analytical method shows good performance as an analytical method for determining methanol content in kitchen detergents.

Transoral removal of a hilo-parenchymal submandibular sialolith.

In sialolithiasis, the lithiasis is often large and located at the junction of the middle and posterior third of the duct, in the hilum region. In such cases, transoral approach for submandibular lithiases (TASL) is a useful treatment of choice in patients with large submandibular stones that can be palpated bimanually.

[Analysis of Organoarsenic Compounds Concentration in Fish with the Newly Developed LC-MS/MS Method].

Most fish contain some kinds of organoarsenic compounds. To assess the health risk for the chronic effects due to intake of these compounds, it is necessary to quantify the concentration of each chemical form, since the toxicity is difference depending on the form. We developed and validated the LC-MS/MS method to determine the concentration of monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), tetramethylarsonium (TeMA), arsenobetaine (AB), and arsenocholine (AC) in fish. Using this method, we quantified the concentration of each organoarsenic compounds and total arsenic in 50 fish samples from across 10 groups. Total arsenic concentration ranged from 0.53 to 25 mg/kg in all samples, except for in thread-sail filefish where the concentration ranged from 8.3 to 25 mg/kg. With the exception of sardines, in all samples AB was found at the highest level in relation to the total arsenic concentration. In sardines, the concentration of DMA was higher than that of AB, accounting for 16 to 24% of total arsenic. In red sea bream, concentrations of total arsenic, AB, and AC in farm-raised fish were lower than those in wild-caught fish.

[Surveillance of Chromium (VI) Concentrations in Mineral Water Products in 2018].

We used ion chromatography with post column derivatization to measure chromium (VI) concentrations in 155 mineral water (MW) products purchased from markets in Japan in 2018. Chromium (VI) recovery in spiked samples of the 155 MW products ranged from 95 to 106%, suggesting that this method is well suited for ensuring that MW products meet the requisite health and food safety standards. Chromium (VI) levels were above detection limits in 54 of the MW products, giving a total detection rate of 35%. The minimum and maximum chromium (VI) concentrations in all of the MW products were 0.0001 mg/L and 0.045 mg/L, respectively. The median chromium (VI) concentration was 0.0003 mg/L, with most MW products having chromium (VI) concentrations in the range of 0.0001 to 0.0002 mg/L. None of the MW products had chromium (VI) concentrations that exceeded the current standard value (0.05 mg/L) for MW in Japan.

[Validation Procedure for a Multi-Element Analytical Method Aiming at Dietary Intake Estimation].

Total diet study (TDS) is a useful way to estimate the dietary intake of hazardous and chemical substances. Regarding the analysis performed in TDS, international guidelines published by the World Health Organization, recommend selecting and confirming the validity of suitable analytical methods to achieve the purpose of TDS. However, concrete procedures and/or approaches for confirming the validity of suitable methods have yet to be established. In the present study, we aimed to develop samples, referred to as SEMPs; Samples to estimate methods performance, that can be used to evaluate the performance of the analytical methods applied to the composite samples prepared in TDS. The concentrations of 14 kinds of elements, including hazardous substances such as arsenic, lead, and cadmium, in SEMPs were measured for use in the validation of a multi-element analytical method for estimating dietary intake. After examining the appropriate amount of relevant elements added to the samples, we established a performance evaluation procedure by repeatedly analyzing five fortified and non-fortified SEMPs each.

Analysis of mineral apposition rates during alveolar bone regeneration over three weeks following transfer of BMP-2/7 gene via in vivo electroporation

Alveolar bone is not spontaneously regenerated following trauma or periodontitis. We previously proposed an animal model for new alveolar bone regeneration therapy based on the non-viral BMP-2/7 gene expression vector and in vivo electroporation, which induced the formation of new alveolar bone over the course of a week. Here, we analysed alveolar bone during a period of three weeks following gene transfer to periodontal tissue. Non-viral plasmid vector pCAGGS-BMP-2/7 or pCAGGS control was injected into palatal periodontal tissue of the first molar of the rat maxilla and immediately electroporated with 32 pulses of 50 V for 50 msec. Over the following three weeks, rats were double bone-stained by calcein and tetracycline every three days and mineral apposition rates (MAR) were measured. Double bone-staining revealed that MAR of alveolar bone was as similar level three days before BMP-2/7 gene transfer as three days after gene transfer. However, from 3 to 6 days, 6 to 9 days, 9 to 12 days, 12 to 15 days, 15 to 18 days, and 18 to 20 days after, MARs were significantly higher than prior to gene transfer. Our proposed gene therapy for alveolar bone regeneration combining non-viral BMP-2/7 gene expression vector and in vivo electroporation could increase alveolar bone regeneration potential in the targeted area for up to three weeks.

