Metabolism of bisphenol A in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) in relation to estrogenic response

The kinetics of bisphenol A (BPA) were investigated in zebrafish (Danio rerio) exposed to 100 microg BPA/l. BPA uptake was measured during a 7-day period followed by an elimination phase of similar duration. After 2, 6, 12, 24, 48, 72, 120 and 168 h of uptake/elimination, fish were analysed for their content of BPA, bisphenol A glucuronic acid (BPAGA) and bisphenol A sulfate (BPAS). Within the first 24 h steady state levels of BPA, BPAGA and BPAS were reached and the total body concentrations were calculated to be 569, 12,600 and 39.9 ng/g fish, respectively. Elimination rates of the three compounds in zebrafish were estimated by fitting the data to a compartment model. An initial rapid elimination phase was observed for BPA and BPAS with total body half lives (T(1/2)) of <1.1 h and 30 min, followed by a slower second elimination phase with T(1/2) values of 139 and 71 h, respectively. Excretion of BPAGA occurred from a single compartment with a T(1/2) of 35 h. The steady state concentration of BPA and its metabolites were investigated in rainbow trout (Oncorhynchus mykiss) exposed to 100 microg BPA/l. The toxicokinetic parameters from zebrafish and rainbow trout were compared; including previously published data on the rainbow trout. The data indicate that the smaller estrogenic sensitivity observed for the zebrafish may be caused by a more rapid metabolism of BPA in the zebrafish liver.     

Detection of differential DNA methylation in repetitive DNA of mice and humans perinatally exposed to bisphenol A

Developmental exposure to bisphenol A (BPA) has been shown to induce changes in DNA methylation in both mouse and human genic regions; however, the response in repetitive elements and transposons has not been explored. Here we present novel methodology to combine genomic DNA enrichment with RepeatMasker analysis on next-generation sequencing data to determine the effect of perinatal BPA exposure on repetitive DNA at the class, family, subfamily, and individual insertion level in both mouse and human samples. Mice were treated during gestation and lactation to BPA in chow at 0, 50, or 50,000 ng/g levels and total BPA was measured in stratified human fetal liver tissue samples as low (non-detect to 0.83 ng/g), medium (3.5 to 5.79 ng/g), or high (35.44 to 96.76 ng/g). Transposon methylation changes were evident in human classes, families, and subfamilies, with the medium group exhibiting hypomethylation compared to both high and low BPA groups. Mouse repeat classes, families, and subfamilies did not respond to BPA with significantly detectable differential DNA methylation. In human samples, 1251 individual transposon loci were detected as differentially methylated by BPA exposure, but only 19 were detected in mice. Of note, this approach recapitulated the discovery of a previously known mouse environmentally labile metastable epiallele, Cabp^IAP. Thus, by querying repetitive DNA in both mouse and humans, we report the first known transposons in humans that respond to perinatal BPA exposure.     

Gender differences in the levels of bisphenol A metabolites in urine

The metabolism of bisphenol A (BPA), a suspected endocrine disruptor, should be considered for monitoring human exposure to BPA, because the conjugation with beta-D-glucuronide and sulfate reduces the estrogenic activity. In this study, BPA levels in 30 healthy Koreans (men, N=15, 42.6+/-2.4 years; women, N=15, 43.0+/-2.7 years) were analyzed from urine treated with/without beta-glucuronidase and/or sulfatase by an RP-HPLC with fluorescence detection. The total BPA concentrations including free BPA and the urinary conjugates were similar in men and women (2.82+/-0.73 and 2.76+/-0.54 ng ml(-1), respectively), but gender differences were found in the levels of urinary BPA conjugates. Men had significantly higher levels of BPA-glucuronide (2.34+/-0.85 ng ml(-1)) than women (1.00+/-0.34 ng ml(-1)), whereas women had higher levels of BPA-sulfate (1.20+/-0.32 ng ml(-1)) than men (0.49+/-0.27 ng ml(-1)).     

Quantitation of efletirizine in human plasma and urine using automated solid-phase extraction and column-switching high-performance liquid chromatography

