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.     

Comparative study of the interactions between bisphenol-A and its endocrine disrupting analogues with bovine serum albumin using multi-spectroscopic and molecular docking studies

Interaction studies of bisphenol analogues; biphenol-A (BPA), bisphenol-B (BPB), and bisphenol-F (BPF) with bovine serum albumin (BSA) were performed using multi-spectroscopic and molecular docking studies at the protein level. The mechanism of binding of bisphenols with BSA was dynamic in nature. SDS refolding experiments demonstrated no stabilization of BSA structure denatured by BPB, however, BSA denatured by BPA and BPF was found to get stabilized. Also, CD spectra and molecular docking studies revealed that BPB bound more strongly and induced more conformational changes in BSA in comparison to BPA. Hence, this study throws light on the replacement of BPA by its analogues and whether the replacement is associated with a possible risk, raising a doubt that perhaps BPB is not a good substitute of BPA.     

Production of hemopoietic growth factors and gamma-interferon by large granular lymphocytes from patients with T gamma lymphocytosis

Two patients with T gamma lymphocytosis in whom expanded large granular lymphocyte (LGL) populations were detected in peripheral blood and bone marrow are described. The surface antigen phenotypes of the LGL from these patients were similar with a major portion of cells carrying T3, T8, T11 and Leu7 markers. However, whereas fresh LGL from both patients demonstrated antibody-dependent cell cytotoxicity (ADCC), natural killer (NK) cell function was present in one case but absent in the other. Supernatants from enriched suspensions of the LGL unstimulated by exogenous antigen or mitogens were shown to contain significant amounts of hemopoietic growth factors colony-stimulating activity (CSA) and burst-promoting activity (BPA). In one case gamma-interferon was also detected. This study contributes to the accumulating evidence that LGL are able to generate factors which have the capacity to influence the proliferation of hemopoietic progenitor cells in vitro.     

Gestational exposure to low dose bisphenol A alters social behavior in juvenile mice

Bisphenol A (BPA) is a man-made compound used to make polycarbonate plastics and epoxy resins; public health concerns have been fueled by findings that BPA exposure can reduce sex differences in brain and some behaviors. We asked if a low BPA dose, within the range measured in humans, ingested during pregnancy, would affect social behaviors in prepubertal mice. We noted sex differences in social interactions whereby females spent more time sitting side-by-side, while males engaged in more exploring and sitting alone. In addition BPA increased display of nose-to-nose contacts, play solicitations and approaches in both sexes. Interactions between sex and diet were found for self grooming, social interactions while sitting side-by-side and following the other mouse. In all these cases interactions were produced by differences between control and BPA females. We examined brains from embryos during late gestation to determine if gene expression differences might be correlated with some of the sexually dimorphic or BPA affected behaviors we observed. Because BPA treatments ended at birth we took the brains during embryogenesis to increase the probability of discovering BPA mediated effects. We also selected this embryonic age (E18.5) because it coincides with the onset of sexual differentiation of the brain. Interestingly, mRNA for the glutamate transporter, Slc1a1, was enhanced by exposure to BPA in female brains. Also we noted that BPA changed the expression of two of the three DNA methyltransferase genes, Dnmt1 and Dnmt3a. We propose that BPA affects DNA methylation of Sc1a1 during neural development. Sex differences in juvenile social interactions are affected by BPA and in particular this compound modifies behavior in females.     

Fetal exposure to bisphenol A affects the primordial follicle formation by inhibiting the meiotic progression of oocytes

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Frequent human exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on germ cell cyst breakdown and primordial follicle formation. Pregnant mice were treated with BPA at doses of 0, 0.02, 0.04, 0.08 mg/kg body weight/day from 12.5 day postcoitum. BPA was delivered orally to pregnant female mice. A dose–response relationship was observed with increased BPA exposure level associated with more oocytes in germ cell cyst and less primordial follicle at postnatal day 3 (P < 0.01). Progression to meiosis prophase I of oocytes was delayed in the 0.08 mg/kg bw/day treated group (P < 0.01). Decreased mRNA expression of specific meiotic genes including Stra8, Dmc1, Rec8 and Scp3 were observed. In conclusion, BPA exposure can affect the formation of primordial follicle by inhibiting meiotic progression of oocytes.     

