Differential activities of the multixenobiotic resistance mechanism in freshwater fishes inhabiting environments of Patagonia Argentina.

Environmental impairment resulted from urbanizations can produce damage on freshwater species including strong physiological effects at individual or population level. The multixenobiotic resistance (MXR) is a defence mechanism which has been demonstrated in several aquatic organisms. The key mediators of MXR activity are ATP-binding cassette (ABC) proteins like P-glycoprotein (P-gp). This system protects aquatic organisms against the accumulation of xenobiotics by extruding them from cells in an energy-dependent manner. MXR has been pointed out as relevant in the ecotoxicological context and has been proposed as a biomarker for pollution assessment. Since fish species are common target in freshwater biomonitoring programs, the purpose of the study was to evaluate the MXR mechanism in native Hatcheria macraei (Patagonian catfish) and exotics Salmo trutta (brown trout), Oncorhynchus mykiss (rainbow trout) and Oncorhynchus tshawytscha (Chinook salmon) freshwater fishes widespread in Argentine Patagonia. We characterized the MXR mechanism using a combination of functional assays and Western blot analysis. Our results in different tissues such as liver, gills, muscle and epidermis indicate that the fishes studied have different species-specific levels of MXR activity, being gills and liver the tissues with greater detoxifying activity. Induction of MXR transport activity was also identified in liver tissue from rainbow trout from urban stream suggesting their suitability in the biomonitoring of aquatic environments subjected to urban contaminants.

Influence of Water Temperature on the MXR Activity and P-glycoprotein Expression in the Freshwater Snail, Physa acuta (Draparnaud, 1805).

Cristina N. Horak and Yanina A. Assef (2017) P-glycoprotein (P-gp) mediated multixenobiotic resistance (MXR) is a mechanism analogous to multidrug resistance, which has been extensively characterized in mammalian tumours. The expression and function of the MXR mechanism has been demonstrated in numerous aquatic organisms and has been proposed as a biomarker for pollution assessment. A close relationship between thermal stress and MXR response has been reported in some aquatic organisms. Seasonal studies in freshwater organisms are scarce and conducted mainly in zebra mussel (Dreissena polymorpha), whose presence has not been reported in South America. The general purpose of the present study was to evaluate seasonal variation of a biomarker, the MXR mechanism, in the worldwide distributed freshwater snail P. acuta. We analyzed the in situ influence of temperature on the biomarker response over an 18-month field study. MXR defence system was evaluated by a combination of functional assays (RB accumulation) and molecular approaches to analyse P-gp expression. The results demonstrated a linear correlation between MXR response, at activity and expression level, and water temperature at sample site, in P. acuta snails. The characterization of the MXR system in worldwide distributed species, including the study of their seasonal fluctuations, could contribute to the increasing interest to incorporate this biomarker to provide an integrated assessment of mussel health status. This work supports the possible use of P. acuta snails with this purpose and also highlights that the occurrence of variations in MXR response related to water temperature has to be taken into account in the interpretation of in situ monitoring studies.

ENaC channels in oocytes from Xenopus laevis and their regulation by xShroom1 protein.

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, ß, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na(+) currents (INa((amil))). We observed a marked reduction in INa((amil)) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant ß-mENaC subunit (ß-S518K) with an open probability of 1 had enhanced INa((amil)) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa((amil)) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, ß and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

Cell migration in BeWo cells and the role of epithelial sodium channels.

Cell migration/proliferation processes associated with wound healing were measured in BeWo cells at 6 h, when mitosis is still scarce. Cells were cultured in medium with 1% fetal bovine serum to minimize proliferation. BeWo cell migration covered 20.6 +/- 7.0%, 38.0 +/- 5.4%, 16.6 +/- 4.8% and 13.7 +/- 3.6% of the wound when cultivated under control, aldosterone (100 nM, 12 h), aldosterone plus amiloride (10 muM) and amiloride treatments, respectively. When BeWo cells were treated with aldosterone, there was an increase in wound healing (P < 0.05), which was prevented by adding the ENaC blocker amiloride (P < 0.05, n = 16). Immunocytochemistry studies showed that the three ENaC subunits showed greater expression at the leading edge of the wound 3 h after injury, supporting the notion that these proteins participate in a postinjury signal. Antisense oligonucleotides directed against the alpha-ENaC subunit decreased the migratory response of the cells compared to the sense treated cells or the cells without oligonucleotides (P < 0.001, n = 16): 30.2 +/- 3.7%, 17.6 +/- 1.3%, 27.5 +/- 1.5% and 20.2 +/- 1.5% reinvasion of the wound with aldosterone, aldosterone plus antisense, aldosterone plus sense treatments and control conditions, respectively. Aldosterone and amiloride influence wound healing in BeWo cells, probably by their effects upon ENaCs, transmitting a signal to the cell cytoplasm for the release of several agents that promote cell migration.

HERG1 currents in native K562 leukemic cells.

The human ether-a-go-go related gene (HERG1) K+ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to -40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC50) of the blocker was 4.69 nM: The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at -120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K+ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H2O2.

Ionic currents in multidrug resistant K562 human leukemic cells.

In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 microM n activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 microM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 microM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 microM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.

Effects of ionomycin and thapsigargin on ion currents in oocytes of Bufo arenarum.

In this study, two electrode voltage clamp technique was used to assess the ionic current of oocytes of the South American toad Bufo arenarum and to study the dependence of these currents on the extracellular and intracellular Ca2+ concentrations. Ca2+ chelators, ionomycin -a calcium ionophore- and thapsigargin, a blocker of the Ca2+ pump of the sarcoplasmic reticulum, were used. The main results were the following: Most oocytes showed a voltage activated rectifying conductance. Ionomycin (1 microM) increased inward and outward currents in control solution. The effect of ionomycin was blocked partially at negative potentials and was blocked completely at positive potentials in absence of extracellular Ca2+. When the oocytes were treated with thapsigargin (2 microM) or BAPTA-am, a membrane-permeant intracellular chelator in control solution (10 microM), ionomycin did not increased either inward nor outward currents. The conclusion of our experiments is that there are two sources of Ca2+ for activation of the current induced by ionomycin, the cytoplasmic stores and the extracellular space. We believe ionomycin directly translocates Ca2+ from the SER into the cytoplasm but not from the extracellular medium. Ca2+ entry probably occurs through store-operated-Ca-channels.

CFTR in K562 human leukemic cells.

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

An outwardly rectifying anion channel in human leukaemic K562 cells.

In this study, an outwardly rectifying anion channel was characterized in the cell line K562 obtained from a chronic human leukaemia. Ion channel activity was recorded in the cell-detached (inside-out) configuration with standard patch-clamp technology. In most of the K562 cells studied, the channel exhibited low spontaneous activity, an outwardly rectifying current/voltage relationship and single-channel conductances of 19 pS and 40 pS for inwards and outwards currents respectively. The channel had a low permeability for gluconate with a relative permeability P(gluconate)/ P(Cl) of 0.14 and was blocked by glibenclamide (50 micro M) or diphenylamine-2-carboxylate (DPC, 1 mM) added to the cytoplasmic side of the patch. These results are characteristic of the outwardly rectifying Cl channel (ORCC) found in other types of cells.