[Implementation and Evaluation of Risk Communication Regarding Residual Pesticides].

In this study, a public seminar on risk communication methods was conducted to raise awareness and disseminate accurate knowledge about residual pesticides to consumers. Additionally, surveys on consumer awareness were conducted on the attendees before and after the seminar to evaluate its effectiveness. Responses were obtained from 84 participants. The paired t-test was used to analyze the changes in awareness before and after the seminar. The results showed significant improvements in "trust in the government" and "understanding of residual pesticides." Furthermore, step-wise multiple regression analysis was performed to explore the factors influencing satisfaction with the risk communication seminar, and the item "understanding of the safety of residual pesticides in food" was extracted. Understanding food safety is a crucial concern in daily life for consumers. To enable consumers to have an accurate understanding of food risks and make appropriate judgments, it is essential to continue implementing risk communication and conveying information about food safety and security in the future.

[Development of Analytical Method and Surveillance ofGibberellic Acid in Banana, Cherry, and Kiwi Fruit].

Gibberellic acid (GA3) is commonly used as a plant growth regulator in many food crops owing to its essential signaling functions during plant growth and development. In Japan, a threshold for administrative action for GA3 content of 0.3 mg/kg applies in produce in which maximum residue limits have not been established. Although the threshold is based on previous studies, the GA3 concentrations in individual foods are still unknown. Thus, we surveyed the concentrations of GA3 in banana, cherry, and kiwi fruit on the Japanese market. We developed and validated a method for the analysis of GA3 using solid-phase extraction and LC-MS/MS in accordance with accepted criteria of trueness, repeatability, and selectivity. The limits of detection and of quantification were determined as 0.005 and 0.05 mg/kg, respectively, in all fruits. Concentrations of GA3 did not exceed 0.3 mg/kg regardless of ripeness, suggesting the reasonability of the current regulation of GA3 in banana, cherry, and kiwi fruit. These findings can support prompt administrative action on these fruits, contributing to the regulation of GA3 in Japan.

[Estimation of the Distribution of Dietary Exposure to Lead Using Two-Dimensional Monte Carlo Simulation: An Attempt to Apply to Total Diet Samples].

The average dietary exposure to lead (Pb) in male and female Japanese individuals >1 year of age was estimated using 280 total diet samples representing 14 food groups from 10 areas over a two-year period. A probabilistic exposure estimation was performed using a two-dimensional Monte Carlo simulation (2D-MCS) with a Bayesian estimation that consided the uncertainty of the estimation process. The Bayesian estimation was performed using the likelihood function with cumulative distribution function between the lower and upper boundary values for no-detected values. The median dietary exposure to Pb was estimated as 5.82 µg/person/day. The 90% interval was 2.51-16.9 µg/person/day. Comparison with previously reported Pb exposure values indicates that the estimation of Pb exposure distribution using total diet samples is reasonable. The contribution to Pb exposure was highest in the order of food group 8 (light-colored vegetables, mushrooms, and seaweeds: 20.0±16.1%)>food group 1 (rice and rice products: 12.3±19.0%)>food group 10 (fish and shellfish: 10.5±13.9%). Owing to the high uncertainties of contribution ratios, it was not possible to identify dominant food groups contributing to Pb exposure. However, it was evident that the uncertainty of the estimation of Pb exposure was influenced by the uncertainty of Pb concentration than the uncertainty of food consumption rate. In particular, the effect of uncertainty from the Pb concentration of the food group 1 was 68.2%. When the margin of exposures were calculated, the estimated probabilities that a value would be <1 were 14.5% for developmental neurotoxicity to children (1-6 years old), 0.13% for blood pressure and 0.93% for kidney disease in Japanese individuals ≥1 year of age. The findings suggest that the health risk due to dietary Pb exposure is small but not negligible.

Evaluation of the effect of Lactiplantibacillus pentosus SN001 fermentation on arsenic accumulation and antihypertensive effect of Sargassum horneri in vivo.

