Effects of selected endocrine disruptors on meiotic maturation,cumulus expansion,synthesis of hyaluronan and progesterone by porcine oocyte–cumulus complexes

In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100 pM–100 μM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte–cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100 μM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44 h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors.     

Xenopus laevis oocytes expressing human P-glycoprotein: Probing trans- and cis-inhibitory effects on [3H]vinblastine and [3H]digoxin efflux

P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 °C. [³H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K_m of 145.5 ± 25.4 μM. [³H]Vinblastine (5.6 ± 0.3 μM) and [³H]digoxin (1.0 ± 0.1 μM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [³H]vinblastine and [³H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [³H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug–drug and/or drug–inhibitor interactions.     

Evaluation of cytotoxicity,genotoxicity and embryotoxicity of insecticide propoxur using flounder gill (FG) cells and zebrafish embryos

Cytotoxicity, genotoxicity and embryotoxicity of carbamate insecticide propoxur were evaluated using flounder gill (FG) cells and zebrafish embryos. The cytotoxicity of propoxur in FG cells was analyzed by MTT, neutral red uptake (NRU), lactate dehydrogenase (LDH) release and Hoechst 33342 and propidium iodide double staining, and acute cytotoxic effects were observed in a concentration-dependent manner. The 24 h-IC₅₀ values of 89.96 ± 1.04, 103.4 ± 1.14 and 86.59 ± 1.13 μg/ml propoxur were obtained by MTT, NRU and LDH assays, respectively. The lethal effects were induced in FG cells mainly through necrosis but not apoptosis as evidenced by double fluorescence staining. Comet assay showed weak genotoxic effects and statistically significant DNA damages were recorded in the cells exposed to highest tested concentration of 75 μg/ml propoxur (p < 0.05). Propoxur exerted obvious acute toxic effects on the survival, spontaneous movement, hatching and heart rate, and development (yolk and pericardial sac edema) of zebrafish embryos in both time- and concentration-dependent manner only at ⩾100 μg/ml. The corresponding 24 h-, 48 h- and 96 h-LC₅₀ values of propoxur in zebrafish embryos were 166.4 ± 1.06, 146.3 ± 1.07 and 134.8 ± 1.06 μg/ml, respectively. The above data obtained suggest a low acute toxicity of propoxur to the in vitro cultured FG cells and zebrafish embryos.     

Reactive oxygen species induced by beauvericin,patulin and zearalenone in CHO-K1 cells

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC₅₀ values obtained on the MTT and NR assays ranged from 0.69 to 79.40 μM and 4.40 to 108.76 μM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H₂-DCFDA was measured. The relative intensity of fluorescence from cells incubated with BEA, PAT and ZEA was approximately 4-, 7- and 4-fold higher in comparison with control cells at 0 min, respectively. Lipid peroxidation induced by ROS generation was assessed using the thiobarbituric acid reactive substances (TBARS) method for 2, 24 and 48 h. The malondialdehyde (MDA) production was increased with BEA and PAT exposure in a concentration- and time-dependent manner. MDA was not increased after 1 and 5 μM ZEA exposures for 2 h, but was slightly increased at 50 μM.In conclusion, PAT was the most cytotoxic mycotoxin to CHO-K1 cells, followed by BEA and ZEA. Mycotoxins reduce cell viability correlated with the increases of ROS generation and MDA formation in concentration- and time-dependent manner. These data suggested that cytotoxicity and ROS generation are mechanisms of mycotoxins mediated toxicity.     

N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine (CP-100,356) as a “chemical knock-out equivalent” to assess the impact of efflux transporters on oral drug absorption in the rat