Non-surgical model for alveolar bone regeneration by bone morphogenetic protein-2/7 gene therapy.

BACKGROUND: Alveolar bone is a critical tissue for tooth retention; however, once alveolar bone is lost, it may not spontaneously regenerate. Currently, bone grafts or artificial bone is commonly used for alveolar bone regeneration therapy. However, these therapies require surgical procedures, which present risks, particularly in elderly patients. Therefore, development of alveolar bone regeneration techniques that do not require surgical procedures is critical. It is well known that stem cells present in the periosteal and periodontal ligament may be induced to differentiate into osteogenic cells. This study hypothesizes that transfer of the bone morphogenetic protein-2/7 (BMP-2/7) gene into periodontal tissues via in vivo electroporation induces exogenous BMP production and causes stem cells in periodontal tissues to differentiate into osteogenic cells, enabling generation of new alveolar bone. METHOD: The BMP-2/7 gene expression vector was introduced via electroporation into the target site in periodontal tissues of the first molar of rat maxillae. RESULTS: Exogenous BMP-2 and -7 were detected in the target areas, and growth of new alveolar bone tissue was observed 5 days after gene transfer. On day 7, new alveolar bone tissues were found to connect to the original bone tissues. Moreover, mineral apposition rates of the alveolar bone after BMP-2/7 gene transfer were significantly higher than those in the control group after LacZ gene transfer. CONCLUSION: The present findings indicate that a combination of the BMP-2/7 non-viral vector and in vivo electroporation represents a promising non-surgical option for alveolar bone regeneration therapy.

[Surveillance of Cadmium Concentration in Chocolate and Cocoa Powder Products Distributed in Japan].

Chocolate and cocoa are manufactured from cacao beans produced by the cacao tree (Theobroma cacao). These products may contain cadmium (Cd), which originates from contaminated soil. Here, we surveyed the Cd concentrations in dark chocolate, milk chocolate, white chocolate and cocoa powder products purchased at retail stores in Japan, using inductively coupled plasma mass spectrometry. The Cd concentrations in these chocolate and cocoa powder products ranged from 0.00021 to 2.3 mg/kg and from 0.015 to 1.8 mg/kg, respectively. A weak positive correlation was found between the Cd concentration and the content of cocoa solids stated on the product labels. A comparison between these results and the maximum levels (MLs) set by the European Union revealed that the Cd concentrations in chocolate and cocoa powder products on the Japanese market exceeded the MLs for eight of the 180 chocolate products and 26 of the 140 cocoa powder products.

Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation.

We previously developed a novel method for gene transfer, which combined a non-viral gene expression vector with transcutaneous in vivo electroporation. We applied this method to transfer the bone morphogenetic protein (BMP) gene and induce ectopic bone formation in rat skeletal muscles. At present, it remains unclear which types of cells can differentiate into osteogenic cells after BMP gene transfer by in vivo electroporation. Two types of stem cells in skeletal muscle can differentiate into osteogenic cells: muscle-derived stem cells, and bone marrow-derived stem cells in the blood. In the present study, we transferred the BMP gene into rat skeletal muscles. We then stained tissues for several muscle-derived stem cell markers (e.g., Pax7, M-cadherin), muscle regeneration-related markers (e.g., Myod1, myogenin), and an inflammatory cell marker (CD68) to follow cell differentiation over time. Our results indicate that, in the absence of BMP, the cell population undergoes muscle regeneration, whereas in its presence, it can differentiate into osteogenic cells. Commitment towards either muscle regeneration or induction of ectopic bone formation appears to occur five to seven days after BMP gene transfer.

Validation Study on a Method for Simultaneous Quantification of Elements in Mineral Water and Surveillance of Element Concentrations.

A validation study was performed on the Japanese official method to determine several elements (B, Cr, Mn, Cu, Zn, As, Se, Cd, Ba, Pb) in mineral water simultaneously using ICP-MS. The trueness, repeatability and reproducibility of the method were estimated to be in the ranges of 95-106, 0.2-1.4 and 0.4-4.2%, respectively. The values of every performance parameter fulfilled the requirements in the guidelines announced by the Ministry of Health, Labour, and Welfare of Japan, and the validity of the method was confirmed. The concentrations of the elements in mineral water products purchased from Japanese markets in 2013 and 2014 were surveyed using the method. All products met the specifications defined in the Japanese Food Sanitation Law. No clear relationship between the element concentrations and the survey year or the producing country was found.

[Surveillance of Chromium(VI) Concentrations in Mineral Water Products].