Sargassum horneri contains water-soluble polysaccharides, which have antihypertensive effects, and arsenic, which is harmful to the human body. Boiling and other treatments are effective in removing arsenic; however, water-soluble polysaccharides are lost during processing. Therefore, a method to remove arsenic and further increase its antihypertensive effect is required. To this end, we investigated fermentation with Lactiplantibacillus pentosus SN001 in this study. Boiled and fermented S. horneri were administered to spontaneously hypertensive rats (SHR), and blood pressure and arsenic accumulation in organs were observed to simultaneously examine the effects of fermentation on hypertension and arsenic accumulation. The ACE (angiotensin-converting enzyme) inhibition rate, an indicator of antihypertensive effects, showed a maximum at 4 days of fermentation. Consecutive dosing studies using S. horneri, boiled S. horneri, and fermented boiled S. horneri in SHR were conducted. Although the boiled group showed high blood pressure values, the fermented boiled group showed lower blood pressure values than the boiled cohort. The amount of arsenic accumulated in the liver, kidney, and spleen of rats was significantly lower in the boiled and fermented boiled groups than that in the S. horneri group. This confirmed the arsenic removal effect of boiling pretreatment and the in vivo safety of fermented boiled S. horneri. These results suggest that fermentation of arsenic-free S. horneri with L. pentosus SN001 can enhance its antihypertensive effect in vivo. This is the first study to simultaneously examine the antihypertensive effect of fermentation of S. horneri and its effect on the arsenic accumulation in vivo.

Presence of nano-sized mercury-containing particles in seafoods, and an estimate of dietary exposure.

The toxicity of nano-sized particles of mercury (NP-Hg), which are thought to be generated during the detoxification of methyl mercury (MeHg), may differ from that of MeHg, elemental Hg (Hg0), and inorganic Hg (I-Hg). From a human health perspective, it is important to evaluate the presence of NP-Hg in seafoods. We investigated the in vivo formation of NP-Hg in fish and shellfish, which are the main sources of Hg exposure in humans. NP-Hg was measured in 90 fish samples with single-particle inductively coupled plasma mass spectrometry (spICP-MS) after enzyme degradation with pancreatin and lipase. In addition to NP-Hg, total Hg (T-Hg), MeHg, and selenium (Se) concentrations were evaluated. Transient Hg signals were detected as nanoparticles from almost all samples by using spICP-MS. Higher particle number concentrations (CPN) were observed in the tuna-swordfish group than in the shellfish group (17.7 × 107 vs. 1.2 × 106 particles/g, respectively). Although the CPN and maximum particle mass increased significantly with increasing T-Hg concentration, the increase in CPN was greater than those in maximum particle mass. Assuming that the NP-Hg detected was HgSe (tiemannite) and spherical based on previous reports, the maximum particle diameter was estimated to be 89 nm. The mean dietary exposures to NP-Hg, T-Hg, and MeHg were estimated to be 0.067, 5.75, and 5.32 µg/person per day, respectively. Generation of NP-Hg was inferred to be widespread in marine animals, with a preferential increase in the number of particles rather than an increase in particle size. The mean dietary exposure to NP-Hg in Japanese people was estimated to be 1.2 ng/kg body weight (BW) per day. Compared to PTWI of 4 µg/kg BW per week (0.57 µg/kg BW per day) derived by JECFA (2011), the health risk from redissolved I-Hg from NP-Hg is small.

[Evaluation of a Risk Communication Program for Pesticide Residues].

To promote and raise the awareness of accurate knowledge on pesticide residues, the symposium program on risk communication on pesticide residues was held by the broadcasted online style. The risk communication program was statistically evaluated using a pre- and post-program online questionnaire survey. We had the questionnaire answers of the 105 valid participants. The analysis of post-program questionnaires shows that the risk communication program was effective in terms of levels of understanding and interest. Pre-program risk perception or awareness of safety assessments was significantly and positively correlated with awareness for establishing standard values of pesticide residues by the analysis of pre-program questionnaires. Risk perception after the program was significantly higher than before the program, suggesting that risk perception increased due to the program by analysis the same question between pre- and post-program questionnaires. Multiple regression analysis suggests that the participants with higher pre-program awareness of safety assessments or pre-program awareness for establishing standard values appeared to have higher levels of understanding and post-program risk perception.

Quantification of progesterone in beef with marbling using liquid chromatography-tandem mass spectrometry with stable isotope-labelled standards.