The utility of the diaminoquinazoline derivative CP-100,356 as an in vivo probe to selectively assess MDR1/BCRP-mediated drug efflux was examined in the rat. CP-100,356 was devoid of inhibition (IC₅₀ >50 µM) against major human P450 enzymes including P4503A4. In human MDR1-transfected MDCKII cells, CP-100,356 inhibited acetoxymethyl calcein (calcein-AM) uptake (IC₅₀ ～0.5 ± 0.07 µM) and digoxin transport (IC₅₀ ～1.2 ± 0.1 µM). Inhibition of prazosin transport (IC₅₀ ～1.5 ± 0.3 µM) in human BCRP-transfected MDCKII cells by CP-100,356 confirmed the dual MDR1/BCRP inhibitory properties. CP-100,356 was a weak inhibitor of OATP1B1 (IC₅₀ ～66 ± 1.1 µM) and was devoid of MRP2 inhibition (IC₅₀ >15 µM). In vivo inhibitory effects of CP-100,356 in rats were examined after coadministration with MDR1 substrate fexofenadine and dual MDR1/BCRP substrate prazosin. Coadministration with increasing doses of CP-100,356 resulted in dramatic increases in systemic exposure of fexofenadine (36- and 80-fold increase in C_max and AUC at a CP-100,356 dose of 24 mg/kg). Significant differences in prazosin pharmacokinetics were also discernible in CP-100,356-pretreated rats as reflected from a 2.6-fold increase in AUC. Coadministration of CP-100,356 and P4503A substrate midazolam did not result in elevations in systemic exposure of midazolam in the rat. The in vivo methodology should have utility in drug discovery in selective and facile assessment of the role of MDR1 and BCRP efflux transporters in oral absorption of new drug candidates. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4914–4927, 2009     

Comparison of bidirectional lamivudine and zidovudine transport using MDCK,MDCK–MDR1, and Caco-2 cell monolayers

Bidirectional transport studies were conducted using Caco-2, MDCK, and MDCK–MDR1 to determine P-gp influences in lamivudine and zidovudine permeability and evaluate if zidovudine permeability changes with the increase of zidovudine concentration and/or by association of lamivudine. Transport of lamivudine and zidovudine separated and coadministrated across monolayers based on these cells were quantified using LC–MS–MS. Drug efflux by P-gp was inhibited using GG918. Bidirectional transport of lamivudine and zidovudine was performed across MDCK–MDR1 and Caco-2 cells. Statistically significant transport decrease in B → A direction was observed using MDCK–MDR1 for zidovudine and MDCK–MDR1 and Caco-2 for lamivudine. Results show increased transport in B → A and A → B directions as concentration increases but data from P_app increase in both directions for both drugs in Caco-2, decrease in MDCK, and does not change significantly in MDCK–MDR1. Zidovudine transport in A → B direction increases when coadministrated with increasing lamivudine concentration but does not change significantly in B → A direction. Zidovudine and lamivudine are P-gp substrates, but results assume that P-gp does not affect significantly lamivudine and zidovudine. Their transport in monolayers based on Caco-2 cells increase proportionally to concentration (in both directions) and zidovudine transport in Caco-2 cell monolayer does not show significant changes with lamivudine increasing concentrations. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4413–4419, 2009     

Inhibition of vascular endothelial growth factor-mediated angiogenesis involved in reproductive toxicity induced by sesquiterpenoids of Curcuma zedoaria in rats

The use of herbal medicine has rapidly increased in recent decades, prompting an increase in toxicity concerns. Here we investigated whether and how essential oil of Curcuma zedoaria may induce reproductive and developmental toxicity. Whole embryo culture in rats revealed that the essential oil produced a concentration-dependent toxicity ex vivo in the embryos on gestation Day 9.5 (GD9.5). Weight loss, abnormal hematological and biochemical effects on dams and embryos were also observed in GD17 pregnant rats orally administrated with 100 mg kg⁻¹ or 200 mg kg⁻¹ essential oil from GD7 onward. Induction of embryotoxicity may be related to placental calcification attributed to inhibition of vascular endothelial growth factor (VEGF)-mediated angiogenesis. Analysis by gas chromatography–mass spectrometry demonstrated that the main toxic compounds in essential oil were sesquiterpenoids. Our results suggest that the reproductive toxicity of C. zedoaria may be caused by sesquiterpenoids in the essential oil blocking VEGF-mediated angiogenesis.     

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1

The impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe ³H-digoxin in air–liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin–Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. ³H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER = 11.4 or 3.0 were measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on ³H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in ³H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.     