We developed and evaluated a method of quantifying chromium(VI) in mineral water (MW). After a performance evaluation, we performed a surveillance of chromium(VI) concentrations in 150 kinds of MW products purchased from markets in 2016. Recovery rates, as examined by using 150 spiked samples prepared from the purchased MW products, ranged from 93 to 107%. These results strongly suggest that the validated method is suitable for quantifying chromium(VI) in MW. Chromium(VI) was detected in 65 kinds of MW products; the detection rate was 43%. The minimum concentration of chromium(VI) was 0.0001 mg/L and the maximum was 0.0019 mg/L. The median value of chromium(VI) concentration was 0.0003 mg/L. In most of the products in which chromium(VI) was detected, the concentration ranged from 0.0001 to 0.0002 mg/L. No products were found with concentrations higher than the value (0.05 mg/L) set by the Food Sanitation Law as the specification for MW.

[Development and Applicability of Analytical Methods for Quantifying Cyanide and Bromic Acid in Mineral Waters].

We developed and evaluated methods of quantifying cyanide (cyanide ion and cyanogen chloride) and bromic acid in mineral waters (MW). After performance evaluation, recovery studies were performed on 110 kinds of MW products to examine the applicability of the methods. The approximate proportion of the MW samples, in which the recovery rate of these anionic compounds was within 90 to 110%, was 95% in the cyanide ion and bromic acid analysis and 45% in the cyanogen chloride analysis. We observed low rates of recovery of cyanogen chloride from some MW products with pH values around neutral. To increase the recovery rate, we propose adding phosphoric acid buffer to adjust the pH of these MW samples. The retention times for bromic acid in some MW products differed from that in standard solution. We concluded that carbonic acid influences the retention times. It may be necessary to to exclude carbon dioxide from the MW samples by degassing to synchronize the retention times of bromic acid in the MW samples and the standard solution.

[Development of ICP-OES, ICP-MS and GF-AAS Methods for Simultaneous Quantification of Lead, Total Arsenic and Cadmium in Soft Drinks].

In this study, we developed methods to quantify lead, total arsenic and cadmium contained in various kinds of soft drinks, and we evaluated their performance. The samples were digested by common methods to prepare solutions for measurement by ICP-OES, ICP-MS and graphite furnace atomic absorption spectrometry (GF-AAS). After digestion, internal standard was added to the digestion solutions for measurements by ICP-OES and ICP-MS. For measurement by GF-AAS, additional purification of the digestion solution was conducted by back-extraction of the three metals into nitric acid solution after extraction into an organic solvent with ammonium pyrrolidine dithiocarbamate. Performance of the developed methods were evaluated for eight kinds of soft drinks.

[Surveillance of cadmium level in octopus, squid, clam, short-necked clam and chocolate].

In order to investigate cadmium contents in foods sold in Japan, cadmium levels in 40 seafood samples and 30 chocolate samples were measured by means of atomic absorption spectrometry and ICP-OES. We first confirmed the validity of the method according to the guidelines of the Ministry of Health, Labour and Welfare. Among 40 seafood samples investigated, cadmium was detected in 31 samples, in which the concentration exceeded half the LOQ (0.025 mg/kg), and the level was ranged from 0.03 to 0.38 mg/kg. We could not find any sample containing cadmium in excess of 2 mg/kg, which the Codex Alimentarius sets as the maximum standard value. Among 30 chocolate samples, cadmium was detected in 21 samples, and the level ranged from 0.025 to 0.54 mg/kg.

Sperm-specific C-terminal processing of the proteasome PSMA1/α6 subunit.

We previously reported that the ascidian sperm proteasome degrades the egg-coat protein extracellularly during fertilization. In order to explore an extracellular transport signal, we purified the proteasome from ascidian sperm and compared its subunit structure with egg and muscle proteasomes. The results showed that PSMA1/α6 subunit of the sperm proteasome is distinct from egg and muscle proteasomes. LC/MS/MS analysis revealed that the C-terminal 16 residues of sperm α6 subunit are processed. Whereas sperm-specific paralogous genes of α subunits are reported, its sperm-specific C-terminal processing is a newly discovered novel post-translational modification of the proteasome.

Functional expression of streptococcal galactosyltransferase in baculovirus/insect cell expression system.

The cpsIaJ gene of Streptococcus agalactiae type Ia codes for beta-1,4-galactosyltransferase. In this study, the functional expression of His-tagged CpsIaJ in a baculovirus expression system was performed, because the efficient functional expression of this enzyme in Escherichia coli had been unsuccessful. Using a partially purified enzyme preparation, we found that the enzyme had a restricted substrate specificity and that the entire structure of the substrate GlcNAc beta1-3Gal beta1-4Glc was required for the activity. Furthermore, mutations in a conserved DXD motif caused the loss of the enzyme's activity.