Impact of reaction conditions on the laccase-catalyzed conversion of bisphenol A

The oxidative conversion of aqueous BPA catalyzed by laccase from Trametes versicolor was conducted in a closed, temperature-controlled system containing buffer for pH control. The effects of medium pH, buffer concentration, temperature and mediators and the impacts of dissolved wastewater constituents on BPA conversion were investigated. The optimal pH for BPA conversion was approximately 5, with greater than half maximal conversion and good enzyme stability in the range of 4-7. The stability of the enzyme was not impacted by buffer concentration, nor was BPA conversion. Despite the observation that the enzyme tended to be inactivated at elevated temperatures, enhanced conversion of BPA was observed up until a reaction temperature of 45 degrees C. Of the mediators studied, ABTS was most successful at enhancing the conversion of BPA. Dissolved wastewater constituents that were studied included various inorganic salts, organic compounds and heavy metal ions. BPA conversion was inhibited in the presence of anions such as sulfite, thiosulfate, sulfide, nitrite and cyanide. The metal ions Fe(III) and Cu(II) and the halogens chloride and fluoride substantially suppressed BPA conversion, but the presence of selected organic compounds did not significantly reduce the conversion of BPA.     

Excretion of bisphenol A-glucuronide into the small intestine and deconjugation in the cecum of the rat

The environmental estrogen bisphenol A (BPA) is regarded as a modulator of endocrine systems and has been reported to have adverse effects on the reproductive organs of animals. In rats, BPA is metabolized to glucuronide by UDP-glucuronosyltransferase UGT2B1 in the liver and excreted into the bile. In the present study, we found that most of the bisphenol A-glucuronide (BPA-GA) excreted into the small intestine was deconjugated in the contents of the cecum. After BPA administration, BPA-GA was (immediately should be 15 min) found in the contents of the upper part of the small intestine, and then it moved to the lower part of the small intestine. However, only free BPA was found in the content of the cecum, and there was smaller amount of free BPA in the colon contents, indicating that BPA had been reabsorbed in the colon. BPA-GA was deconjugated by extract prepared from the cecum content which included highest beta-glucuronidase (beta-Gase) observed in Western blot analysis using antibodies against bacterial beta-Gase.These results indicate enterohepatic circulation of BPA and suggest that the adverse effects of BPA are enhanced by repeated exposure.     

The interaction of bovine plasma albumin with cationic detergents at pH9

The interaction of bovine plasma albumin (BPA) with tetradecyltri-methylammonium bromide (TTAB) was studied at pH 9.0. When the system BPA-TTAB was analyzed by the gel electrophoresis, the pattern changed with the molar mixing ratio (TTAB/BPA). At molar mixing ratio 12, for example, zones 1, 2, 3, 4, and 5 were observed. Component 1 is a monomer and component 2 is a dimer of BPA. Components 3–5 are further aggregates of BPA. Thus, the intermolecular SH - SS exchange reaction occurs between BPA molecules unfolded by cationic detergent, leading to the formation of a series of lower aggregates of BPA. Under some conditions, partial precipitation of BPA occurred. Components 1′ and 1″, which are modified monomeis, were observed at certain concentrations of detergent.Studies were also made using a series of cationic detergents differing in the length of hydrocarbon chain C₆C₁₂. Including TTAB, the longer the hydrocarbon chain, the more remarkable was the effect on BPA.The effect of cationic detergent on BPA resembles that of urea insofar as gel electrophoresis is concerned. Furthermore the denatureation of BPA by cationic detergent resembles that by heat and by high pressure. These four agents are initiators of SH - SS exchange reaction for the protein.The effect of cationic detergent differs entirely from that of the anionic detergent such as sodium dodecyl sulfate (SDS). The anionic detergent does not initiate the intermolecular exchange reaction at pH 9.0 even when the molar mixing ratio SDS/BPA is high enough to make BPA unfold.     