Association of the MDR1 (ABCB1) gene 3435C> T polymorphism with male infertility

Infertility is a common problem affecting one in six couples, and in 30% of infertile couples, the male factor is a major cause due to defective sperm quality. P-glycoprotein (P-gp), a product of the MDR1 (ABCB1) gene, may be a link between genetic and environmental factors contributing to the development of male infertility because pesticides (P-gp substrates) are well established factors of male infertility. The aim of the present study was to examine the effect of the MDR1 gene 3435C>T polymorphism on male infertility. In total, 162 male patients undergoing semen analysis due to initial infertility workup were included in the study. The control group consisted of 191 healthy males with proven fertility. MDR1 3435C>T genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Assessment of MDR1 genotypes among the infertile men showed that 17.9% of subjects were carriers of the CC genotype, 58.0% were CT and 24.1% were TT. Among fertile men, 30.4% of subjects were characterised by the CC genotype, 49.7% were CT and 19.9% were TT. In addition, the frequency of carriers of at least one T allele (i.e., CT and TT genotypes) among infertile and fertile subjects was 82.1% and 69.6%, respectively. The risk of infertility was significantly elevated by two-fold in individuals carrying at least one T allele (CT and TT genotypes: p = 0.009, OR = 2.00, 95% CI: 1.20–3.32). Furthermore, this elevated risk was still found when considering each of the CT and TT genotypes alone (TT genotype: p = 0.027, OR = 2.05, 95% CI: 1.09–3.86; CT genotype: p = 0.013, OR = 1.98, 95% CI: 1.16–3.36). This preliminary report suggests that P-gp may play some role in male infertility, mediating detrimental effects of environmental factors.     

Emerging Fusarium mycotoxins in organic and conventional pasta collected in Spain

One of the main sources of emerging Fusarium mycotoxins in human nutrition is the cereals and cereal products. In this study, an analytical method to determine enniatins A, A1, B and B1 (ENs), beauvericin (BEA) and fusaproliferin (FUS) based on Ultra-Turrax extraction followed by liquid chromatography coupled to triple quadrupole mass spectrometer detector (MS/MS QqQ), was applied for the analysis of pasta. For this purpose, 114 commercial samples of pasta were acquired from supermarkets located in Valencia. The results showed higher frequencies of contamination in organic pasta than in conventional pasta, while the concentration levels were variable for both types of pasta. In positive samples, BEA levels varied from 0.10 to 20.96 μg/kg and FUS levels varied from 0.05 to 8.02 μg/kg. ENs levels ranged from 0.25 to 979.56 μg/kg, though the majority of the values were below 25 μg/kg. Besides, it was observed the simultaneous presence of two or more mycotoxins in a high percentage of the samples. Finally, an evaluation of the dietary exposure of the emerging Fusarium mycotoxins was performed in the Spanish population. The prevalence of ENs, BEA and FUS in cereal products suggests that the toxins may pose a health risk to Spanish population.     

Transport of 5,5-diphenylbarbituric acid and its precursors and their effect on P-gp,MRP2 and CYP3A4 in Caco-2 and LS180 cells

AimTo examine the transport of 5,5-diphenylbarbituric acid sodium (T2007) and its mono- (MMMDPB) and di- (T2000) methoxymethylated precursors and their inducibility potential in Caco-2 and LS180 cells.MethodsTransport studies of T2000, MMMDPB and T2007 in Caco-2 cells were performed in Transwells. P-gp and CYP3A4 activities were assayed by [³H]digoxin and rhodamine 123 cellular retention and testosterone 6β-hydroxylation, respectively. Expressions of PXR, VDR and CAR mRNA and CYP3A4, MDR1/P-gp and MRP2 mRNA and protein were determined by qPCR and Western blotting, respectively. PXR siRNA was used to assess the involvement of PXR.ResultsThe P_app(A→B)s and P_app(B→A)s of T2000, MMMDPB and T2007 were similar (30–35 × 10^?6 cm/s) in Caco-2 cells. Treatment for 3 days with T2000 (15 μM), MMMDPB (70 μM) and T2007 (300 μM) generally furnished a greater induction in LS180 cells over the Caco-2 cells due to the higher, natural abundance of PXR. Changes in expression were confined mostly to MDR1 and CYP3A4: in LS180 cells, treatment for 3 days increased MDR1 and CYP3A4 but not MRP2 mRNA, and elevated P-gp and CYP3A4 protein expression that led to decreased cellular accumulation of [³H]digoxin and rhodamine 123, and enhanced testosterone 6β-hydroxylase activity towards T2007, respectively. The silencing of PXR by PXR siRNA in LS180 cells significantly attenuated the induction of MDR1 and CYP3A4.ConclusionsT2000, MMMDPB, and T2007 exhibited high permeabilities but are not P-gp substrates. T2007 and its analogs upregulated CYP3A4 and MDR1 modestly via the PXR.     

Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression

The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression.     