A heart-cut column-switching, ion-pair, reversed-phase HPLC system was used for the quantitation of efletirizine (EFZ) in biological fluids. The analyte and an internal standard (I.S.) were extracted from human EDTA plasma by C₁₈ solid-phase extraction (SPE) using a RapidTrace^® workstation. The eluent from the SPE was evaporated, reconstituted and injected onto the HPLC column. Urine samples were diluted and injected directly without the need of extraction. The compounds of interest were separated from most of the extraneous matrix materials by the first C₁₈ column, and switched onto a second C₁₈ column for further separation using a mobile phase of stronger eluting capability. Linearity range was 10–2000 ng ml⁻¹ for plasma and 0.05–10 μg ml⁻¹ for urine. The lower limit of quantitation (LOQ) was 10 ng from 1 ml of plasma, with a signal-to-noise ratio of 15:1. Inter-day precision and bias of quality control samples (QCs) were <5% for plasma and <7% for urine. Selectivity was established against six other antihistamines, three analogs of efletirizine, and on 12 control plasma lots and nine control urine lots. Recovery was 90.0% for EFZ and 89.5% for I.S. from plasma. One hundred samples can be processed in every 2.75 h on a 10-module RapidTrace^® workstation with minimal human attention. Method ruggedness were tested on three brands of SPE and six different lots of one SPE brand. Performance ruggedness was demonstrated by different analysts on multiple HPLC systems. Analyte stability through sample storage, extraction process (benchtop, freeze–thaw, refrigeration after extraction) and chromatography (on-system, reinjection) was established.     

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml⁻¹ using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile–aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 μg ml⁻¹. Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 μg ml⁻¹ and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

Determination of midazolam and two metabolites of midazolam in human plasma by gas chromatography—negative chemical-ionization mass spectrometry

A method is described for measuring midazolam, a new anesthesia induction agent and hypnotic, and its hydroxymethyl and desmethyl metabolites in human plasma. Deuterated analogues of each compound are added to plasma as internal standards. The compounds are extracted from plasma with benzene containing 20% 1, 2-dichloroethane and after removal of the extracting solvent are dissolved in a solution of bis-(trimethylsilyl)acetamide and acetonitrile. An aliquot of this solution is analyzed by gas chromatography—mass spectrometry with the mass spectrometer set to monitor in the gas chromatographic effluent the M$$$ ions of drug, metabolites and internal standards generated by methane electron-capture negative chemical ionization. For all three compounds, the limit of quantitation is 1 ng ml⁻¹, and the precision (relative standard deviation) at a concentration of 5 ng ml⁻¹ is less than 6%. Measurable amounts of the hydroxymethyl, but not the desmethyl, metabolite of midazolam could be found in the plasma of humans given either an intravenous or an oral dose of midazolam maleate.     

Determination of bisphenol A in river water and body fluid samples by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry

A new method, based on stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water, urine, plasma, and saliva samples. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volumes and extraction time are investigated. Then, the stir bar is subjected to TD followed by GC-MS. The detection limits of BPA in river water, urine, plasma, and saliva samples are 1-5, 20, 100, and 20pgml(-1) (ppt), respectively. Calibration for BPA was shown to be linear with a correlation coefficient of >0.99. The average recoveries of BPA in all samples are higher than 95% (R.S.D. < 10%) with correction using an added surrogate standard, 13C12-bisphenol A. This simple, accurate, sensitive, and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Miniaturized hollow fiber assisted liquid-phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for analysis of bisphenol A in human urine sample

A new method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in human urine samples. The detection limit and the quantification limit of BPA in human urine sample are 0.02 and 0.1 ng ml(-1) (ppb), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 0.1-50 ng ml(-1). The average recoveries of BPA in human urine samples spiked with 1 and 5 ng ml(-1) BPA are 101.0 (R.S.D.: 6.7%) and 98.8 (R.S.D.: 1.8%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C12. This simple, accurate, sensitive and selective analytical method can be applicable to the determination of trace amounts of BPA in human urine samples.     

双酚A对斑马鱼胚胎的毒性作用

Bisphenol A(BPA)is a chemical estrogen-like sub-stance with properties that are of environmental concern.It is widely used in the chemical industry to manufactureepoxy-and polyester-styrene resins.It has been reportedthat BPA ranges between0and33μg in each plasticcup[1].After atwo-weekexposureto0.5%bisphenol Aithas beenreportedthat disattachments betweensertoli cellsand spermatogonia were observed while spermatogoniawere arrangedin disorder and displacement of spermatogo-nia away fromthe basement membrance ...     

Application of liquid chromatography-mass spectrometry to the quantification of bisphenol A in human semen

The potential risks to human health and reproduction from the xenoestrogen bisphenol A (BPA) have not been well established. This is due in part to the absence of accurate analytical methods to quantify BPA in biological samples. In this study we establish an accurate, sensitive and selective analytical method for the quantification of BPA in human semen. To quantify BPA we compared the techniques of liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). In addition we have taken steps to eliminate BPA contamination during sample extraction and preparation. Results show that the ELISA method gives an over-estimate of BPA concentration, which may be due, at least in part, to non-specific interactions with the BPA-antibodies. LC-MS gave much more accurate results and proved to be more sensitive with a detection limit of 0.5 ng ml(-1) compared to 2.0 ng ml(-1) by ELISA.     