Extending phylogenetic studies of coevolution: secondary Brooks parsimony analysis, parasites, and the Great Apes

Dowling recently compared the empirical properties of Brooks parsimony analysis (BPA) and the leading method for studying phylogenetic aspects of coevolution, reconciled tree analysis (using the computer program TreeMap), based on a series of simulations. Like the majority of authors who have compared BPA with other methods, however, Dowling considered only the form of BPA proposed in 1981 and did not take into account various modifications of the method proposed from 1986 to 2002. This leaves some doubt as to the robustness of his assessments of both the superiority of BPA and its shortcomings. We provide a précis of the principles of contemporary BPA, including ways to implement it algorithmically, using either Wagner algorithm‐based or Hennigian argumentation‐based approaches, followed by an empirical example. Our study supports Dowling's fundamental conclusions about the superiority of primary BPA relative to TreeMap. However, his conclusions about the shortcomings of BPA due to inclusive ORing (i.e., the production of ghost taxa) are incorrect, as secondary BPA eliminates inclusive ORing from the method. Secondary BPA provides a more complete account of the evolutionary associations between the parasite groups and their hosts than does primary BPA, without sacrificing any indirectly generated information about host phylogeny. Secondary BPA of two groups of nematodes inhabiting Great Apes shows that TreeMap analysis underestimated the amount of cospeciation in the evolution of the nematode genus Enterobius.     

Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects

Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. This compound is a building block of polycarbonate plastics often used for food and beverage storage, and BPA is also a component of epoxy resins that are used to line food and beverage containers. Studies have shown that BPA can leach from these and other products in contact with food and drink, and as a result, routine ingestion of BPA is presumed. This compound is also found in an enormous number of other products that we come into contact with daily, and therefore it is not surprising that it has been detected in the majority of individuals examined. BPA is a known endocrine disruptor. Although initially considered to be a weak environmental estrogen, more recent studies have demonstrated that BPA may be similar in potency to estradiol in stimulating some cellular responses. Moreover, emerging evidence suggests that BPA may influence multiple endocrine-related pathways. Studies in rodents have identified adverse effects of BPA at levels at or below the current acceptable daily intake level for this compound. The various reported adverse effects of BPA are reviewed, and potential mechanisms of BPA action are discussed. Much more investigation is needed to understand the potential adverse health effects of BPA exposure in humans and to understand the multiple pathways through which it may act. Although many questions remain to be answered, it is becoming increasingly apparent that exposure to BPA is ubiquitous and that the effects of this endocrine disruptor are complex and wide-ranging.     

Electrochemical cell-based chip for the detection of toxic effects of bisphenol-A on neuroblastoma cells

A cell-based chip was fabricated for the electrochemical detection of the dose-dependent effects of bisphenol-A (BPA) on neuroblastoma cells (SH-SY5Y), which showed dual-mode correlation as a standard curve. Toxicity assessment of BPA became very important in environmental toxicants detection since BPA can be reached out easily from various common plastic-based product and give negative cellular effects on living organism. Cell chip was fabricated by immobilizing cells on C(RGD)(4) peptide coated electrode to detect the cytotoxicity of BPA electrochemically. Redox properties in living cells were determined by cyclic voltammetry using a home-made three-electrode system, and the cathodic peak current (I(pc)) was used as a parameter for measurement of the effect of BPA on cell viability. The peak current, I(pc) value increased with the concentration of BPA up to 300 nM and then decreased because of the stimulation of cancer cell activity at the concentration of BPA below 300nM and cytotoxicity at the concentration of BPA above 300 nM, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and optical microscopy-based morphological analysis confirmed the results of electrochemical study. This dual-mode correlation between the concentration of BPA and voltammetric signal intensity should be firstly considered to analyze its dose-dependent stimulus and cytotoxic effects on neuroblastoma cells by cell chip.     

Bisphenol A inhibits testicular functions and increases luteinizing hormone secretion in adult male rats

Effects of a xenobiotic estrogen, bisphenol A (BPA), on reproductive functions were investigated using adult male rats. BPA was dissolved into sesame oil and injected s.c. every day (1 mg/rat) for 14 days. Animals were killed by decapitation after the final administration of BPA, and the trunk blood, pituitary, and testes were collected. Plasma concentrations of prolactin were dramatically increased and pituitary contents of prolactin were slightly increased in the BPA group compared to the control group. Plasma concentrations of testosterone were decreased and plasma concentrations of LH were increased in BPA-treated rats compared to control rats. Testicular contents of inhibin were decreased in BPA-treated rats compared to control rats, although plasma concentrations of inhibin were not changed after administration of BPA. The testicular response to hCG for progesterone and testosterone release was decreased in BPA-treated rats. Administration of BPA did not change the pituitary response to luteinizing hormone-releasing hormone (LH-RH) in castrated male rats treated with testosterone. Male sexual behavior also was not changed as a result of BPA treatment. These results suggest that BPA directly inhibits testicular functions and the increased level of plasma LH is probably due to a reduction in the negative feedback regulation by testosterone. The testis is probably a more sensitive site for BPA action than the hypothalamus-pituitary axis.     