Identification of pathology from diesel exhaust particles in the bladder in a rat model by aspiration of particles from the pharynx

To determine whether diesel exhaust particles (DEPs) could be a toxic agent to the bladder, rats were exposed to different concentrations of DEPs for one month or three months. When the rats were sacrificed, morphologic changes of the urothelium were investigated. The antioxidase activity and the levels of lipid peroxidation in the bladder were assayed. In the three-month group, DEPs at doses of 21.03 μg/μl insulted the structural integrity of surface glycosaminoglycans, widened the gap between urothelial cells, increased levels of lipid peroxidation, and decreased antioxidase activities in the urinary bladder (p < 0.05). Furthermore, DEPs at a dose of 5.61 μg/μl decreased glutathione, catalase, and glutathione peroxidase activities (p < 0.05). These results led to the conclusion that DEPs were a toxic agent in the bladder. The toxic effects might be attributed to oxidative damage mediated by pro-oxidant/antioxidant imbalance or excessive free radicals.     

Glyphosate induces cardiovascular toxicity in Danio rerio

Glyphosate is a broad spectrum herbicide used aggressively in agricultural practices as well as home garden care. Although labeled “safe” by the chemical industry, doses tested by industry do not mimic chronic exposures to sublethal doses that organisms in the environment are exposed to over long periods of time. Given the widespread uses of and exposure to glyphosate, studies on developmental toxicity are needed. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate on the developing heart. Treatment by embryo soaking with 50 μg/ml glyphosate starting at gastrulation results in structural abnormalities in the atrium and ventricle, irregular heart looping, situs inversus as well as decreased heartbeats by 48 h as determined by live imaging and immunohistochemistry. Vasculature in the body was also affected as determined using fli-1 transgenic embryos. To determine if the effects noted at 48 h post fertilization are due to early stage alterations in myocardial precursors, we also investigate cardiomyocyte development with a Mef2 antibody and by mef2ca in situ hybridization and find alterations in the Mef2/mef2ca staining patterns during early cardiac patterning stages. We conclude that glyphosate is developmentally toxic to the zebrafish heart.     

In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells

The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7–8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun–Emmet–Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 μg/mL for 6, 24 and 48 h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50–100 μg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.     

Evaluation of dalbavancin,tigecycline, minocycline,tetracycline, teicoplanin and vancomycin against community-associated and multidrug-resistant hospital-associated meticillin-resistant Staphylococcus aureus

Dalbavancin is an investigational semisynthetic lipoglycopeptide that is structurally related to teicoplanin. We examined the activity of dalbavancin (DAL) compared with tigecycline (TIG), minocycline (MIN), tetracycline (TET), teicoplanin (TEC) and vancomycin (VAN) against community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) and multidrug-resistant hospital-associated meticillin-resistant S. aureus (MDR HA-MRSA). Two hundred and twenty clinical isolates of CA-MRSA and MDR HA-MRSA were utilised. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined according to Clinical and Laboratory Standards Institute guidelines. Selective time–kill studies were performed against CA-MRSA and MDR HA-MRSA in triplicate. Overall, DAL exhibited low MIC values against all strains of MRSA. Time–kill studies with CA-MRSA demonstrated DAL = VAN > TIG > MIN = TEC > TET (P < 0.006), and with MDR HA-MRSA demonstrated DAL = VAN = TEC > TIG = MIN > TET (P > 0.05). DAL demonstrated potent activity against clinical strains of CA-MRSA and MDR HA-MRSA, including tetracycline-resistant S. aureus.     

Naringenin (NAR) and 8-prenylnaringenin (8-PN) reduce the developmental competence of porcine oocytes in vitro

Flavanones such as naringenin (NAR) and 8-prenylnaringenin (8-PN) are increasingly used as dietary supplements despite scientific concern regarding adverse effects on female reproduction upon excessive intake. In the present study, NAR and 8-PN (0.3–1 μM) significantly affected porcine oocyte maturation in vitro by decreasing cumulus expansion. In addition, NAR and 8-PN decreased percentages of meiotic spindle formation, oocyte cleavage and blastocyst formation. The effects of NAR and 8-PN were different from estradiol (3.12 μM)-induced effects. Still, the flavanone-induced effects were observed at concentrations that can be found in human plasma upon supplement intake and that resemble physiological estrogen equivalence levels in follicular fluids. Considering that abnormal oocyte maturation can cause subfertility, our study warrants that precautions are in place and excessive intake of NAR and 8-PN e.g. via dietary supplements should be avoided by women.