Highly sensitive reporter gene assay for dioxins in human plasma by using cycloheximide as an enhancer substance.

A reporter gene assay (RGA) that uses a mouse liver recombinant Hepa1c1c7 containing the firefly luciferase gene was developed to screen for dioxins in human plasma. For a high-sensitivity method, the addition of cycloheximide to the culture medium brought about a fivefold increase in the sensitivity. The detection limit was 0.1 pg/microL/well. Aryl hydrocarbon receptor (AhR) binding affinity factors (AhR-BAF), calculated from the effect concentration 50 (EC(50)) value, showed approximately the same values as those in WHO-TEF (2006). A significant correlation between RGA and the conventional gas chromatography/mass spectrometry (GC/MS) method was obtained.

Impact of determination of hepatitis B virus subgenotype and pre-core/core-promoter mutation for the prediction of acute exacerbation of asymptomatic carriers.

AIM: A large cohort study in Japan revealed that the specific viral profile may influence the fulminant outcome in acute hepatitis B virus (HBV) infections, while the genetic influence on outcome has not been clarified in patients with acute exacerbation of chronic liver disease caused by HBV. We experienced a case of fatal liver failure that developed as the result of chronic HBV infection. To determine possible genetic factor involving acute exacerbation, genetic analysis of serum from the patient and his siblings was performed. METHODS: HBV subgenotype as well as pre-core/core-promoter mutations of samples mentioned above were determined. RESULTS: Patient had HBV-Bj with pre-core (1896/1899) and core-promoter (1762/1764) mutations, the genomic profile frequently seen in fulminant hepatitis caused by acute HBV infection. CONCLUSION: This result suggests that determination of the HBV subgenotype and pre-core/core promoter mutations could provide a rationale for development of a treatment strategy in asymptomatic HBV carriers.

Application of a novel large-volume injection method using a stomach-shaped inlet liner in capillary gas chromatographic trace analysis of dioxins in human milk and plasma.

A newly developed large-volume injection (LVI) technique that employs a unique stomach-shaped inlet liner (SSIL) inside of a programmable temperature vaporizer was used for the determination of trace amounts of dioxins in human milk and plasma. The initial temperature and the initial dwelling time of the inlet and the kind of solvent used were found to be critical in determining the analytical sensitivity of dioxins due to the loss of these relatively volatile compounds during solvent vaporization. Human milk and plasma were purified and fractionated by pre-packed multi-layered silica-gel chromatography and activated carbon silica-gel column chromatography. A 20-microL aliquot of the fraction collected from the chromatography with toluene was directly applied to the LVI system in high-resolution gas chromatography/high-resolution mass spectrometry. Excellent correlation (r > 0.97) between the values obtained by the LVI method using the SSIL device and those by the conventional regular-volume splitless injection method was obtained for PCDDs, PCDFs and non-ortho PCBs in human milk and plasma samples.

Internal dose-effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in gonadotropin-primed weanling rat model.

Single sc injection of 5 IU equine chorionic gonadotropin (eCG) induces ovulation in weanling female rats 3 days later. It has been shown that treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24 h before eCG injection reduces eCG-stimulated ovarian hypertrophy and inhibits ovulation. The present study intended to compare internal dose-effects of TCDD between these endpoints and representative endpoints for TCDD toxicity, such as weights of the liver and thymus, in weanling female rats given orally 0, 1, 4 or 16 microg/kg TCDD 24 h before eCG injection on postnatal day 25. Measurement of plasma TCDD concentrations by ELISA at 6, 72 and 96 h after TCDD revealed that significant levels of TCDD were maintained in systemic circulation until 96 h (on the day of induced ovulation) with the highest level at 6 h after TCDD treatment. Ovarian TCDD concentrations varied similarly and tended to be higher than those in the thymus at all time points, whereas hepatic concentrations of TCDD were the highest among the tissues. Although > or = 4 microg/kg TCDD affected the weights of the thymus and liver, no differences were observed in ovarian weights at any time point or in ovulation between corn oil-treated and TCDD-treated groups. Furthermore, ovarian levels of representative mRNAs in follicles were not affected by TCDD treatment. Since TCDD increased the amount of cytochrome P450 1A1 mRNA in the ovary, the administered TCDD stimulated the aryl hydrocarbon receptor-signaling pathway. From these results, we concluded that thymus weights of weanling female rats responded to TCDD at a lower internal dose as compared with that ovarian hypertrophy and follicular growth from early antral stage to ovulation would respond to.

Systematic analysis and overall toxicity evaluation of dioxins and hexachlorobenzene in human milk.

