Insights into Three Whole-Genome Duplications Gleaned from the Paramecium caudatum Genome Sequence

Paramecium has long been a model eukaryote. The sequence of the Paramecium tetraurelia genome reveals a history of three successive whole-genome duplications (WGDs), and the sequences of P. biaurelia and P. sexaurelia suggest that these WGDs are shared by all members of the aurelia species complex. Here, we present the genome sequence of P. caudatum, a species closely related to the P. aurelia species group. P. caudatum shares only the most ancient of the three WGDs with the aurelia complex. We found that P. caudatum maintains twice as many paralogs from this early event as the P. aurelia species, suggesting that post-WGD gene retention is influenced by subsequent WGDs and supporting the importance of selection for dosage in gene retention. The availability of P. caudatum as an outgroup allows an expanded analysis of the aurelia intermediate and recent WGD events. Both the Guanine+Cytosine (GC) content and the expression level of preduplication genes are significant predictors of duplicate retention. We find widespread asymmetrical evolution among aurelia paralogs, which is likely caused by gradual pseudogenization rather than by neofunctionalization. Finally, cases of divergent resolution of intermediate WGD duplicates between aurelia species implicate this process acts as an ongoing reinforcement mechanism of reproductive isolation long after a WGD event.     

The macronuclear genome of anaerobic ciliate Entodinium caudatum reveals its biological features adapted to the distinct rumen environment

Entodinium caudatum is an anaerobic binucleated ciliate representing the most dominant protozoal species in the rumen. However, its biological features are largely unknown due to the inability to establish an axenic culture. In this study, we primally sequenced its macronucleus (MAC) genome to aid the understanding of its metabolism, physiology, ecology. We isolated the MAC of E. caudatum strain MZG-1 and sequenced the MAC genome using Illumina MiSeq, MinION, and PacBio RSII systems. De novo assembly of the MiSeq sequence reads followed with subsequent scaffolding with MinION and PacBio reads resulted in a draft MAC genome about 117 Mbp. A large number of carbohydrate-active enzymes were likely acquired through horizontal gene transfer. About 8.74% of the E. caudatum predicted proteome was predicted as proteases. The MAC genome of E. caudatum will help better understand its important roles in rumen carbohydrate metabolism, and interaction with other members of the rumen microbiome.     

Perinatal Exposure to a Low Dose of Bisphenol A Impaired Systemic Cellular Immune Response and Predisposes Young Rats to Intestinal Parasitic Infection

Perinatal exposure to the food contaminant bisphenol A (BPA) in rats induces long lasting adverse effects on intestinal immune homeostasis. This study was aimed at examining the immune response to dietary antigens and the clearance of parasites in young rats at the end of perinatal exposure to a low dose of BPA. Female rats were fed with BPA [5 µg/kg of body weight/day] or vehicle from gestational day 15 to pup weaning. Juvenile female offspring (day (D)25) were used to analyze immune cell populations, humoral and cellular responses after oral tolerance or immunization protocol to ovalbumin (OVA), and susceptibility to infection by the intestinal nematode Nippostrongylus brasiliensis (N. brasiliensis). Anti-OVA IgG titers following either oral tolerance or immunization were not affected after BPA perinatal exposure, while a sharp decrease in OVA-induced IFNγ secretion occurred in spleen and mesenteric lymph nodes (MLN) of OVA-immunized rats. These results are consistent with a decreased number of helper T cells, regulatory T cells and dendritic cells in spleen and MLN of BPA-exposed rats. The lack of cellular response to antigens questioned the ability of BPA-exposed rats to clear intestinal infections. A 1.5-fold increase in N. brasiliensis living larvae was observed in the intestine of BPA-exposed rats compared to controls due to an inappropriate Th1/Th2 cytokine production in infected jejunal tissues. These results show that perinatal BPA exposure impairs cellular response to food antigens, and increases susceptibility to intestinal parasitic infection in the juveniles. This emphasized the maturing immune system during perinatal period highly sensitive to low dose exposure to BPA, altering innate and adaptative immune response capacities in early life.     

Ionic Control of the Reversal Response of Cilia in Paramecium caudatum : A calcium hypothesis

The duration of ciliary reversal of Paramecium caudatum in response to changes in external ionic factors was determined with various ionic compositions of both equilibration and stimulation media. The reversal response was found to occur when calcium ions bound by an inferred cellular cation exchange system were liberated in exchange for externally applied cations other than calcium. Factors which affect the duration of the response were (a) initial amount of calcium bound by the cation exchange system, (b) final amount of calcium bound by the system after equilibration with the stimulation medium, and (c) concentration of calcium ions in the stimulation medium. An empirical equation is presented which relates the duration of the response to these three factors. On the basis of these and previously published data, the following hypothesis is proposed for the mechanism underlying ciliary reversal in response to cationic stimulation: Ca⁺⁺ liberated from the cellular cation exchange system activates a contractile system which is energized by ATP. Contraction of this component results in the reversal of effective beat direction of cilia by a mechanism not yet understood. The duration of reversal in live paramecia is related to the time course of bound calcium release.     