The rapid degradation of bisphenol A induced by the response of indigenous bacterial communities in sediment

In the present study, sediment was spiked with bisphenol A (BPA) solution to explore the interaction between indigenous bacterial communities and BPA biodegradation in sediment. Results showed that BPA could be adsorbed to the sediment and then biodegraded rapidly. Biodegradation efficiency of BPA in treatments with 10 and 50 mg/L BPA reached 64.3 and 61.8% on the first day, respectively. Quantitative polymerase chain reaction and denaturing gradient gel electrophoresis analysis indicated that BPA affected the densities, species, and diversities of bacteria significantly. The response of bacterial community to BPA favored BPA biodegradation by promoting the growth of BPA-reducing bacteria and inhibiting other competitors. According to the results of sequencing, Pseudomonas and Sphingomonas played vital roles in the degradation of BPA. They presented over 73% of the original bacterial community, and both of them were promoted by BPA comparing with controls. Laccase and polyphenol oxidase contributed to the degradation of BPA and metabolic intermediates, respectively. This paper illustrates the rapid biodegradation of BPA induced by the response of indigenous bacterial communities to the BPA stress, which will improve the understandings of BPA degradation in sediment.

     

Interdependence between the aerobic degradation of BPA and readily biodegradable substrates by activated sludge in semi-continuous reactors

The objective of the present work was to analyze the interrelationship between the aerobic degradation of BPA and readily biodegradable substrates by activated sludge (AS) in semi-continuous reactors (SCRs). AS were obtained from three SCRs fed with glucose, acetate or peptone. AS from these reactors were used as inocula for three SCRs that were fed with each biogenic substrate, and for three SCRs that were fed with the biogenic substrate and BPA. In all cases, dissolved organic carbon (DOC), BPA, total suspended solids (TSS) and respirometric measurements were performed. Although BPA could be removed in the presence of all the tested substrates, AS grown on acetate exhibited the longest acclimation to BPA. Reactors fed with peptone attained the lowest TSS concentration; however, these AS had the highest specific BPA degradation rate. Specific DOC removal rates and respirometric measurements demonstrated that the presence of BPA had a negligible effect on the removal of the tested substrates. A mathematical model was developed to represent the evolution of TSS and DOC in the SCRs as a function of the operation cycle. Results suggest that the main effect of BPA on AS was to increase the generation of microbial soluble products. This work helps to understand the relationship between the biodegradation of BPA and readily biodegradable substrates.     

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.     

Modeling the physisorption of bisphenol A on graphene and graphene oxide

The physisorption of bisphenol A (BPA) on pristine and oxidized graphene was studied theoretically via calculations performed at the PBE-D3 level (including dispersion force corrections). Three stable conformations of BPA on graphene were found. A lying-down configuration was energetically favored because the presence of π–π stacking and dispersion forces increased interactions. In addition, the adsorption of BPA on the edges of graphene oxide was enhanced when adsorption occurred on carboxyl and carbonyl groups, whereas the adsorption strength decreased when adsorption occurred on hydroxyl groups. The highest physisorption strength was obtained on the surface of graphene oxide due to the presence of π–π stacking and dispersion forces (which provided the greatest contribution to the adsorption energy) as well as hydrogen bonds (which provided a smaller contribution), indicating that oxidized graphene is a better candidate than pristine graphene for BPA removal. On the other hand, an increase in electrophilicity was observed after the physisorption of BPA in all systems (with respect to graphene and BPA in their isolated forms), with the adsorbent acting as the electron acceptor. Finally, molecular dynamics simulations performed using the PM6 Hamiltonian showed that the adsorption of BPA on graphene is stable.     