Progesterone (P4) is contained naturally in animal tissue, and it is also used as a veterinary drug in cattle for treatment purposes. To assess the risk from P4 residues in beef derived from treated cattle, it is essential to quantify the P4 contained naturally in cattle tissue (endogenous P4). Therefore, we performed a method validation for the quantification of endogenous P4 (method quantification limit = 0.06 ng g-1) by using isotope-labelled P4s, and investigated the P4 contents in Japanese beef (n= 112; 0.07 to 121 ng g-1). The P4 contents in cattle muscle ranged from 0.07 to 54.3 ng g-1 in males, and from 0.27 to 121 ng g-1 in females. Our investigation also indicated that the developed method using both 13C- and deuterium-labelled P4 standards could be used to certify the recovery of P4 from cattle muscle containing various amounts of intramuscular fat, and enabled the determination of the P4 content in all Japanese beef samples that exceeded the method quantification limit.

Assignment of PM2.5 sources in western Japan by non-negative matrix factorization of concentration-weighted trajectories of GED-ICP-MS/MS element concentrations.

Rapid economic growth in Asian countries has raised concerns about the influence of air pollutants transported to Japan by westerly winds. We coupled a gas exchange device (GED) with a tandem inductively coupled plasma mass spectrometer (ICP-MS/MS) to enable direct introduction of PM2.5 to ICP and thus provide better data than could be obtained from samples collected by conventional filter methods. We used the GED-ICP-MS/MS system in Matsue City in western Japan to monitor in real time 29 elements in PM2.5 at 10-min intervals and to estimate the pollutant sources by non-negative matrix factorization (NMF) of concentration-weighted air-mass trajectories. The trajectory analysis identified high V, As, Sn, and Sb concentrations over the ocean from Taiwan to Tsushima Strait. NMF analysis revealed that these elements could be decomposed to multiple factors that indicated a large contribution from oceanic areas. The elemental contributions of these factors were high for metals/metalloids with low melting points as oxides, strongly suggesting that they were sourced from combustion of ship fuel. Our results demonstrate that both emissions from ships at sea and land-based emissions from Japan and continental Asia contribute to PM2.5 in Matsue City.

Attempt of Bayesian Estimation from Left-censored Data Using the Markov Chain Monte Carlo Method: Exploring Cr(VI) Concentrations in Mineral Water Products.

Hexavalent chromium (Cr(VI)) is toxic, carcinogenic, and mutagenic substances. Oral exposure to Cr(VI) is thought to be primarily from drinking water. However, under the certain reporting limit (~0.1 µg/L), percentage of Cr(VI) concentration in mineral water products under the reporting limit were estimated higher than 50%. Data whose values are below certain limits and thus cannot be accurately determined are known as left-censored. The high censored percentage leads to estimation of Cr(VI) exposure uncertain. It is well known that conventional substitution method often used in food analytical science cause severe bias. To estimate appropriate summary statistics on Cr(VI) concentration in mineral water products, parameter estimation using the Markov chain Monte Carlo (MCMC) method under assumption of a lognormal distribution was performed. Stan, a probabilistic programming language, was used for MCMC. We evaluated the accuracy, coverage probability, and reliability of estimates with MCMC by comparison with other estimation methods (discard nondetects, substituting half of reporting limit, Kaplan-Meier, regression on order statistics, and maximum likelihood estimation) using 1000 randomly generated data subsets (n = 150) with the obtained parameters. The evaluation shows that MCMC is the best estimation method in this context with greater accuracy, coverage probability, and reliability over a censored percentage of 10-90%. The mean concentration, which was estimated with MCMC, was 0.289×10-3 mg/L and this value was sufficiently lower than the regulated value of 0.05 mg/L stipulated by the Food Sanitation Act.

Changes in copper, zinc and cadmium distributions in the liver of Formosan squirrels with characteristic high copper accumulation.

We discovered previously that Formosan squirrels (Callosciurus erythraeus) accumulate copper (Cu) in their livers at levels averaging 1700 µg per dry g (approximately 420 µg per wet g). In the current study, we investigated the relationship between Cu accumulation and hepatic injury, and we determined the distribution and chemical form of Cu in the liver supernatant. In particular, we explored the role of metallothionein in the liver supernatant. We observed no significant differences in hepatic Cu concentration between squirrels that showed pathological changes in the liver and those that did not. Serum alanine aminotransferase activity did not increase with increasing hepatic Cu concentration. These results suggest that abnormal Cu accumulation in the livers of Formosan squirrels does not induce severe hepatic injury. We found that 26.7% of the Cu in the liver was distributed to the supernatant, and only 11.0% of the Cu in the liver was bound to metallothionein, suggesting that metallothionein in the hepatic supernatant does not contribute to detoxification of excess Cu in Formosan squirrels.

