Hiroyuki Nakazawa

Decreased Serum Free Testosterone in Workers Exposed to High Levels of Di-n-butyl Phthalate (DBP) and Di-2-ethylhexyl Phthalate (DEHP): A Cross-Sectional Study in China

Background Observations of adverse developmental and reproductive effects in laboratory animals and wildlife have fueled increasing public concern regarding the potential for various chemicals to impair human fertility. Objective Our objective in this study was to assess the effect of occupational exposure to high levels of phthalate esters on the balance of gonadotropin and gonadal hormones including luteinizing hormone, follicle-stimulating hormone, free testosterone (fT), and estradiol. Methods We examined urine and blood samples of 74 male workers at a factory producing unfoamed polyvinyl chloride flooring exposed to di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) and compared them with samples from 63 male workers from a construction company, group matched for age and smoking status. Results Compared to the unexposed workers, the exposed workers had substantially and significantly elevated concentrations of mono-n-butyl phthalate (MBP; 644.3 vs. 129.6 μg/g creatinine, p < 0.001) and mono-2-ethylhexyl phthalate (MEHP; 565.7 vs. 5.7 μg/g creatinine, p < 0.001). fT was significantly lower (8.4 vs. 9.7 μg/g creatinine, p = 0.019) in exposed workers than in unexposed workers. fT was negatively correlated to MBP (r = −0.25, p = 0.03) and MEHP (r = −0.19, p = 0.095) in the exposed worker group. Regression analyses revealed that fT decreases significantly with increasing total phthalate ester score (the sum of quartiles of MBP and MEHP; r = −0.26, p = 0.002). Conclusion We observed a modest and significant reduction of serum fT in workers with higher levels of urinary MBP and MEHP compared with unexposed workers.

Correlations between Prenatal Exposure to Perfluorinated Chemicals and Reduced Fetal Growth

Background Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are man-made, ubiquitous, and persistent contaminants in the environment, wildlife, and humans. Although recent studies have shown that these chemicals interfere with fetal growth in humans, the results are inconsistent. Objectives Our goal was to investigate the correlation between relatively low levels of PFOS and PFOA in maternal serum and birth weight and birth size. Methods We conducted a hospital-based prospective cohort study between July 2002 and October 2005 in Sapporo, Japan. A total of 428 women and their infants were involved in the study. We obtained characteristics of the mothers and infants from self-administered questionnaire surveys and from medical records. We analyzed maternal serum samples for PFOS and PFOA by liquid chromatography–tandem mass spectrometry (LC/MS/MS). Results After adjusting for confounding factors, PFOS levels negatively correlated with birth weight [per log10 unit: β = −148.8 g; 95% confidence interval (CI), −297.0 to −0.5 g]. In addition, analyses stratified by sex revealed that PFOS levels negatively correlated with birth weight only in female infants (per log10 unit: β = −269.4 g; 95% CI, −465.7 to −73.0 g). However, we observed no correlation between PFOA levels and birth weight. Conclusion Our results indicate that in utero exposure to relatively low levels of PFOS was negatively correlated with birth weight.

Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection.

A simple and sensitive method using high-performance liquid chromatography with multi-electrode electrochemical detection (HPLC-ED) including a coulometric array of four electrochemical sensors has been developed for the determination of bisphenol A in water and human serum. For good separation and detection of bisphenol A, a CAPCELL PAK UG 120 C18 reversed-phase column and a mobile phase consisting of 0.3% phosphoric acid-acetonitrile (60:40) were used. The detection limit obtained by the HPLC-ED method was 0.01 ng/ml (0.5 pg), which was more than 3000-times higher than the detection limit obtained by the ultraviolet (UV) method, and more than 200-times higher than the detection limit obtained by the fluorescence (FL) method. Bisphenol A in water and serum samples was pretreated by solid-phase extraction (SPE) after removing possible contamination derived from a plastic SPE cartridges and water used for the pretreatment. A trace amount (ND approximately 0.013 ng/ml) of bisphenol A was detected from the parts of cartridges (filtration column, sorbent bed and frits) by extraction with methanol, and it was completely removed by washing with at least 15 ml of methanol in the operation process. The concentrations of bisphenol A in tap water and Milli-Q-purified water were found to be 0.01 and 0.02 ng/ml, respectively. For that reason, bisphenol A-free water was made to trap bisphenol A in water using an Empore disk. In every pretreatment, SPE methods using bisphenol A-free water and washing with 15 ml of methanol were done in water and serum samples. The yields obtained from the recovery tests using water to which 0.5 or 0.05 ng/ml of bisphenol A was added were 83.8 to 98.2%, and the RSDs were 3.4 to 6.1%, respectively. The yields obtained from the recovery tests by OASIS HLB using serum to which 1.0 ng/ml or 0.1 ng/ml of bisphenol A was added were 79.0% and 87.3%, and the RSDs were 5.1% and 13.5%, respectively. The limits of quantification in water and serum sample were 0.01 ng/ml and 0.05 ng/ml, respectively. The method was applied to the determination of bisphenol A in healthy human serum sample, and the obtained detection was 0.32 ng/ml. From these results, the HPLC-ED method should be the most useful in the determination of bisphenol A at low concentration levels in water and biological samples.

