Search

CuO NPs have previously been reported as toxic to a range of cell cultures including kidney epithelial cells from the frog, Xenopus laevis (A6). Here we examine the molecular mechanisms affecting toxicity of Cu in different forms and particle sizes. A6 cells were exposed to ionic Cu (Cu2+) or CuO particles of three different sizes: CuO NPs of 6 nm (NP6), larger Poly-dispersed CuO NPs of

Cisplatin is a platinum-containing antitumour agent, the usefulness of which is limited by nephrotoxicity. In the present study, we examined the effects of cisplatin on the established renal epithelial A6 cell line, which forms a polarized monolayer in vitro with active transmembrane ion transport. The effect of cisplatin (0–800 μ m ) on transepithelial ion transport and transepithelial resistance (TER) was monitored by the short-circuit current (SSC) technique. Cell integrity was determined by monitoring TER at increasing concentration of cisplatin during 24 hours. The half-maximal inhibition concentration was 49 and 540 μ m when applied to either the basolateral or apical surface, respectively. This suggests that cisplatin-mediated deterioration of cell integrity is far more pronounced when cisplatin is applied basolaterally than apically. Continuous measurements of TER demonstrated a dose- and time-dependent decline in TER/TER0 (TER at time zero). In addition, cisplatin from the basolateral side had no prompt effect on transepithelial ion transport. Instead a slow, but dose-dependent decline which at the highest concentration resembled the decline observed when ouabain was added. Inhibition of Na-K-ATPase by cisplatin was examined by the use of nystatin, a membrane permeabilizer, and data suggest that cisplatin at 800 μ m inhibits Na-K-ATPase by about 50% after 60 minutes of exposure. Morphological examinations of subcultured cisplatin treated cells indicate that cell death is probably due to apoptosis rather than necrosis.

A cultured epithelial cell line from toad kidney (A6) was used to investigate side-specific toxicity related to the apical (outer) and basolateral (inner) membranes of epithdia. Well-known inhibitors and stimulators of ion transport were used to show that the ion transport proteins are asymmetrically distributed: the apical membrane contains sodium and chloride channels and the basolateral membrane contains Na+/K+ pumps, Na+/Cl- co-transporters, potassium channels and receptors for antidiuretic hormone The data demonstrate that the cellular toxicity of chemicals decreases when they are added to the apical side, illustrating that the epithelium acts as a functional barrier. However, the side-specific toxicity was more pronounced for ions and water-soluble molecules than for organic solvents, indicating that A6 epitheha can be used to distinguish between drugs that target specific membrane proteins and those that target membrane lipids. Furthermore, the cell line could be used to pick up chemicals that, at low concentrations inhibit sodium absorption and chloride secretion, without having any effect on cellular toxicity.

Nanoparticles (NPs) have unique chemical and physical properties caused by their small size (1-100 nm) and high surface to volume ratio. This means that the NPs are potentially more toxic than their bulk counterparts. In the present study a cultured epithelial cell line from Xenopus laevis (A6) was used to investigate toxicity of copper (Cu) in 3 different forms; Cu ions (Cu(2+)), CuO NPs (6 nm) and poly-dispersed CuO NPs (100 nm, poly-CuO). Continuous exposures at concentrations of 143-200 μM demonstrated that cytotoxicity differed among the 3 Cu forms tested and that the effects depend on cell state (dividing or differentiated). Dividing cells treated with poly-CuO, CuO NPs (6 nm) or Cu(2+) showed cell cycle arrest and caused significant increase in cell death via apoptosis after 48 h, 6 and 7 days of treatment, respectively. Treatment with either CuO NPs (6 nm) or Cu(2+) caused significant decrease in cell proliferation. Treatments of differentiated cells, revealed the same patterns of toxicity for Cu forms tested, but after shorter exposure periods.

An established epithelial cell line (A6) from toad kidney was used to study in vitro cytotoxicity. When grown on permeable support, A6 cells form a monolayer epithelium with a high electrical resistance and a transepithelial potential. These two easily measured electrophysiological endpoints showed a dose-related decrease after exposure of the cells for 24 hr to 21 selected chemicals. It was demonstrated that both transepithelial potential and transepithelial resistance correlated well with acute cytotoxicity data obtained using human lymphocytes and with calculated human lethal dose values. The polarity of the epithelial cells was demonstrated by specific chemicals that targeted the basolateral membrane. The results show that electrophysiological measurements of A6 epithelia could be used as a general cell model to study cytotoxicity and as a specific model to evaluate toxic affects on tight epithelia.

