Quantitative Measurement of Phthalate Exposure Biomarker Levels in Diaper-Extracted Urine of Japanese Toddlers and Cumulative Risk Assessment: An Adjunct Study of JECS Birth Cohort.

Phthalate exposure monitoring and risk assessment in non-toilet-trained children are rarely reported. This adjunct study of the Japan Environment and Children's Study assessed cumulative health risks in 1.5-year-old toddlers in the Aichi regional subcohort by biomonitoring 16 urinary metabolites of eight phthalate plasticizers. Overnight urine was extracted from toddlers' diapers (n = 1077), and metabolites were quantified using ultraperformance liquid chromatography coupled with tandem mass spectrometry. The analyses' quality was assured by running quality control samples. The highest geometric mean concentration was found for mono-(2-ethyl-5-carboxypentyl) phthalate, followed by mono-isobutyl phthalate (23 and 21 µg/L, respectively). Di-2-ethylhexyl phthalate (DEHP) and di-butyl phthalate exhibited higher risks [hazard quotient (HQ) > 1] than the cutoff level in a small proportion of toddlers; 8 and 14% of toddlers were at cumulative risk of multiple phthalates beyond the cutoff level [hazard index, (HI) > 1], based on the tolerable daily intake of the European Food Safety Authority and the United States Environmental Protection Agency Reference Dose. HI > 1 for antiandrogenicity in creatinine-unadjusted and -adjusted estimations were exhibited by 36 and 23% of the children, respectively. Thus, identifying exposure sources and mitigating exposure are necessary for risk management. Additionally, continuous exposure assessment and evaluation of health outcomes, especially antiandrogenic effects, are warranted.

Residue levels of organophosphate pesticides and dialkylphosphates in agricultural products in Japan.

High urinary levels of dialkylphosphates (DAPs), which are common structures of organophosphate pesticides (OPs), have been associated with several adverse health outcomes in human biomonitoring studies. Previous studies have indicated that dietary OP exposure and ingestion of environmentally degraded DAP, which is inactive with acetylcholinesterase, can lead to an increase in urinary DAP levels in the general population. However, the specific food sources contributing to the intake of OPs and DAPs have not been identified. In this study, we analyzed the levels of OPs and preformed DAPs in various food items. DAP levels were markedly high in certain fruits, such as persimmon, apple juice, kiwi, and mandarin. In contrast, only moderate levels of OPs were detected in these foods. Furthermore, the levels of OPs and DAPs were positively associated with vegetables, whereas no such association was observed in fruits. Increased consumption of certain fruits presumably leads to a marked increase in urinary DAP levels in individuals despite limited exposure to OPs, resulting in reduced reliability of urinary DAPs as a marker of OP exposure. Therefore, the possible effects of dietary habits and the resulting intake of preformed DAPs should be considered when interpreting biomonitoring data of urinary DAPs. Additionally, DAP levels in most organic foods were much lower than those in conventional foods, suggesting that the reduction in urinary DAPs by organic diet intervention may be mainly attributed to the reduced intake of preformed DAPs rather than reduced exposure to OPs. Therefore, urinary DAP levels may not be suitable indicators for evaluating ingested OP exposure.

[Internal Quality Control of Residual Pesticides Analysis Using Multiple Stable Isotope Labeled Compounds].

Internal quality control (IQC) is essential to ensure the reliability of the results of chemical analysis. In this study, we propose a novel method of IQC for multiresidue analysis of pesticides. A total of seven stable isotope labeled compounds (SILC) were added to analytical samples and were used to monitor and evaluate the quality of analytical results. In contrast to conventional IQC method in which only a limited number of control materials were analyzed to ensure the reliability of the results for an entire batch, the developed method can monitor the analytical quality of all the samples in the batch. It was shown that the developed method could achieve better performance than that of conventional method. Therefore, the developed method is considered to be promising for practical applications.(Received January 27, 2022; Accepted July 4, 2022).

Increased levels of renal damage biomarkers caused by excess exposure to trivalent chromium in workers in tanneries.