Quantification of proteins by measuring the sulfur content of their constituent peptides by means of nano HPLC-ICPMS.

The sulfur (S) concentrations of three peptides were determined by using nano HPLC-ICPMS under a flow of O2 in an octapole reaction cell, and the determined values showed a good agreement with theoretical values. This method was then applied to trypsin-digested peptides from human albumin for protein quantification. Assigning of the number of S atoms in each peptide/peak and the tryptic digestion efficiency were important for protein quantification. The number of S atoms in each peptide/peak was assigned by using verification scores that gave the lowest standard deviation of the peptide S concentration and the highest S recovery. The peptide concentrations were calculated as the ratio of the S concentration/the number of S atoms in the peptide/peak. The tryptic digestion efficiency was calculated as the sum of the S concentration in the mono-peptides divided by the total S concentration in a native polyacrylamide gel electrophoresis (PAGE) band before tryptic digestion. Our result indicates that a protein can be quantified through peptide quantification, after taking into account the tryptic digestion efficiency, via S quantification using ICPMS.

Selenium metabolism and excretion in mice after injection of (82)Se-enriched selenomethionine.

The organic Se compounds (particularly selenomethionine [SeMet]) in plants and yeasts are very effective chemoprotectants for mammalian cancer. To characterize the dynamics of selenomethionine utilization pathways, we intravenously injected (82)Se-enriched SeMet into mice under different nutritional states (Se-adequate and Se-deficient mice) and then measured their endogenous and exogenous (82)Se levels. Furthermore, we quantified Se compounds and selenoproteins in liver, kidneys, plasma, and urine. The average recoveries of exogenous (82)Se from solid tissues, urine, and feces were 81% for Se-adequate mice and 84% for Se-deficient mice. Exogenous (82)Se was distributed in the hepatic and renal cytosols as cellular glutathione peroxidase (cGPx), selenosugar, and SeMet within 1 h after injection. Synthesis of cGPx was maintained until 72 h after injection, regardless of the Se nutritional status. Whereas plasma levels of exogenous (82)Se as selenoprotein P (Sel-P) peaked at 6 h after injection, those of Se-containing albumin (SeAlb), extracellular GPx, and SeMet peaked at 1 h after injection. These results suggest three Se transport pathways in mice injected with SeMet: SeAlb (within 1 h after injection); SeMet (from 1 to 72 h after injection); and Sel-P (from 6 to 72 h after injection). The amount of Sel-P in Se-deficient mice was 1.5 times that of Se-adequate mice, and this increase was much larger than Se-containing compounds other than Sel-P. Our results indicate that Sel-P has an important role in Se transport when the nutritional supply of Se is insufficient.

Scanning protein analysis of electrofocusing gels using X-ray fluorescence.

Recently, "metallomics," in addition to genomics and proteomics, has become a focus as a novel approach to identify sensitive fluctuations in homeostasis that accompany metabolic processes, such as stress responses, differentiation, and proliferation. Cellular elements and associated protein behavior provide important clues for understanding cellular and disease mechanism(s). It is important to develop a system for measuring the native status of the protein. In this study, we developed an original freeze-dried electrofocusing native gel over polyimide film (native-gel film) for scanning protein analysis using synchrotron radiation excited X-ray fluorescence (SPAX). To our knowledge, this is the first report detailing the successful mapping of metal-associated proteins of electrofocusing gels using X-ray fluorescence. SPAX can provide detection sensitivity equivalent to that of LA-ICP-MS. In addition to this increased sensitivity, SPAX has the potential to be combined with other X-ray spectroscopies. Our system is useful for further applications in proteomics investigating cellular element-associated protein behaviors and disease mechanisms.

Isolation of selenoprotein-P and determination of Se concentration incorporated in proteins in human and mouse plasma by tandem heparin affinity and size-exclusion column HPLC-ICPMS.