Stability of microcystins from cyanobacteria—II. Effect of UV light on decomposition and isomerization

Microcystins are very potent hepatotoxins and strong liver tumor promoters produced by cyanobacteria, and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, we examined the stability of microcystins during photolysis with UV light. The toxins were easily decomposed by UV light at wavelengths around the absorption maxima of the toxins and the decomposition depended on the intensity of the light. The half-life of microcystin LR by 147 microW/cm2 UV irradiation was 10 min, and the toxin was completely decomposed by 2550 microW/cm2 UV after 10 min. When the toxins were irradiated with weaker UV light, isomerization was also observed by a different mechanism from that during photolysis by sunlight and pigment, and several products including three geometrical isomers of the conjugated diene of Adda were detected. Microcystin RR showed almost the same behavior as that of microcystin LR under the same conditions. Since no noxious products were formed in the present study, a water treatment including UV irradiation is very possible for removing microcystins from raw water.

Determination of bisphenol A in canned vegetables and fruit by high performance liquid chromatography

A high performance liquid chromatograph y (HPLC) method was developed for the determination of bisphenol A (BPA) that had migrated into canned fruit and vegetables. BPA was extracted with acetonitrile from the solid portion of canned food, and with an OASIS HLB cartridge from the aqueous portion, respectively. Both extracts were cleaned up on a Florisil cartridge. The HPLC separation was carried out on a Wakosil II 3C18 RS column (4.6 x 150mm) with acetonitrile-water (40:60, v/v) as a mobile phase with a flow rate of 0.8 ml/min. BPA was detectable by UV detector at 228 nm and determined with the similarity of chromatographic peak spectrum by multiwavelength detector (similarity index was 0.99 or above). The quantification limits were 10 ng/g for the solid portion and 5 ng/ml for the aqueous portion, respectively. BPA was mainly detected in the solid portion of canned food and found at the maximum level of 11 μg per can. To verify migration into the solid portion of canned food, a partitioning experiment was carried out.

Measurement of bisphenol A levels in human blood serum and ascitic fluid by HPLC using a fluorescent labeling reagent

A sensitive column-switching HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) in human blood serum and ascitic fluid samples. 4-(4,5-Diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was used as the fluorescent label, and the excess reagent was removed by a column-switching technique. Liquid-liquid extraction with chloroform was used for the pretreatment of serum and ascitic fluid samples. BPA in both the samples could be determined in the concentration range of 0.1-7.0 ppb with the detection limit of 0.04 ppb at a signal-to-noise ratio of 3. The recoveries of BPA spiked to serum and ascitic fluid were 78.6 and 77.7%, respectively. The mean concentrations of BPA (n=9) in maternal and umbilical cord blood sera obtained from healthy pregnant women were 0.46+/-0.20 and 0.62+/-0.13 ppb, respectively. BPA levels (n=21) in blood sera and ascitic fluid obtained from the patients with sterility were also determined to be 0.46+/-0.20 and 0.56+/-0.19 ppb, respectively. Relationships of BPA concentrations were observed between maternal and umbilical cord blood serum samples (r=0.626), as well as blood serum and ascitic fluid samples (r=0.785).