BACKGROUND: The process for leather material production is carried out in developing countries using a large amount of trivalent chromium [Cr(III)]. Assesment of health risks for millions of workers in tanneries worldwide that are highly polluted with Cr(III) is needed. METHODS: Levels of total Cr and its chemical species in wastewater samples from tannery built-up areas of Bangladesh were investigated. Cr-mediated renal damage was assessed in 100 male tannery workers by epidemiological analysis consisting of questionnaires and measurements of levels of urinary Cr and urinary renal damage markers [urinary levels of total protein and kidney injury molecule-1 (KIM-1)]. RESULTS: High levels of total Cr (mean ± standard deviation = 1,908,762 ± 703,450 µg/L) were detected in wastewater samples from 13 sites of tanneries. More than 99.99% of total Cr in the wastewater was Cr(III), indicating that workers in the tanneries were exposed to large concentrations of Cr(III). Cr levels (mean ± standard, 2.89 ± 4.23 µg/g creatinine) in urine samples from the workers in tanneries were >24-fold higher than the levels in a general population previously reported. Multivariate analysis showed significant correlations between urinary levels of Cr and urinary levels of renal damage biomarkers. Nagelkerke Pseudo R2 values also showed that Cr level is the strongest contributor to the levels of renal damage biomarkers in the workers. CONCLUSION: Our results newly suggest that excess exposure to Cr(III) could be a risk for renal damage in humans.

Multidisciplinary approach to assess the toxicities of arsenic and barium in drinking water.

Well water could be a stable source of drinking water. Recently, the use of well water as drinking water has been encouraged in developing countries. However, many kinds of disorders caused by toxic elements in well drinking water have been reported. It is our urgent task to resolve the global issue of element-originating diseases. In this review article, our multidisciplinary approaches focusing on oncogenic toxicities and disturbances of sensory organs (skin and ear) induced by arsenic and barium are introduced. First, our environmental monitoring in developing countries in Asia showed elevated concentrations of arsenic and barium in well drinking water. Then our experimental studies in mice and our epidemiological studies in humans showed arsenic-mediated increased risks of hyperpigmented skin and hearing loss with partial elucidation of their mechanisms. Our experimental studies using cultured cells with focus on the expression and activity levels of intracellular signal transduction molecules such as c-SRC, c-RET, and oncogenic RET showed risks for malignant transformation and/or progression arose from arsenic and barium. Finally, our original hydrotalcite-like compound was proposed as a novel remediation system to effectively remove arsenic and barium from well drinking water. Hopefully, comprehensive studies consisting of (1) environmental monitoring, (2) health risk assessments, and (3) remediation will be expanded in the field of environmental health to prevent various disorders caused by environmental factors including toxic elements in drinking water.

Discovery, primary, and crystal structures and capacitation-related properties of a prostate-derived heparin-binding protein WGA16 from boar sperm.

Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface.

Novel SPE purification approach using the direct adsorption of vaporised propionic acid in food for rapid HPLC determination.

Solid phase extraction (SPE) is a technique widely used in food analysis for the isolation of analytes. Herein, we proposed a novel application of SPE to extract vaporised propionic acid, a common preservative, from a heated sample solution. A sample was heated under acidified conditions and the resulting steam was directly passed through an SPE column to extract the propionic acid, followed by elution and HPLC analysis. Here, the extraction on the SPE column ensures direct capture of propionic acid. The results demonstrated excellent linearity (R2 greater than 0.999) and recoveries of 89.9%-97.6% with intra- and inter-day precisions lower than 3.9%. To the best of our knowledge, no study has investigated the applicability of SPE to an analyte vaporised in the headspace of food products. The proposed method is promising in its application to various volatile compounds and in the routine analysis of propionic acid in food.

Unexpected associations of long-term and excessive exposure to trivalent chromium with hypertension and glycosuria in male tannery workers.

Serious health hazards including renal, skin and hearing disorders have been reported in Bangladeshi tannery workers (TWs) who were chronically exposed to a large amount of trivalent chromium [Cr(III)]. However, the effects of Cr(III) exposure on the prevalence of hypertension and the prevalence of glycosuria in TWs remain unknown. Since the Cr level in toenails is an established marker reflecting long-term exposure to Cr(III) in humans, the associations of Cr levels in toenails with the prevalence of hypertension and the prevalence of glycosuria in male tannery and non-tannery office workers (non-TWs) in Bangladesh were investigated in this study. The mean toenail Cr level in non-TWs (0.5 µg/g, n = 49) was comparable to that in the general population reported previously. Mean Cr levels in TWs with a low toenail Cr level (5.7 µg/g, n = 39) and those with a high toenail Cr level (298.8 µg/g, n = 61) were >10-fold and >500-fold higher, respectively, than that in non-TWs. Our univariate and multivariate analyses indicated that the prevalence of hypertension and the prevalence of glycosuria in TWs with a high toenail Cr level, but not in TWs with a low toenail Cr level, were significantly lower than those in non-TWs. This study showed for the first time that long-term and excessive exposure to Cr(III) that is more than >500-fold but not >10-fold higher than the usual exposure level could decrease the prevalence of hypertension and the prevalence of glycosuria in TWs. Thus, this study revealed unexpected effects of exposure to Cr(III) on health.