Adsorption of bisphenol A by lactic acid bacteria,Lactococcus, strains

Ten strains of the genus Lactococcus were examined for their ability to remove bisphenol A [2, 2-bis(4-hydroxyphenyl)propane; BPA], which is known as an endocrine disrupter. Nine strains of the lactococci tested could remove BPA from media during growth, although the removal ratio was below 9%. When BPA was incubated with lyophilized cells of lactococci for 1 h, the concentration of BPA in the media was decreased by 9–62%. Especially, the highest removal ratio of BPA was observed for Lactococcus lactis subsp. lactis 712. The lactococci could adsorb BPA but not degrade it, because the lactococci maintained the ability to remove BPA from the medium after autoclaving. When the lyophilized cells of L. lactis subsp. lactis 712 were also incubated with six analogues of BPA, they effectively adsorbed hydrophobic compounds such as 2, 2′-diphenylpropane and bisphenol A dimethylether. The BPA-adsorbing ability of lactococci could be due to the hydrophobic binding effect. The removal ratio of BPA by L. lactis subsp. lactis 712 was increased after treatment with sodium dodecyl sulfate and decreased after digestion with trypsin. These results suggest that the hydrophobic proteins on cell surface may be involved in the BPA-adsorbing ability of lactococci.     

Sample clean-up with sol-gel enzyme and immunoaffinity columns for the determination of bisphenol A in human urine

The paper describes the development of a simple and highly selective analytical method for the determination of free and total bisphenol A in urine samples. Free bisphenol A levels can be determined after sample clean-up using sol-gel immunoaffinity columns containing anti-bisphenol A antibodies. In determining total bisphenol A levels, the sample pre-treatment procedure consists of sample preparation using an on-line combination of two sol-gel columns, an enzyme column containing glucuronidase and arylsulfatase, and an immunoaffinity column. Bisphenol A can then be quantified by high-performance liquid chromatography and fluorescence detection. The mean recovery was found to be 78% with a standard deviation of 3.4%, the LOD (S/N=3) was 0.2 ng/ml. The method was applied to determine free and total urinary BPA levels of healthy adults and dialysis patients.     

Assessment of human contamination of estrogenic endocrine-disrupting chemicals and their risk for human reproduction

There is broad human exposure to estrogenic endocrine-disrupting chemicals (EDCs), but the data sets that exist are primarily for various environmental media such as food and water rather than the most relevant internal exposure. We have detected various kind of EDC contamination in humans including dioxin and bisphenol A (BPA) widely used for the production of plastic products. BPA was present in serum and follicular fluid at approximately 1–2 ng/ml, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. An approximately five-fold higher concentration, 8.3 ± 8.7 ng/ml, was revealed in amniotic fluid at 15–18 weeks of gestation, compared to other fluids showing increased exposure at the critical developmental period in humans. Interestingly, serum BPA concentrations were significantly higher in normal men and in women with polycystic ovary syndrome (PCOS) compared with normal women possibly due to differences in the androgen-related metabolism of BPA. These findings may provide some insight into the metabolism of EDCs in human and the pathophysiology of endocrine disorders such as PCOS. Dioxin contamination in relationship to development of endometriosis is also discussed.     

Analysis of the imidazoacridinone C1311 by high-performance liquid chromatography

The imidazoacridinone C1311 has shown anti-tumour activity both in vitro and in vivo, prompting its acceptance for Phase I clinical trials. A high-performance liquid chromatography method using fluorescence detection has been developed for the analysis of C1311 in mouse and human plasma and mouse tissue samples. This method is selective, sensitive (limit of detection of 1 ng ml⁻¹) and reproducible, with recoveries of>90%. C1311 was stable over 8 h, at 25°C, in plasma, tumour homogenate, saline and a range of buffers (pH 3.0–8.0). The compound was highly protein bound (>90%) in plasma which may have important consequences in the pharmacokinetics of the drug.     

ipso-substitution: a general biochemical and biodegradation mechanism to cleave alpha-quaternary alkylphenols and bisphenol A