Biodegradation of bisphenol A and its halogenated analogues by <Emphasis Type="Italic">Cunninghamella elegans</Emphasis> ATCC36112

Bisphenol A and its halogenated analogues are commonly used industrial chemicals with strong toxicological effects over many organisms. In this study, metabolic fate of bisphenol A and its halogenated analogues were evaluated with Cunninghamella elegans ATCC36112. Bisphenol A and related analogues were rapidly transformed into several metabolites by C. elegans within 2–4 days. Detailed analysis of metabolites reveals that both phase I and II metabolism occurred in C. elegans. Cytochrome P450-dependent hydroxylation was observed in BPA. However, major reaction with bisphenol A and analogues with 1-2 halogen atoms were the formation of glucose-conjugate, not being inhibited by cytochrome P450 inhibitor. Overall metabolic rates decreased with increasing number of substitution at 2- and 6-position of BPA structures, which may be consequences of limited bioavailability or steric hindrance to conjugate-forming reaction. Information from the current study will provide detailed insights over the fungal metabolism of BPA and analogues.     

Perinatal Exposure to Bisphenol-A Impairs Spatial Memory through Upregulation of Neurexin1 and Neuroligin3 Expression in Male Mouse Brain

Bisphenol-A (BPA), a well known endocrine disruptor, impairs learning and memory in rodents. However, the underlying molecular mechanism of BPA induced impairment in learning and memory is not well known. As synaptic plasticity is the cellular basis of memory, the present study investigated the effect of perinatal exposure to BPA on the expression of synaptic proteins neurexin1 (Nrxn1) and neuroligin3 (Nlgn3), dendritic spine density and spatial memory in postnatal male mice. The pregnant mice were orally administered BPA (50 µg/kgbw/d) from gestation day (GD) 7 to postnatal day (PND) 21 and sesame oil was used as a vehicle control. In Morris water maze (MWM) test, BPA extended the escape latency time to locate the hidden platform in 8 weeks male mice. RT-PCR and Immunoblotting results showed significant upregulation of Nrxn1 and Nlgn3 expression in both cerebral cortex and hippocampus of 3 and 8 weeks male mice. This was further substantiated by in-situ hybridization and immunofluorescence techniques. BPA also significantly increased the density of dendritic spines in both regions, as analyzed by rapid Golgi staining. Thus our data suggest that perinatal exposure to BPA impairs spatial memory through upregulation of expression of synaptic proteins Nrxn1 and Nlgn3 and increased dendritic spine density in cerebral cortex and hippocampus of postnatal male mice.     

Bisphenol A Modulates Calcium Currents and Intracellular Calcium Concentration in Rat Dorsal Root Ganglion Neurons

The endocrine-disrupting chemical bisphenol A (BPA) is used to manufacture plastics including food containers, and it may leach into these containers. Consumption of BPA that has leached out of plastics may be harmful as recent research highlighted that BPA can induce alterations in the nervous system. In the present work, we studied the effects of BPA on Ca²⁺ channels in dorsal root ganglion (DRG) neurons. Using whole-cell patch-clamp recordings, we found that I _Ca could be reduced by BPA in a concentration-dependent manner. Additionally, BPA shifted the activation curve of calcium currents toward a depolarizing direction and increased the slope factor of the curve. The inactivation curve for the currents was also assessed, and the curve shifted toward the depolarizing direction, although it was not significant. Moreover, inhibitory effects of BPA on the increments of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by 50 mM KCl were observed in DRG neurons using a laser scanning confocal microscopy assay. Further work revealed that the PKA and PKC pathways may be involved in the inhibitory effects of BPA since the PKA antagonist GÖ-6983 and the PKC antagonist H-89 significantly alleviated the inhibitory effects of BPA on I _Ca. As such, the results of the present study provide direct evidence that BPA decreases I _Ca and impairs calcium homeostasis, which may be involved in any toxic effects of BPA on DRG neurons.     

Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (Esr1, Il-6st, and Stat3) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, Stat3, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating Stat3 as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within STAT3 varied with liver tissue BPA level in human fetal liver samples as well, suggesting STAT3 may be a translationally relevant candidate biomarker. These data implicate Stat3 as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.     