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein.

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca(2+) regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca(2+) oscillation without changing the basal intracellular Ca(2+) concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca(2+) concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca(2+) regulation may be a common feature among animal sperm.

Decreased hearing levels at frequencies for understanding speech in tannery workers exposed to a high level of trivalent chromium in Bangladesh.

Hexavalent chromium [Cr(VI)], which has a strong corrosive effect, has been reported to cause perforation of the eardrum. Trivalent chromium [Cr(III)] also has a weak corrosive effect. However, there has been no study on the effects of exposure to Cr, either Cr(VI) or Cr(III), on hearing levels in animals or humans. In this study, the effect of Cr(III) exposure on hearing levels was determined in a human study. Then the reproducibility of the results obtained in the human study and the etiology were investigated in an animal study. The mean levels of total chromium (t-Cr) in hair and toenails from 100 Bangladeshi tannery workers were >20-fold and >360-fold higher, respectively, than those in hair and toenails from 49 Bangladeshi non-tannery workers (office workers). Multivariate analysis revealed decreases of hearing levels (DHLs) at 1 k and 4 k Hz, frequencies that are crucial for understanding language, but not at 8 k and 12 k Hz, in the tannery workers. Since >99.99% of t-Cr in the wastewater that the workers were in direct contact with in the tanneries was Cr(III), the epidemiological results suggest Cr(III)-mediated DHLs in the tannery workers. The results of animal experiments in this study further showed that treatment with eardrops but not intraperitoneal injection with the same amount of Cr(III) that tannery workers might be exposed to resulted in DHL with a damaged eardrum in mice. Previous studies suggested that Cr(III) can directly reach the eardrums of tannery workers via droplets in the air. Cr(III) could also reach the eardrum via picking an ear canal with a finger contaminated with tannery wastewater including Cr(III). Taken together, the results of both human and animal studies suggest the risk of DHLs caused by damage of the eardrum through external exposure to Cr(III) via the ear canal.

Quantitative analysis of organophosphate pesticides and dialkylphosphates in duplicate diet samples to identify potential sources of measured urinary dialkylphosphates in Japanese women.

Increased levels of dialkylphosphates (DAP) in maternal urine are associated with a variety of adverse developmental outcomes in children. Although urinary DAP levels are usually considered to be a marker of exposure to organophosphate (OP) pesticides, excretion of DAP may also increase by ingesting preformed DAP. To date, no study has quantitatively assessed the possible contribution of the dietary intake of preformed DAP and OP pesticides to urinary levels of DAP. Therefore, we aimed to estimate the levels of 6 DAPs and 84 OP pesticides in duplicate diet samples and urine samples collected from 73 women living in urban areas of Japan in 2018. DAP and OP pesticides were detected in 94% and 45% of diet samples, while DAP was detected in 100% of urinary samples, respectively. The average daily intake of preformed DAP was significantly higher than that of parent OP pesticides in our participants. Dimethylphosphate and diethylphosphate were predominant in the preformed DAP, and the estimated average daily intake of total amount of DAP was 78.3 nmol. Fruits and vegetables were the major dietary sources of DAP. Dietary intake of DAP was positively associated with urinary DAP levels, suggesting that a considerable amount of urinary DAP was derived from ingesting preformed DAP. Our results show that attributing urinary DAP levels exclusively to OP pesticide exposure would result in a substantial overestimation of the exposure level. Therefore, the urinary levels of DAP may not be suitable for evaluating OP pesticide exposure in the general urban population.

Combined use of a modifier gas generator, analyte protectants and multiple internal standards for effective and robust compensation of matrix effects in gas chromatographic analysis of pesticides.

In the analysis of pesticides performed with gas chromatography, the quantitative performance of measurements can be severely compromised by phenomena known as matrix effects. In seeking a solution to the problem of matrix effects, the application of a modifier gas generator (MGG) was investigated in this study, together with analyte protectants and multiple internal standards. Ethylene glycol (EG) was used as modifier gas and matrix effects in GCMS analysis were then evaluated by using the extracts of various food commodities. MGG was used in combination with other known methods of matrix effect compensation and its performance in reducing matrix effects tested. We have found that by combining MGG with conventional analyte protectants, matrix effects were substantially reduced for most of pesticides. Use of EG was especially effective for organophosphate pesticides and those with amino groups. Using this approach, the shortcomings of conventional analyte protectants were remedied. Although neither EG nor analyte protectants could sufficiently reduce the matrix effects for certain classes of pesticides, this limitation could be overcome with the use of multiple internal standards (IS) in the analysis. Finally, it was shown that the method we developed could achieve better analytical performance than the matrix-matched calibration method. Our method was robust with respect to the variation of food matrix components, so its application to real-world analyses would be practical and promising.

Compensation of matrix effects in gas chromatography-mass spectrometry analysis of pesticides using a combination of matrix matching and multiple isotopically labeled internal standards.

In the multi-residue analysis of pesticides using GC-MS, the quantitative results are adversely affected by a phenomenon known as the matrix effect. Although the use of matrix-matched standards is considered to be one of the most practical solutions to this problem, complete removal of the matrix effect is difficult in complex food matrices owing to their inconsistency. As a result, residual matrix effects can introduce analytical errors. To compensate for residual matrix effects, we have developed a novel method that employs multiple isotopically labeled internal standards (ILIS). The matrix effects of ILIS and pesticides were evaluated in spiked matrix extracts of various agricultural commodities, and the obtained data were subjected to simple statistical analysis. Based on the similarities between the patterns of variation in the analytical response, a total of 32 isotopically labeled compounds were assigned to 338 pesticides as internal standards. It was found that by utilizing multiple ILIS, residual matrix effects could be effectively compensated. The developed method exhibited superior quantitative performance compared with the common single-internal-standard method. The proposed method is more feasible for regulatory purposes than that using only predetermined correction factors and is considered to be promising for practical applications.

Observed distribution of radiocaesium contamination in shiitake lots and variability of test results.

Shiitake mushrooms (Lentinula edodes) cultivated on bed-log are known to accumulate radiocaesium. Since the Fukushima-Diichi nuclear power plant accident (2011), the violation rate has been higher for log-cultivated shiitake than that for agricultural products or other foodstuffs. When testing shiitake mushrooms for radionuclide contamination, the validation of the sampling plan can be severely compromised by the heterogeneous contamination within shiitake lots. Currently, few data are available on the statistical properties of the radiocaesium contamination of log-cultivated shiitake. In this paper, shiitake lots contaminated by radiocaesium were identified and the distribution of the radiocaesium concentration within the lots investigated. The risk of misclassifying shiitake lots was predicted from the operating characteristic curve generated from Monte Carlo simulations and the performance of various sampling plans was evaluated. This study provides useful information for deciding on an acceptable level of misclassification risk.

[Examination of radioactive contamination in foods].

Following the Fukushima nuclear plant accident in Mar. 2011, the examination of radioactive contamination in foods is being carried out in Nagoya. During the period between 30 Mar. 2011 and 31 Oct. 2012, a total of 300 food samples were collected and the concentrations of radioactive nuclides were determined by means of γ-ray spectrometry using a high-purity germanium semiconductor detector. The results of analysis indicate that the concentrations of radioactive iodine (I) and cesium (Cs) were below the regulatory limits. Radioactive I ((131)I) was detected in 7 samples which belonged to the categories of green and yellow vegetables and other vegetables. Radioactive Cs ((134)Cs and (137)Cs) was detected in 60 samples which belonged to the categories of rice and its processed products, potatoes and its processed products, nuts and seeds, green and yellow vegetables, other vegetables, fruits, mushrooms, fishes and shellfishes, processed sea foods, meat, milk and dairy products and other beverages.

[Estimation of the intake of radioactive cesium based on analysis of total diet samples in Nagoya].

Food samples were purchased in Nagoya based on daily intake in the Tokai region, and prepared as total diet samples according to the market basket method. The contents of radioactive cesium (Cs) were determined by using a γ-ray spectrometer with a germanium semiconductor detector, and a committed effective dose was estimated. Radioactive Cs was not detected in samples collected in 2006 before the Fukushima nuclear plant accident. Radioactive Cs was detected in samples prepared in August, 2011, five months after the accident. The sources were sugar and confectioneries (3rd food group), other vegetables, seaweeds and mushrooms (8th food group) and fishes, shellfishes and processed seafoods (10th food group). Only Cs-137 was detected in samples prepared in August, 2012, one year and five months after the accident. The sources were the 8th and the 10th food groups. The estimated committed effective dose for radioactive Cs was 0.0015 mSv in 2011 and 0.00016 mSv in 2012.