Sel-P is considered to be the most important selenoprotein for evaluating the selenium (Se) status in the body. To isolate and determine Sel-P in plasma, we have developed an analytical method combining heparin affinity (AF) and size-exclusion column (SEC) high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). We used this method to validate the adsorption efficiency of selenoproteins on a heparin AF column, and to then determine the Se concentrations incorporated in proteins in human and mouse plasma. The adsorption efficiency of Sel-P on a heparin column was more than 90% for both human and mouse plasma. Tandem AF and SEC separation proved to be useful for determining the Se concentrations incorporated in Sel-P in mouse plasma, but not in human plasma, because of nonspecific adsorption of plasma-extracellular glutathione peroxidase (eGPx) and albumin on the heparin AF column. Ultimately, we used the tandem AF and SEC separation method for mouse plasma and SEC separation alone for human plasma. The Se concentration incorporated in selenoproteins determined by our method showed good agreements with the total Se concentration determined following acid digestion.

Cycling of rare earth elements in the atmosphere in central Tokyo.

Concentrations of 14 rare earth elements (REEs) in six size classes of airborne particulate matter (APM) (<0.43, 0.43-0.65, 0.65-1.1, 1.1-2.1, 2.1-11, and >11 µm) and in two different phases (suspended particulate and dissolved) in rainwater were determined by inductively coupled plasma mass spectrometry (ICP-MS). Positive Eu and Tb anomalies were observed in size-classified APM. These anomalies may be due to large emissions of Eu and Tb to the atmosphere resulting from the recent change in Japan from the use of cathode-ray tubes to plasma displays in television sets (Eu and Tb) and from the widespread use of magneto-optical disks (Tb). The light REEs were enriched in fine APM particles (diameter < 1.1 µm). Because compositions of La/Ce/Sm in fine APM (diameter < 1.1 µm) were similar to those in automobile catalyst, the light REE enrichment was attributed to automobile emissions. In contrast, the REE distribution pattern in the suspended particulate phase in rainwater was similar to that in coarse APM (diameter > 2.1 µm), and a positive Tb anomaly was observed, suggesting that coarse particles easily become trapped in rain droplets. A negative Eu anomaly was observed in the dissolved phase in rainwater, but not in APM or in the suspended particulate phase in rainwater. Unlike other REEs, Eu can exist as both bivalent and trivalent ions in nature, and Eu-selective dissolution from or adsorption onto the trapped particles of Eu might account for the negative anomaly. These results show that atmospheric REE cycling is affected by the physico-chemical properties of APM.

Dynamic pathways of selenium metabolism and excretion in mice under different selenium nutritional statuses.

The selenoprotein, cellular glutathione peroxidase (cGPx), has an important role in protecting organisms from oxidative damage through reducing levels of harmful peroxides. The liver and kidney in particular, have important roles in selenium (Se) metabolism and Se is excreted predominantly in urine and feces. In order to characterize the dynamics of these pathways we have measured the time-dependent changes in the quantities of hepatic, renal, urinary, and fecal Se species in mice fed Se-adequate and Se-deficient diets after injection of (82)Se-enriched selenite. Exogenous (82)Se was transformed to cGPx in both the liver and kidney within 1 h after injection and the synthesis of cGPx decreased 1 to 6 h and continued at a constant level from 6 to 72 h after injection. The total amount of Se associated with cGPx in mice fed Se-deficient diets was found to be less than in mice fed Se-adequate diets. This finding indicated that cGPx synthesis was suppressed under Se-deficient conditions and did not recover with selenite injection. Excess Se was associated with selenosugar in liver and transported to the kidney within 1 h after injection, and then excreted in urine and feces within 6 h after injection. Any excess amount of Se was excreted mainly as a selenosugar in urine.

Determination of rare earth elements (REES) in airborne particulate matter (APM) collected in Tokyo, Japan, and a positive anomaly of europium and terbium.

The determination of rare earth elements (REEs) in airborne particulate matter (APM) was conducted, and the distribution pattern of atmospheric REEs was evaluated in this study. The APM was collected in the center of Tokyo, Japan, where serious air pollution is always of concern. A cellulose acetate membrane filter was used to collect the APM because Ba and REEs contamination is lower than that in a quartz glass fiber filter. The REEs measurement was conducted by ICP-MS after the digestion of the APM by a microwave acid digestion procedure. The standard reference material (SRM) of NIST 1648 urban particulate matter was used to validate the accuracy of the analytical method. The analytical results for SRM well agreed with those of the reference and reported values. Consequently, the analytical method established in this study was applied to the determination of REEs in APM collected in Tokyo, Japan. The obtained REEs distribution pattern in the APM showed a positive anomaly of Tb and Eu. The La/Sm ratio, which is considered to be as a good indicator of the anthropogenic effect, in size-classified APM showed a high degree of the anthropogenic effect in fine APM with a diameter of <1.1 µm. Emission sources of Tb, Eu and other REEs are discussed.

Distribution and dynamic pathway of selenium species in selenium-deficient mice injected with (82)Se-enriched selenite.

In order to elucidate Se metabolism in a living body, (82)Se-enriched selenite was injected intravenously into mice fed Se-adequate and -deficient diets. We studied the time-dependent changes in the distribution of the labeled Se in organs, red blood cells, and plasma. The total Se was determined by flow-injection ICPMS, and Se speciation analysis was conducted by micro-affinity chromatography coupled with low-flow ICPMS. Total Se in almost all organs, including liver, showed the maximum at 1 h after injection. From speciation analysis, exogenous (82)Se as Se-containing proteins other than selenoprotein P (Sel-P) (selenium containing albumin (SeAlb) and extra cellular glutathione peroxidase (eGPx)), peaked at 1 h and quickly decreased from 1 to 6 h after injection, whereas that as Sel-P, peaked at 6 h, and gradually decreased from 6 to 72 h after injection. We found that there were two pathways for the transfer of Se in mice; one was as SeAlb until 1 h after injection, and the other was as Sel-P from 6 to 72 h after injection.

Accumulation of trace elements used in semiconductor industry in Formosan squirrel, as a bio-indicator of their exposure, living in Taiwan.

Concentrations of 17 trace elements were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS) in Formosan squirrels (Callosciurus erythraeus) of Taiwan and Japan to document trace element pollution in Taiwan. High concentrations of elements used to produce semiconductors - Ga, As, Cd, In and Tl - were found in animals captured in Miaoli County, which is the nearest site to Hsinchu City, a chief city of Taiwan's semiconductor industry. Significant correlations between Ga, As, In and Tl were found in the kidney, liver, lung and muscle tissues of Taiwanese squirrels. Hierarchical cluster analysis indicated that Ga, As, In and Tl were of the same clade, indicating that Ga, As, In and Tl were discharged from an identical origin. Molar ratios of Ga/As concentration in lungs of animals captured in Miaoli resembled those of animals after intratracheal administration of particulate gallium arsenide (GaAs). This result might indicate that the higher concentrations of Ga and As in the specimens in Miaoli resulted from atmospheric exposure to GaAs.

Pharmacokinetics of arsenic species in Japanese patients with relapsed or refractory acute promyelocytic leukemia treated with arsenic trioxide.

PURPOSE: To investigate the pharmacokinetics of arsenic species in Japanese patients with relapsed or refractory acute promyelocytic leukemia (APL) treated with arsenic trioxide (ATO) at a daily dose of 0.15 mg/kg. METHODS: Inorganic arsenic (AsIII and AsV) and the major metabolites monomethylarsonic acid (MAA(V)) and dimethylarsinic acid (DMAA(V)) in plasma and urine collected from 12 Japanese patients were quantified by HPLC/ICP-MS. RESULTS: The plasma concentrations of AsIII and AsV on day 1 reached the similar Cmax (12.4 +/- 8.4 and 10.2 +/- 3.9 ng/ml) immediately after completion of administration followed by a biphasic elimination. The AUC(0-infinity) of AsV was about twice that of AsIII. The appearance of methylated metabolites in the blood was delayed. During the repeated administration, the plasma concentrations of inorganic arsenic reached the steady state. In contrast, the MAA(V) and DMAA(V) concentrations increased in relation to increased administration frequency. The mean total arsenic excretion rate including inorganic arsenic and methylated arsenic was about 20% of daily dose on day 1 and remained at about 60% of daily dose during week 1-4. CONCLUSIONS: This study demonstrates that ATO is metabolized when administered intravenously to APL patients and methylated metabolites are promptly eliminated from the blood and excreted into urine after completion of administration, indicating no measurable accumulation of ATO in the blood.