Differential Effects of Bisphenol A and Diethylstilbestrol on Human,Rat and Mouse Fetal Leydig Cell Function

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10⁻¹² to 10⁻⁵ M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5–10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1–3 days. BPA concentrations as low as 10⁻⁸ M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10⁻⁵ M BPA were required. Similarly, 10⁻⁸ M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10⁻⁵ and 10⁻⁶ M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10⁻⁸ M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.     

Use of NHANES Data to Link Chemical Exposures to Chronic Diseases: A Cautionary Tale

Background

The National Health and Nutrition Examination Survey (NHANES) is one example of cross-sectional datasets that have been used to draw causal inferences regarding environmental chemical exposures and adverse health outcomes. Our objectives were to analyze four NHANES datasets using consistent a priori selected methods to address the following questions: Is there a consistent association between urinary bisphenol A (BPA) measures and diabetes, coronary heart disease (CHD), and/or heart attack across surveys? Is NHANES an appropriate dataset for investigating associations between chemicals with short physiologic half-lives such as BPA and chronic diseases with multi-factorial etiologies? Data on urinary BPA and health outcomes from 2003–2004, 2005–2006, 2007–2008, and 2009–2010 were available.

Methodology and Findings

Regression models were adjusted for creatinine, age, gender, race/ethnicity, education, income, smoking, heavy drinking, BMI, waist circumference, calorie intake, family history of heart attack, hypertension, sedentary time, and total cholesterol. Urinary BPA was not significantly associated with adverse health outcomes for any of the NHANES surveys, with ORs (95% CIs) ranging from 0.996 (0.951–1.04) to 1.03 (0.978–1.09) for CHD, 0.987 (0.941–1.04) to 1.04 (0.996–1.09) for heart attack, and 0.957 (0.899–1.02) to 1.01 (0.980–1.05) for diabetes.

Conclusions

Using scientifically and clinically supportable exclusion criteria and outcome definitions, we consistently found no associations between urinary BPA and heart disease or diabetes. These results do not support associations and causal inferences reported in previous studies that used different criteria and definitions. We are not drawing conclusions regarding whether BPA is a risk factor for these diseases. We are stating the opposite–that using cross-sectional datasets like NHANES to draw such conclusions about short-lived environmental chemicals and chronic complex diseases is inappropriate. We need to expend resources on appropriately designed epidemiologic studies and toxicological explorations to understand whether these types of chemicals play a causal role in chronic diseases.     

A Mechanism of Male Germ Cell Apoptosis Induced by Bisphenol-A and Nonylphenol Involving ADAM17 and p38 MAPK Activation

Germ cell apoptosis regulation is pivotal in order to maintain proper daily sperm production. Several reports have shown that endocrine disruptors such as Bisphenol-A (BPA) and Nonylphenol (NP) induce germ cell apoptosis along with a decrease in sperm production. Given their ubiquitous distribution in plastic products used by humans it is important to clarify their mechanism of action. TACE/ADAM17 is a widely distributed extracellular metalloprotease and participates in the physiological apoptosis of germ cells during spermatogenesis. The aims of this work were: 1) to determine whether BPA and NP induce ADAM17 activation; and 2) to study whether ADAM17 and/or ADAM10 are involved in germ cell apoptosis induced by BPA and NP in the pubertal rat testis. A single dose of BPA or NP (50 mg/kg) induces germ cell apoptosis in 21-day-old male rats, which was prevented by a pharmacological inhibitor of ADAM17, but not by an inhibitor of ADAM10. In vitro, we showed that BPA and NP, at similar concentrations to those found in human samples, induce the shedding of exogenous and endogenous (TNF-α) ADAM17 substrates in primary rat Sertoli cell cultures and TM4 cell line. In addition, pharmacological inhibitors of metalloproteases and genetic silencing of ADAM17 prevent the shedding induced in vitro by BPA and NP. Finally, we showed that in vivo BPA and NP induced early activation (phosphorylation) of p38 MAPK and translocation of ADAM17 to the cell surface. Interestingly, the inhibition of p38 MAPK prevents germ cell apoptosis and translocation of ADAM17 to the cell surface. These results show for the first time that xenoestrogens can induce activation of ADAM17 at concentrations similar to those found in human samples, suggesting a mechanism by which they could imbalance para/juxtacrine cell-to-cell-communication and induce germ cell apoptosis.     