A systematic method for analyzing dioxins (PCDDs, PCDFs and dioxin-like PCBs), hexachlorobenzene (HCB), heptachlor epoxide and beta-hexachlorocyclohexane (HCH) in human milk was developed to determine the residual amount of HCB in human milk and to evaluate the overall toxicity of both dioxins and HCB in human milk. The fractionation behavior of HCB on chromatography with silica gel, alumina, and activated carbon/silica gel, and the concentrated sulfuric acid decomposition method, which is widely used as a dioxin cleanup method, were studied in order to make the preprocessing operation for HCB measurement compatible with that for conventional dioxin measurement. HCB was found to be eluted in the 2% dichloromethane (DCM)/hexane 60 ml fraction from an alumina column. Heptachlor epoxide and a part of beta-HCH were eluted in the 10% DCM/hexane 50 ml fraction from a silica gel column, while the remaining beta-HCH was eluted in the 25% DCM/hexane 60 ml fraction from an activated carbon/silica gel column. Moreover, HCB showed significant correlation with dioxin congeners having high toxicity equivalence factors (TEFs). The results suggest that the exposure route to HCB and its accumulation behavior in the human body are similar to those of the dioxins.

Development of a accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis.

A new analytical method has been developed for the quantification of 59 different persistent organohalogen compounds, such as polybrominated diphenyl ethers (PBDEs), polychlorinated naphthalenes (PCNs), polychlorinated biphenyls (PCBs), PCB metabolites, organochlorine pesticides (OCPs) in biological organ tissues. The optimum extraction and cleanup procedures were examined using accelerated solvent extraction (ASE), automated gel permeation chromatography (GPC) on Biobeads S-X3 and automated solid phase extraction (SPE) on silica-gel. The target compounds were divided into two fractions, non-polar compounds and more polar compounds, which in the latter fraction was subsequently methylated using diazomethane. Detection can be achieved by GC/MS in negative chemical ionization (NCI) mode. The average recoveries of the compounds spiked in swine liver, heart, kidney, and cattle adipose tissues were considered satisfactory, and it was confirmed that the method could be used in routine analysis.

Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.

To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.

Extraction and cleanup methods of dioxins in house dust from two cities in Japan using accelerated solvent extraction and a disposable multi-layer silica-gel cartridge.

A simple and rapid method for the extraction and cleanup of dioxins from house dust was developed using an accelerated solvent extraction (ASE) method and a multi-layer silica-gel cartridge. It was found that the WHO-TEQ levels of dioxins extracted from the house dust obtained by both a conventional soxhlet extraction and the ASE were almost equal, when the data obtained by both methods were compared. As for the cleanup method, a multi-layer silica-gel cartridge yielded higher dioxin recoveries than the alkaline digestion method. The average values of the dioxins in house dust from Kumagaya city and Sendai city in Japan (Sendai city is bigger than Kumagaya city with respect to the population and industry), were 15.6 pg TEQ/g (8.6-26.0 pg TEQ/g, n=5, Kumagaya city) and 16.0 pg TEQ/g (5.9-30.5 pg TEQ/g, n=5, Sendai city), respectively.

[Estimation of daily oral exposure to phthalates derived from soft polyvinyl chloride baby toys].

Daily oral exposure of babies to phthalate was estimated on the basis of the mouthing time of infants and the oral concentration of diisononyl phthalate (DINP) released from polyvinyl chloride (PVC) toy specimens. Total mouthing time, including the use of pacifiers, ranged widely from 11.4 min to 351.8 min with the mean of 105.3 +/- 72.1 min. The mean of the total mouthing time without pacifiers was 73.9 +/- 32.9 min. The average amount of DINP in saliva was 92.4 +/- 56.8 micrograms/10 cm2/hr, ranging from 13.2 micrograms/10 cm2/hr to 240.4 micrograms/10 cm2/hr. The exposure of phthalate in two different trials was estimated by the method of Monte Carlo simulation, one with the total mouthing time with pacifiers and the other with the total mouthing time without pacifiers. The average exposure in the former trial was 21.4 micrograms/kg/day and the 95th percentile was 65.8 micrograms/kg/day, while in the latter it was 14.8 micrograms/kg/day and the 95th percentile was 35.7 micrograms/kg/day.

Development of dioxin toxicity evaluation method in human milk by enzyme-linked immunosorbent assay--assay validation for human milk.

In this study, the development of a toxicity evaluation method for dioxins in human milk by enzyme-linked immunosorbent assay (ELISA) was reported. A total of 17 human milk samples were tested by ELISA and by gas chromatography/mass spectrometry (GC/MS) to assess whether the ELISA performed on samples obtained from primiparas could be considered as reliable enough for identifying a dioxins contamination in human milk. The concept of toxicity equivalent quantity (TEQ) screening was validated by comparing TEQ values for a set of human milk samples to the ELISA responses predicted for those samples. A fairly good correlation (r = 0.920) between immunoassay and GC/MS was achieved for human milk. This ELISA should be useful for biological samples monitoring.