Sphingobium xenophagum Bayram is capable of metabolizing 4-alkoxyphenols and endocrine disruptive alpha-quaternary 4-nonylphenols by an ipso-substitution mechanism that involves ring hydroxylation at the site of the substituent. Here, we show that Bayram's ipso-hydroxylating activity was able to transform also bisphenol A (= dimethyl-4,4'-methylenediphenol; BPA) and 4-isopropylphenol. We identified six metabolites when resting cells of strain Bayram were incubated with BPA. They were unambiguously characterized by HPLC-UV, HPLC/MS, and HPLC/MS/MS as hydroquinone, 2-(4-hydroxyphenyl)isopropanol, 4-isopropenylphenol, 4-isopropylphenol, 4-hydroxy-4-isopropenylcyclohexa-2,5-dien-1-one, and 4-hydroxy-4-isopropylcyclohexa-2,5-dien-1-one. In experiments with 4-isopropylphenol as a substrate, 4-hydroxy-4-isopropylcyclohexa-2,5-dien-1-one, one of the metabolites from BPA, accumulated to a high degree. We could rationalize the formation of all metabolites by invoking ipso-hydroxylation and ipso-substitution mechanisms. On closer view, also classical bacterial metabolism of BPA can be well rationalized by an ipso-substitution mechanism, albeit with ipso-attack of an internal alkyl radical instead of an activated oxygen species. This highlights the important role of ipso-substitution as a versatile degradative principle utilized by diverse organisms to degrade alpha-quaternary 4-nonylphenols, 4-alkoxyphenols, and BPA.     

Gender difference in serum bisphenol A levels may be caused by liver UDP-glucuronosyltransferase activity in rats

Gender difference in human bisphenol A (BPA) concentrations was revealed by determining serum BPA. We studied the serum concentrations and the metabolism of BPA in rats by an HPLC system. Rat serum BPA concentrations were significantly higher in males (24.9+/-7.38 ng/ml, P=0.026, n=10) than in females (8.27+/-3.11 ng/ml, n=10), as in humans. The resultant enzyme reaction products of BPA glucuronidation in the rat liver microsomes fraction were analyzed by an HPLC system. The ratio of BPA glucuronidation in the microsome reaction was significantly higher (P=0.015) in female than in male rats. The mRNA expression of UDP-glucuronosyltransferase 2B1 (UGT2B1), an isoform of UGT related to BPA glucuronidation, in the rat liver was analyzed by a real-time quantitative RT-PCR. The relative expression level of UGT2B1 mRNA was significantly higher (P<0.001) in female than in male rat livers. The gender difference in serum BPA concentrations may be explained by the difference in clearance based on the UGT activities.     

Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10⁻⁷ to 1×10⁻⁵ M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10⁻⁸ M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua^R) phenotypic mutation was also found in cells treated with 1×10⁻⁷ and 1×10⁻⁵ M of bisphenol A. The induction of K-ras codon 12 mutations and Oua^R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10⁻⁶ M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Pre- and postnatal bisphenol A treatment results in persistent deficits in the sexual behavior of male rats, but not female rats, in adulthood

Perinatal administration of the endocrine disruptor bisphenol A (BPA) reportedly inhibits the sexual behavior of sexually naïve adult male rats. In order to evaluate the effects of BPA administration during early development on later reproductive behavior, we administered one of five doses of bisphenol A daily to pregnant female rats throughout gestation and lactation, and quantified the appetitive and consummatory sexual behaviors of the resultant male and female offspring over multiple sexual encounters in adulthood. Males receiving low dose perinatal BPA (50 μg/kg bw/day) showed persistent deficits in sexual behavior in adulthood. Males receiving the highest dose (5 mg/kg bw/day), however, were indistinguishable from controls with respect to consummatory sexual behaviors but showed decreased latencies to engage in those behaviors when sexually naïve, with significant non-linear, or U-shaped, dose-response relationships observed on the first and last day of testing. Adult female sexual behavior was not affected by early BPA administration at any dose tested. These results are consistent with previous reports that BPA exerts behavioral effects especially at low doses, and further indicates that BPA can cause lasting impairment of sexual behavior in males, but does not alter the normal development of female appetitive or consummatory sexual behaviors. To our knowledge, this is the first report indicating that adult sexual performance is impaired in sexually experienced animals following perinatal exposure to bisphenol A.     

Serum bisphenol a concentrations showed gender differences, possibly linked to androgen levels

To investigate human exposure to bisphenol A (BPA), a widely used endocrine disruptor, we measured serum BPA concentrations and analyzed the interrelation of BPA with sex-related hormones. BPA was detected in all human sera by a novel enzyme-linked immunosorbent assay. Serum BPA concentrations were significantly higher in normal men (1.49 +/- 0.11 ng/ml; P < 0.01) and in women with polycystic ovary syndrome (1.04 +/- 0.10 ng/ml; P < 0.05) compared with normal women (0.64 +/- 0.10 ng/ml). There were significant positive correlations between serum BPA and total testosterone (r = 0.595, P < 0.001) and free testosterone (r = 0.609, P < 0.001) concentrations in all subjects and likewise between serum BPA and total testosterone (r = 0.559, P < 0.01) and free testosterone (r = 0.598, P < 0.001) concentrations in all female subjects, but not between serum BPA and other sex-related hormone concentrations in any group. These findings showed that there are gender differences in serum BPA concentrations, possibly due to differences in the androgen-related metabolism of BPA.