Photoreversal of nuclear and cytoplasmic effects of short ultraviolet radiation on Paramecium caudatum

1. Irradiation with three short ultraviolet (UV) wave lengths, 226, 233, and 239 mµ rapidly immobilizes Paramecium caudatum, the dosage required being smaller the shorter the wave length. 85 per cent of paramecia immobilized with wave length 226 mµ recover completely. Recovery from immobilizing doses is less the longer the wave length. 2. Irradiation continued after immobilization kills the paramecia in a manner which is markedly different for very short (226, 233, and 239 mµ) and longer (267 mµ) wave lengths. 3. An action spectrum for immobilization in P. caudatum was determined for the wave lengths 226, 233, 239, 248, and 267 mµ, and found to resemble the absorption of protein and lipide in the wave length region below 248 mµ. Addition of these data to those of Giese (1945 b) gives an action spectrum resembling the absorption by albumin-like protein. 4. Division of P. caudatum is delayed by doses of wave lengths 226, 233, and 239 mµ which cause immobilization, the longest wave length being most effective. 5. Immobilization at any of the wave lengths tested (226, 233, 239, 248, 267 mµ) is not photoreversible when UV-treated paramecia are concurrently illuminated. 6. Division delay resulting from immobilizing doses of 226, 233, and 239 mµ is photoreversible by exposure to visible light concurrently with the UV. 7. Division delay induced by exposure to wave length 267 mµ is reduced by exposure to visible light applied concurrently with UV or immediately afterwards. 8. The data suggest that the shortest UV wave length tested (226 mµ) affects the cytoplasm selectively, because it is absorbed superficially as indicated by unilateral fluorescence in UV. Consequently it immobilizes paramecia rapidly but has little effect on the division rate because little radiation reaches the nucleus. 9. The data support the view that nuclear effects of UV are readily photoreversed but cytoplasmic effects are not.     

Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1–3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

An Investigation of the Endocrine-Disruptive Effects of Bisphenol A in Human and Rat Fetal Testes

Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5–17.5 days post-coitum) and humans (8–12 gestational weeks) and under different culture conditions. BPA concentrations of 10^-8M and 10^-5M for 72h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10^-5M suppressed it in the Wistar strain. The suppressive effects at 10^-5M were seen as early as 24h and 48h in both strains. BPA at 10^-7-10^-5M for 72h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10^-8M, 10^-7M, and 10^-5M for 72h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.     

Relationship Between the Environmental Endocrine Disruptor Bisphenol a and Dyslipidemia: A Five-Year Prospective Study

Objective: To investigate whether serum bisphenol A (BPA) concentration is related to the occurrence of dyslipidemia.Methods: A total of 574 adults were enrolled at baseline and followed up for 5 years. Concentrations of serum BPA, triglycerides (TGs), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were measured. Dyslipidemia was defined as the existence of one or more of the following conditions: high-LDL-cholesterolemia (LDL ≥140 mg/dL), hypertriglyceridemia (TGs ≥150 mg/dL), or low-HDL-cholesterolemia (HDL <40 mg/dL). Participants were stratified into tertiles according to low, median, and high baseline serum BPA levels. Multivariable linear and logistic regression models were used. Data from baseline and follow-up were used for cross-sectional and longitudinal analyses, respectively.Results: In the cross-sectional analysis, compared to subjects in the low BPA tertile, those in the high BPA tertile showed a higher level of LDL cholesterol (108.1 ± 24.4 mg/dL versus 119.5 ± 26.9 mg/dL; P<.05) and a lower level of HDL cholesterol (46.2 ± 11.7 mg/dL versus 39.5 ± 7.5 mg/dL; P<.05). In multivariable linear regression models, Z-transformed BPA was positively associated with LDL cholesterol (β= 0.13, P = .002) and negatively associated with HDL cholesterol (β= -0.28; P<.001). After cross-sectionally adjusting for confounders, subjects in higher BPA exposure was associated with a higher prevalence of low-HDL-cholesterolemia. Longitudinally, in subjects without low-HDL-cholesterolemia at baseline, each SD increment in baseline BPA was associated with a higher incidence of low-HDL-cholesterolemia after adjustment for confounders (odds ratio [95% confidence interval; CI] 2.76, 95% CI 1.21, 6.29).Conclusion: Cross-sectionally, higher BPA exposure is associated with a higher prevalence of low-HDL-cholesterolemia. Longitudinally, baseline BPA is an independent predictor of the 5-year incidence of low-HDL-cholesterolemia.Abbreviations: BMI = body mass index; BPA = bisphenol A; CI = confidence interval; CVD = cardiovascular disease; EIMDS = environment, inflammation and metabolic diseases study; HDL = high density lipoprotein; LDL = low density lipoprotein; OR = odds ratio; PPAR = peroxisome proliferator-activated receptor; SBP = systolic blood pressure; TG = triglyceride; Z-BPA = Z-transformed bisphenol A     

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.