Stability of microcystins from cyanobacteria-IV. Effect of chlorination on decomposition

Microcystins, the cyclic heptapeptide toxins produced by cyanobacteria such as Microcystis, show tumor-promoting activity through inhibition of protein phosphatases 1 and 2A. They potentially threaten human health, and are increasing the world-wide interest in the health risk associated with cyanobacterial toxins. In this study, the effect of chlorination on the decomposition of microcystins-LR and -RR was examined. The toxins were easily decomposed by chlorination with sodium hypochlorite, and the decomposition depended on the free chlorine dose. In this operation, many reaction products were formed, one of which was determined to be dihydroxymicrocystin formed through the chloronium ion at the conjugated diene of Adda [3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E), 6(E)-dienoic acid], followed by hydrolysis. Other products may be its stereoisomers and/or regioismers. No noxious products were detected from the chlorination process of microcystin-LR. Although these results suggested that chlorination at an adequate chlorine dose is very effective for the removal of microcystin in raw water, preoxidation of the cell itself with chlorine must be avoided, because it frequently causes toxin release from algae and produce trihalomethanes during water treatment.

Stir bar sorptive extraction with in situ derivatization and thermal desorption–gas chromatography–mass spectrometry in the multi-shot mode for determination of estrogens in river water samples

A novel method for the trace analysis of natural and synthetics estrogens, such as estrone (E1), 17beta-estradiol (E2) and 17alpha-ethynylestradiol (EE), in river water sample was developed, which involved stir bar sorptive extraction (SBSE) with in situ derivatization followed by thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volume and extraction time were investigated. In addition, the single and multi-shot modes in TD were investigated. The detection limits of E1, E2 and EE in river water sample were 0.2, 0.5 and 1 pg ml(-1) (ppt), respectively, in the multi-shot mode using five stir bars. The calibration curves for E1, E2 and EE were linear and had correlation coefficients >0.99. The average recoveries of E1, E2 and EE from all sample volumes were higher than 90% (R.S.D. < 10%) with correction using an added surrogate standard such as estrone-13C4, 17beta-estradiol-13C4 or 17alpha-ethynylestradiol-13C4. This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of estrogens in water samples.

Simultaneous determination of residual tetracyclines in foods by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

We established a method for precisely determining residual tetracycline antibiotics (TCs) in foods by atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry (APCI LC-MS-MS) using selected reaction monitoring with an internal standard. By setting the nebulizer probe temperature to 475 degrees C, we were able to use a mobile phase containing oxalic acid without clogging problems at the APCI interface, since oxalic acid decomposes to carbon dioxide and water at high temperature. DMCTC was very effective as an internal standard for determining TCs in various foods. TCs were cleaned up using a Bond Elut ENV cartridge and analysed by APCI LC-MS-MS. The recovery of TCs from various foods including animal tissues, honey, milk, eggs, and fish fortified at levels of 0.05, 0.10, and 0.50 ppm averaged 60.1-88.9%, with an RSD of 1.2-8.7%. The detection limits were 0.001 ppm for OTC and TC, 0.004 ppm for CTC, and 0.002 ppm for DC. The present method was also successfully used to determine TCs in swine kidney samples that were previously found by microbiological assay.

Measurement of bisphenol A levels in human urine

We report a new approach for assessing human exposure to bisphenol A (BPA) by measuring BPA in urine after enzymatic deglucuronidation. This method involves addition of 13C 12-labeled BPA, enzymatic deconjugation, solid-phase extraction, and derivatization with pentafluorobenzyl bromide. The product of the derivatization is separated by gas chromatography followed by mass spectrometric detection using negative chemical ionization and selected ion monitoring. Using this analysis method, urine samples fortified with both a constant level of labeled BPA and a range of unlabeled BPA levels (0.27–10.6 ng/ml) demonstrated constant percentage recovery. In addition, a range of urine sample volumes (0.25–10.0 ml) with constant amounts of added internal standard produced a linear response (r 2=0.99). The method limit of detection was 0.12 ng/ml. This method was validated by duplicate analyses using gas chromatography coupled to a high-resolution mass spectrometer.