Effects of the xenoestrogen bisphenol A on expression of vascular endothelial growth factor (VEGF) in the rat

Bisphenol-A (BPA) is used to produce polymers for production of polycarbonate and epoxy resins that are used in food containers and dental appliances. BPA binds to estrogen receptors and induces estrogenic activity in a number of biological systems. We recently reported that although Fisher 344 (F344) and Sprague-Dawley (S-D) rat strains exhibit different sensitivities to BPA at the level of vaginal epithelial cell proliferation, there was no difference in immediate early proto-oncogene expression between the two animal strains. In the present study we investigated the effects of BPA on expression of another estrogen-target gene, vascular endothelial growth factor (VEGF), in the uterus, vagina, and pituitary of F344 and S-D rats. Adult rats were ovariectomized and treated with BPA by intraperitoneal injection at concentrations of 0.02 to 150 mg/kg body wt. Expression of VEGF was monitored by RNase protection assay at 2 hr after treatment. There was a significant effect of dose of BPA on the type of VEGF isoform expressed in the uterus, vagina, and pituitary. BPA induced greater (P < 0.01) levels of VEGF164 and VEGF120+188 than VEGF110 levels. The lowest BPA dose that had a significant (P< 0.05) effect on VEGF expression compared with vehicle treatment was 37.5 mg/kg body wt.; dose-response curves did not differ between strains. This is the first report that the primary response of the uterus, vagina, and pituitary to BPA includes rapid induction of VEGF expression. Due to the capacity of VEGF to engage pleiotropic signaling pathways in other cellular systems, we suggest that modulation of VEFG may play a role in establishing the response of estrogen-target organs to estrogenic xenobiotics.     

Bisphenol A in combination with insulin can accelerate the conversion of 3T3-L1 fibroblasts to adipocytes

The confluent cultures of 3T3-L1 fibroblasts were treated with or without bisphenol A (BPA) for 2 days and subsequently treated with insulin (INS) alone for 9 days. When BPA was absent during the first 2-day treatment period, the cultures contained 1.6 microg/microg DNA of triacylglycerol (TG), 202 mU/mg DNA of lipoprotein lipase (LPL) activity, and 462 nmol/min/mg DNA of glycerol-3-phosphate dehydrogenase (GPDH) activity. The presence of BPA during the same period caused a 150% increase in the TG content, a 60% increase in the LPL activity, and a 500% increase in the GPDH activity. Thus, BPA by itself can trigger 3T3-L1 fibroblasts to differentiate into adipocytes. Next, the confluent cultures were treated with BPA for 2 days and subsequently treated with a combination of INS and BPA for 9 days. The simultaneous presence of BPA with INS caused a 370% increase in the TG content, a 200% increase in the LPL activity, and a 225% increase in the GPDH activity compared with the cultures treated with INS alone. The amount of [(3)H]thymidine incorporated into DNA was lower in the cultures treated with INS in the presence of BPA than in those treated with INS alone, indicating that BPA has an anti-proliferative activity on 3T3-L1 cells. Taken together, our results indicate that BPA in combination with INS can accelerate the adipocyte conversion.     

Transgenerational effects of prenatal bisphenol A on social recognition

Bisphenol A (BPA) is a man-made endocrine disrupting compound used to manufacture polycarbonate plastics. It is found in plastic bottles, canned food linings, thermal receipts and other commonly used items. Over 93% of people have detectable BPA levels in their urine. Epidemiological studies report correlations between BPA levels during pregnancy and activity, anxiety, and depression in children. We fed female mice control or BPA-containing diets that produced plasma BPA concentrations similar to concentrations in humans. Females were mated and at birth, pups were fostered to control dams to limit BPA exposure to gestation in the first generation. Sibling pairs were bred to the third generation with no further BPA exposure. First (F1) and third (F3) generation juveniles were tested for social recognition and in the open field. Adult F3 mice were tested for olfactory discrimination. In both generations, BPA exposed juvenile mice displayed higher levels of investigation than controls in a social recognition task. In F3 BPA exposed mice, dishabituation to a novel female was impaired. In the open field, no differences were noted in F1 mice, while in F3, BPA lineage mice were more active than controls. No impairments were detected in F3 mice, all were able to discriminate different male urine pools and urine from water. No sex differences were found in any task. These results demonstrate that BPA exposure during gestation has long lasting, transgenerational effects on social recognition and activity in mice. These findings show that BPA exposure has transgenerational actions on behavior and have implications for human neurodevelopmental behavioral disorders.     

Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546-553]. In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase II oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition. With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure.