Development of an Analytical Method for Detection of Anesthetics and Sedatives in Fish.

BACKGROUND: Anesthetics and sedatives are frequently used to prevent abrasions caused by stress and to facilitate fish management. However, drug residues may persist and cause changes in fish conditions and induce side effects. In addition, drugs that are not permitted for use in edible fish are sometimes potentially used in fish. The drugs can also be found in wastewater and are likely to be detected in fish. OBJECTIVE: The purpose of this study was to establish a quantitative analytical method for 10 anesthetic and sedative (azaperone, chlorpromazine, diazepam, estazolam, haloperidol, nitrazepam, nordiazepam, oxazepam, perphenazine, and temazepam) residues in fish sold in Korean markets. METHOD: Shrimp, flounder, and eel samples were selected as matrices. Acetonitrile (ACN) containing 0.1% formic acid was selected as an extraction solvent for shrimp and 100% ACN for flounder and eel. The QuEChERS method with C18 and primary secondary amine (PSA) was used as the extraction procedure, and the analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Limit of quantitation, recovery, accuracy, and precision were validated, and satisfactory results were obtained for the drugs. All results applied to the real samples were negative. CONCLUSIONS: An optimal validation method was studied. Since the results for all samples were negative, it is considered that additional studies are needed by increasing the number of drugs. HIGHLIGHTS: The most effective QuEChERS pretreatment method and conditions of LC-MS/MS for the analysis of anesthetics and sedatives in fish were established.

Authentication of tejocote (Crataegus mexicana) dietary supplements based on DNA barcoding and chemical profiling.

Tejocote (Crataegus mexicana, Mexican hawthorn), known as a weight-loss supplement, has been marketed online and is easily available for overseas direct purchase. Alipotec (brand name) is known as one of the most popular products containing tejocote in Mexico and other countries. However, adverse effects have been reported by users of these supplements. Therefore it is necessary to find the reason for the side effect. Dietary supplement samples labelled as containing tejocote were analysed using mass spectrometry and DNA barcoding analysis. Our results demonstrate that Alipotec samples contained ingredients from different species, yellow oleander instead of tejocote. The rpoB barcode region was able to differentiate between tejocote and yellow oleander species. Moreover, it was also observed that three compounds, including thevetin B, neriifolin, and digitoxigenin, clearly distinguish between tejocote and yellow oleander samples. This is the first and preliminary investigation to use an integrated approach of both chemical and genomic profiling for the authentication of dietary supplement containing tejocote.

Multi-Class Determination of 64 Illicit Compounds in Dietary Supplements Using Liquid Chromatography-Tandem Mass Spectrometry.

In this work, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for screening and confirmation of 64 illicit compounds in dietary supplements. The target compounds were illegally used pharmaceutical drugs, prohibited compounds, and not authorized ingredients for different therapeutics (sexual enhancement, weight loss, muscular strengthening, and relaxing products). The validation procedure was performed to evaluate selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision according to the Association of Official Analytical Chemists guidelines. The linearity was >0.98 in the range of 0.5-200 µg L-1. The LOQs were in the range 1-10 µg kg-1 for all target compounds. The accuracy (expressed as recovery) was 78.5-114%. The precision (expressed as the relative standard deviation) was below 9.15%. The developed method was applied for the determination of illicit compounds in dietary supplements collected from websites. As a result, the total detection rate was 13.5% (27 samples detected in 200 samples). The concentrations of detected samples ranged from 0.51 to 226 mg g-1. The proposed methodology is suitable for monitoring the adulteration of illicit compounds in dietary supplements.

Comparison of Sample Preparation and Determination of 60 Veterinary Drug Residues in Flatfish Using Liquid Chromatography-Tandem Mass Spectrometry.

This study was performed to optimize the analytical method for multi-residues of 60 compounds in flatfish samples. Three sample preparation methods were tested to identify the optimal recovery conditions for target analytes. As a result, 10 mL of water/acetonitrile (1:4, v/v) was used to extract analytes from fish samples. For purification, C18 and 10 mL of acetonitrile saturated hexane were used to treat the samples. After evaporation and reconstitution, the fish samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The proposed method was validated according to the CODEX guidelines (CAC/GL-71). Our results showed the recoveries of 73.2%-115% and coefficients of variation of 1.6%-22.1%. The limit of quantification was 0.0005-0.005 mg/kg in the fishery products. In analysis of real samples, no samples exceeded the limit of quantification. This analytical method can be used for multi-residue screening and confirmation of the residues of veterinary drugs in fishery products.

Association of urinary acrylamide concentration with lifestyle and demographic factors in a population of South Korean children and adolescents.

Acrylamide (AA) has been identified as probably carcinogenic to humans and thus represents a potential public health threat. This study aimed to determine the urinary concentrations of AA and N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) in a nationally representative sample (n = 1025) of children and adolescents (age range 3-18 years) in South Korea. The AA and AAMA detection rates and geometric mean concentrations were 97%, 19.1 ng/mL, and 98.7%, 26.4 ng/mL, respectively. Although urinary AA levels did not vary widely by age (17.2 ng/mL at 3-6 years, 19.9 ng/mL at 7-18 years), the urinary concentration of AAMA increased with age (18.3 ng/mL at 3-6 years, 30.4 ng/mL at 7-18 years). A multiple linear regression analysis revealed that the urinary levels of AA and AAMA varied significantly by sex, with the adjusted proportional changes indicating rates of 1.47- to 1.48-fold higher at 3-6 years and 1.36- to 1.68-fold higher at 7-18 years among males relative to females. Furthermore, the urinary levels of AA and AAMA correlated with the consumption of certain foods (doughnuts, hotdogs, popcorn, and nachos) among male subjects aged 7-18 years. The urinary concentrations of AA and AAMA increased significantly with the smoking status and passive smoking exposure, with adjusted proportional changes of 1.51 to 1.71-fold higher among smokers relative to non-smokers in the age range of 7-18 years. Exposure to smoking for > 30 min led to adjusted proportional increases in AA and AAMA of 1.51 and 1.77 times in the non-smoking group aged 3-6 years and a 1.52-fold increase in AAMA in the non-smoking group aged 7-18 years. In conclusion, the urinary levels of AA and AAMA were found to associate with age, sex, smoking, and food consumption in a population of Korean children and adolescents.

Assessment of human estrogen receptor agonistic/antagonistic effects of veterinary drugs used for livestock and farmed fish by OECD in vitro stably transfected transcriptional activation assays.

The presence of veterinary drug residues in foods and the environment could potentially cause adverse effects on humans and wildlife. Several veterinary drugs were reported to exhibit endocrine disrupting effects via binding affinities to sexual hormone receptors such as estrogen and androgen receptors. Therefore, we confirmed the human estrogen receptor (ER) agonistic/antagonistic effects of 135 chemicals that were used as veterinary drugs in Korea by the official Organization for Economic Cooperation and Development (OECD) in vitro ER transcriptional activation (TA) assay using the VM7Luc4E2 cell line. In the case of ER agonist screening, 7 veterinary drugs (cefuroxime, cymiazole, trenbolone, zeranol, phoxim, altrenogest and nandrolone) were determined to be ER agonists. In addition, only zeranol was found to exhibit weak ER antagonistic activity. These 7 veterinary drugs, which were determined as ER agonists and/or antagonists by an OECD in vitro assay, were also found to have binding affinity to ERs. These results indicate that various veterinary drugs possess potential (anti-)estrogenic effects. However, further study is needed to determine the precise endocrine-disrupting effects of these compounds.

Web-based Korean maximum residue limit evaluation tools: an applied example of maximum residue limit evaluation for trichlorfon in fishery products.

To ensure public safety against veterinary drug residues in food products from animal sources, maximum residue limits (MRLs) should be established by scientific evidence and a transparent estimation process. The Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) developed an Excel workbook-based tool for MRLs evaluation in 2003. In this study, we developed a web-based tool for MRL evaluation, called Korean MRL evaluation tools (KMET). While KMET used algorithms of JECFA workbook, it added some databases (e.g., Korean food consumption database) and provided additional functions (e.g., selection of target marker residue). Web-based KMET enabled regulatory policy makers to update the database. All input data and output results related to MRL evaluation based on residue depletion and food consumption datasets were archived and provided overall processes from the initial depletion data entry to MRL establishment with user-friendly interface. Our results demonstrated the stepwise processes whereby MRL for trichlorfon in the muscle of Paralichthys olivaceus was established with functional descriptions of KMET. MRL for trichlorfon derived from KMET was proposed and notified by the Ministry of Food and Drug Safety in 2018.

Prevalence of Antibiotic Residues and Antibiotic Resistance in Isolates of Chicken Meat in Korea.

The aim of study was to investigate the correlation between the level of 17 antibiotic residues and 6 antibiotic resistances of Escherichia coli isolates in chicken meats. A total of 58 chicken meats were collected from retail grocery stores in five provinces in Korea. The total detection rate of antibiotic residues was 45% (26 out of 58). Ten out of 17 antibiotics were detected in chicken meats. None of the antibiotics exceeded the maximum residue level (MRLs) in chicken established by the Ministry of Food and Drug Safety (MFDS). The most detected antibiotics were amoxicillin (15.5%), followed by enrofloxacin (12.1%) and sulfamethoxazole (10.3%). In a total of 58 chicken meats, 51 E. coli strains were isolated. E. coli isolates showed the highest resistance to ampicillin (75%), followed by tetracycline (69%), ciprofloxacin (65%), trimethoprim/ sulfamethoxazole (41%), ceftiofur (22%), and amoxicillin/clavulanic acid (12%). The results of study showed basic information on relationship between antibiotic residue and resistance for 6 compounds in 13 chicken samples. Further investigation on the antibiotic resistance patterns of various bacteria species is needed to improve food safety.

Veterinary drug, 17ß-trenbolone promotes the proliferation of human prostate cancer cell line through the Akt/AR signaling pathway.

Trenbolone acetate (TBA) is a synthetic anabolic steroidal growth factor that is used for rapid muscle development in cattle. The absorbed TBA is hydrolyzed to the active form, 17ß-trenbolone (17 TB; 17ß-hydroxy-estra-4,9,11-trien-3-one) in meat and milk products, which can cause adverse health effects in humans. Similar to 5α-dihydrotestosterone (DHT), 17 TB was reported to exhibit endocrine disrupting effects on animals and humans due to its androgenic effect via binding to the androgen receptor. The purpose of this study is to investigate the molecular mechanism of cell proliferation in prostate cancer (PCa) cells treated with 17 TB. We found that 17 TB induces AR-dependent cell proliferation in the human prostate cancer cell line, 22Rv1 in a concentration dependent manner. Treatment with 17 TB increased the expression of cell cycle regulatory proteins, cyclin D2/CDK-4 and cyclin E/CDK-2, whereas the expression of p27 was down-regulated. Furthermore, phosphorylation of Rb and activation of E2F were also induced, which suggests the activation of cyclin D2/CDK-4 and cyclin E/CDK-2 in the cells. When 22Rv1 cells were exposed to 30 pM of 17 TB, which is the effective concentration (EC50) value required to observe proliferative effects on 22Rv1 cells, the expression levels of the phosphorylated forms of Akt and GSK3ß were increased. This study demonstrates that 17 TB induces AR-dependent proliferation through the modulation of cell cycle-related proteins in the Akt signaling pathway. The present study provides an effective methodology for identifying cell proliferation signaling of veterinary drugs that exert AR agonistic effects.

Assessment of androgen receptor agonistic/antagonistic effects on 25 chemicals in household applicants by OECD in vitro stably transfected transcriptional activation assays.

The aim of this study is to assess the androgen receptor (AR) agonistic/antagonistic effects on various chemicals, which are used in household products including cleaning agents and wetted tissues by in vitro OECD test guideline No. 458 (using AR-EcoScreen™ cell line) and the me-too test method (using 22Rv1cell line), which was adopted as OECD project No. 4.99. All chemicals were not determined as AR agonists. However α-dodecyl-ω-hydroxypoly (oxyethylene) and 3-iodo-2-propynyl butylcarbamate have shown a weak AR antagonistic effects with IC50 values of 2.18 ±â€¯0.12 and 4.26 ±â€¯0.17 µg/ml via binding affinity to AR in only 22Rv1/mouse mammary tumor virus using AR transcriptional activation assay, because of their different cytotoxicity on each applied cell line. This report firstly provides information about agonistic/antagonistic effects against human AR of various chemicals including surfactants and biocides by OECD in vitro stably transfected transcriptional activation assays. However, further in vivo and human model studies are needed to confirm their adverse effects.

In vitro OECD test methods applied to screen the estrogenic effect of chemicals, used in Korea.

In this study, 27 chemicals found in household products, which became an issue in Korea were screened for the agonistoc and antagonistic effects against human estrogen receptor using official Organization for Economic Cooperation and Development (OECD) in vitro assays, STTA assay using ERα-HeLa-9903 cell line and BG1Luc ER TA assay. In the case of human ER agonist screening by two assays, all tested chemicals did not show agonist effect against ER. In ER antagonist test by BG1Luc ER TA assay, five surfactants α-dodecyl-ω-hydroxypoly(oxyethylene), alcohols C16-18 ethoxylated, nonylphenol, ethoxylated, 3,6,9,12,15,18,21-heptaoxatritriacontan-1-ol, and α-dodecyl-ω-hydroxypoly(oxy-1,2-ethanediyl)) were found to exhibit weak antagonistic activities. The agonist/antagonist effects against human estrogen receptor of various chemicals, used in Korea by OECD test guideline are reported in this study. These results indicated that two OECD in vitro assays will can be applied in Korea by screening of agonistic/antagonistic effects against human ER of various chemicals.

Urinary benzophenone concentrations and their association with demographic factors in a South Korean population.

Benzophenone (BP) and its derivatives are widely used in various cosmetics, personal care products, and food packaging ink. The use of BP has raised concerns about the potential health risks associated with its endocrine-disrupting effects. This study evaluated urinary concentrations of BP derivatives in a national sample of the South Koreans population aged 6-89 years. From July to September in each 2010 and 2011, 1576 urine samples were collected. Urinary concentrations of benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), benzophenone-4 (BP-4), benzophenone-8 (BP-8), and 4-hydroxybenzophenone (4-OH-BP) were analyzed using liquid chromatography-mass spectrometry. The detection rate for BP-1 and 4-OH-BP were 56% [limit of detection (LOD) 0.59ng/mL] and 88% (LOD 0.04ng/mL), respectively, whereas those for BP-2, BP-3, BP-4, and BP-8 were all below 25%. The geometric means of urinary BP-1 and 4-OH-BP concentrations were 1.24ng/mL and 0.45ng/mL, respectively. Multiple linear regression analysis indicated that concentrations of BP-1 in and of 4-OH-BP in adults were associated with sex and age. The BP-1 and 4-OH-BP concentration of children and adolescents was associated with sex, age, income, and current area of residence. The correlation was observed between urinary concentrations of BP derivatives, which is an important indication of exposure biomarkers and the metabolic pathways from BP-3. This is the first national study to evaluate the presence of BP derivatives in urine samples from the South Korean population, stratified by demographic factors.

Risk assessment based on urinary bisphenol A levels in the general Korean population.

Bisphenol A (BPA) is a high-volume industrial chemical used in the global production of polycarbonate plastics and epoxy resins, which are used in food and drink containers, such as tableware (plates and mugs). Due to its broad applications, BPA has been detected in human blood, urine and breast milk as well as environmental substances, including water, indoor and outdoor air, and dust. Indeed, exposure to high concentrations of BPA can result in a variety of harmful effects, including reproductive toxicity, through a mechanism of endocrine disruption. Our comparison of reported BPA urinary concentrations among different countries revealed that exposures in Korea may be higher than those in other Asian countries and North America, but lower than or similar to those in European countries. The current study included a total of 2044 eligible subjects of all ages. The subjects were evenly divided between males and females (48.58% and 51.42%, respectively). The geometric mean (GM) of pre-adjusted (adjusted) urinary BPA concentrations was 1.83µg/L (2.01µg/g creatinine) for subjects of all ages, and there was no statistically difference in BPA concentrations between males (1.90µg/L, 1.87µg/g creatinine) and females (1.76µg/L, 2.16µg/g creatinine). Multiple regression analysis revealed only one positive association between creatinine pre-adjusted urinary BPA concentration and age (ß=-0.0868, p<0.001). The 95th percentile levels of 24-hour recall (HR), food frequency questionnaires (FFQ) and estimated daily intake (EDI) through urinary BPA concentrations were 0.14, 0.13, and 0.22µg/kg bw/day, respectively. According to the Ministry of Food and Drug Safety (MFDS), a tolerable daily intake (tDI) of 20µg/kg bw/day was established for BPA from the available toxicological data. Recently, the European Food Safety Authority (EFSA) established a temporary TDI of 4µg/kg bw/day based on current toxicological data. By comparing these TDIs with subjects' exposure, we conclude that there are no health concerns for any age group as a result of current levels of dietary exposure to BPA.

Urinary concentrations of parabens and their association with demographic factors: A population-based cross-sectional study.

Parabens are broad-spectrum antimicrobial agents used in a range of consumer products, including personal care products, cosmetics, and food. Recently, the widespread use of parabens has raised concerns about the potential health risks associated with their endocrine-disrupting effect. In the present study, 2541 urine samples were collected and analyzed by liquid chromatography-mass spectrometry for the determination of the concentrations of methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP). The detection rate and geometric mean concentrations of parabens in the general population followed the order MeP (97.7%; 116ng/mL)>EtP (97.2%; 24.7ng/mL)>PrP (96.7%; 11.0ng/mL)>BuP (83.5%; 1.13ng/mL). The composition profiles showed that MeP and EtP accounted for >90% of the urinary paraben concentration. We performed statistical analysis in order to evaluate differences between demographic variables and urinary concentrations. Our results showed that adjusted proportional change of MeP, PrP, and BuP in adults were 2.67-6.13 times higher in females than in males. The urinary concentrations of PrP in adults increased significantly with age. The adjusted proportional changes of MeP and PrP in adults were associated with increased body mass index (BMI). The adjusted proportional changes of BuP and PrP in children and adolescents were 1.44 and 1.69 times higher in females than in males. However, there was no clear association between paraben concentrations and demographic variables in the children and adolescents groups. The estimated daily intake (EDIurine) of MeP and EtP in adults were 301µg/kg bw/day, which is lower than the acceptable daily intake (ADI; 10mg/kg bw/day). In summary, our results revealed that the general population in Korea was exposed to parabens during 2009-2010, and most Koreans are exposed to parabens. The urinary levels of parabens varied by age group with demographic factors in the Korean population. The results of study may be used to establish a nationally representative baseline of exposure to parabens in risk assessment.

The Association Between Urinary Benzophenone Concentrations and Personal Care Product Use in Korean Adults.

Benzophenone (BP) derivatives are widely used in personal care products (PCPs) for protection from ultraviolet radiation. Because of their broad applications, BP derivatives have been found in various human bodily fluids. In the present study, we investigated the relationship between urinary concentrations of BP derivatives and PCP use in Korean adults. A urinary BP biomonitoring survey was conducted in Korea by the Ministry of Food and Drug Safety in 2014. BP derivatives (BP-1, BP-3, and 4-OH-BP) were measured in urine samples from 168 Korean adults (mean age, 43.2 ± 15.4 years) by high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry. Information about the use of PCPs in the past 7 days was obtained by direct interviews. The mean levels of BP-1, BP-3, and 4-OH-BP were 0.87, 5.87, and 0.13 ng/g creatinine, respectively. The geometric mean levels of BP-1, BP-3, and 4-OH-BP were significantly higher in female than those in male. The medians of the urinary concentration of BP derivatives were significantly higher among users of the following PCPs than those in non-users; the PCPs included sunscreen, skin care products, functional cosmetics, makeup base, makeup, lip cosmetics, eye cosmetics, color cosmetics, perfume products, and nail products. A regression analysis revealed a significant linear association between urinary BP-3 concentrations and the number of additional cosmetic products used. These findings provide evidence of a positive association between exposure to PCPs and urinary BP derivative concentrations in Korean adults.

Total mercury concentrations in the general Korean population, 2008-2011.

The purpose of this study was to evaluate the blood mercury levels of the general Korean population. The data from subjects of all ages were pooled from recent national surveys. In the combined surveys, the geometric means (GMs) of the blood mercury concentrations in subjects aged 0-7, 8-18, and 19years and above were 2.05 (2010-2011), 2.12 (2010-2011), and 3.74µg/L (2008-2011), respectively. There was an increasing trend in blood mercury levels with age until 59years and then a slight decline in the group above 60years. The time trend for exposure to mercury in Korea also showed a marked decline. In comparing estimated methylmercury exposure to the Korean health-based guidance value (tolerable weekly intake [TWI]: 2.0µg/kgbw/week), the GMs of methylmercury exposure for subjects aged 0-7, 8-18, and 19years and above were 0.30, 0.31, and 0.43µg/kgbw/week, respectively, while methylmercury exposure did not exceed the TWI (15.0%, 15.5%, and 21.5% compared to the TWI, respectively). The 95th percentiles of estimated methylmercury exposure ranged from 0.71 to 1.61µg/kgbw/week, which was not above the TWI (range, 35.5-80.5%).

Bisphenol A exerts estrogenic effects by modulating CDK1/2 and p38 MAP kinase activity.

Bisphenol A (BPA) is considered to be an endocrine disruptor, but the mechanisms by which it disrupts endocrine functions are poorly understood. Here, we have shown that BPA binds both estrogen receptor (ER)-α and ER-beta (ER-ß) using a fluorescence polarization competitive binding assay. In addition, we found that BPA induced cell proliferation by modulating cell cycle-related genes in the MCF-7 human mammary cancer cell line. Moreover, using a BG1 luciferase ER transactivation assay, we found that BPA has estrogenic activity. Modulating the MAPK pathway by using an ERK inhibitor (PD98059) or a JNK inhibitor (SP600125) had no effect on the ability of BPA to induce estrogenic activity. However, the antiestrogen, ICI 182,780, and the p38 inhibitor, PD 169316 successfully blocked BPA-induced estrogenic activity. Our findings suggest that BPA mimics ER-dependent estrogenic activity by targeting proteins that regulate the cell cycle and p38 MAPK.

Association between Urinary Bisphenol A and Waist Circumference in Korean Adults.

Bisphenol A (BPA) is widely used in the production of polycarbonate plastics, epoxy resins, and food and beverage containers. In the present study, we aimed to investigate the relationship between urinary concentrations of BPA and waist circumference in Korean adults. A total of 1,030 Korean adults (mean age, 44.3 ± 14.6 years) were enrolled in the study on the integrated exposure to hazardous materials for safety control, conducted by the Ministry of Food and Drug Safety from 2010 to 2012. Abdominal obesity was defined as having a waist circumference of at least 90 cm and 85 cm for men and women, respectively. The participants were divided into 4 groups according to the urinary BPA concentration quartile. Waist circumference was significantly higher among subjects with a urinary BPA concentration in the highest quartile relative to those in the lowest quartile (p = 0.0071). Linear regression analysis revealed a significant positive association between urinary BPA concentrations and body mass index, body fat, after adjusting for potential confounders. Moreover, subjects with urinary BPA concentrations in the fourth quartile were more likely to be obese compared to those with urinary BPA concentrations in the first quartile (odds ratio, 1.938; 95% CI: 1.314~2.857; p for trend = 0.0106). These findings provide evidence for a positive association between urinary BPA concentration and waist circumference in Korean adults.