The influence of reactive oxygen metabolites on ion transport across the plasma membrane was investigated by measuring the effect of hydrogen peroxide (H(2)O(2)) on short-circuit current (SCC) and transepithelial conductance (G(t)) in isolated frog skin. This cellular system gives access to the apical (outer) and basolateral (inner) membranes of the polarized epithelial cells. Both apical and basolateral addition of H(2)O(2) (10 mum to 100 mm) induced a dose-dependent stimulation of SCC. This stimulation could be blocked by amiloride in the apical solution, showing that the H(2)O(2)-induced stimulation of SCC was a result of increased active transepithelial sodium (Na) transport. The increase in Na transport was prevented by addition of catalase, consistent with a role for H(2)O(2) in producing this effect. The mechanisms for H(2)O(2)-stimulated Na transport localized in the apical and basolateral membranes differ markedly. Basolateral H(2)O(2)-stimulated Na transport was inhibited by indomethacin, indicating that increased prostaglandin synthesis was responsible for this effect. Apical H(2)O(2) stimulation of Na transport was not affected by indomethacin, nor did H(2)O(2) interfere with the Na self-inhibition of the Na channels. It is concluded that apical H(2)O(2) increases the Na permeability of the apical membrane, either through direct interaction with the apical Na channels or indirectly through products of lipid peroxidation.

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

Cadmium (Cd) is an important industrial and environmental pollutant, and the kidney is the primary organ to be affected. To elucidate the effects of Cd on cell proliferation, an epithelial cell line (A6) originally derived from the distal part of the Xenopus laevis kidney was cultured in media containing 10% fetal bovine serum. The effects of Cd (added as CdCl(2)) on cellular growth and differentiation from single cells to confluent epithelia were investigated by visual inspection and by measurement of the degree to which living cells covered a unit area. Over a concentration range from 5 to 50 microM, Cd did not affect the settling and adherence of single cells to the bottom of the culture well. The addition of 5 microM Cd for 4 days did not affect the ability of the A6 cells to develop confluent epithelia, measured as the area covered by adherent living epithelial cells (99 +/- 4% of the control value). However, 10 microM Cd did effectively inhibit development of confluent epithelia to 13 +/- 5% compared to control. Visual inspection of adherent cells exposed to 50 microM Cd for 7 days revealed no increase in cell number or in cell death, which indicated the induction of cell cycle arrest. Flow cytometric analysis showed that treatment of cells with Cd (0.4mM) for 24 hours induced a significant increase in the proportion of G1 phase cells from 58.6 +/- 3.9 to 80.6 +/- 3.7%, and a corresponding reduction in the proportion of cells in both the S and G2 phases from 24.0 +/- 3.6 to 13.4 +/- 3.3% and 17.2 +/- 1.7 to 5.8 +/- 2.1%, respectively. This study showed that Cd stopped cell proliferation in a very narrow concentration range, between 5 and 10 microM, and cell cycle analysis indicated that Cd arrested the cells in the G1 phase of the cell cycle.

The aim of the present study was to investigate the mechanism by which the calcium ionophore A23187 stimulates Cl and water secretion from exocrine glands in the frog skin. The Cl secretion was visualized as changes in short-circuit current (SCC) in skins where the Na absorption was blocked by amiloride applied to the apical membrane. Measurements of A23187 stimulated ion fluxes showed that the ionophore induced a net secretion of Cl, Na and K. The active Cl secretion was enhanced more than the Na and K secretion, resulting in a net secretion of negative ions which closely resembled the A23187-stimulated SCC. The effect of A23187 was abolished in skins pretreated with indomethacin, implying the involvement of prostaglandins in the response. Furthermore, the effect of A23187 was inhibited in the presence of quinacrine, indicating that the activation of the cyclooxygenase pathway is dependent on phospholipase A2 activity. In addition, the A23187, but not the arachidonic acid stimulated Cl secretion was abolished in the presence of trifluoperazine, suggesting that the effect of the ionophore may be mediated via a Ca2+ -calmodulin-dependent step located before the activation of the cyclooxygenase. The net water flow and the Cl secretion were measured simultaneously under the conditions outlined above. The stimulation, inhibition, and time-course of the water secretion were similar to the changes observed for the Cl secretion. The A23187 stimulated Cl secretion was enhanced by the phosphodiesterase inhibitor, theophyllin, indicating that the effect of A23187 was caused by an increase in the intracellular cAMP level in the gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)

When added to the apical solution of isolated frog skin, the calmodulin antagonist trifluoperazine (TFP)* stimulated the short-circuit current (SCC) in a dose-dependent manner. The increase in SCC was due to an enhanced active transepithelial Na transport. After addition of TFP (15 microM) the intracellular voltage depolarized, showing that TFP acts by increasing the sodium (Na) permeability of the apical membrane. The TFP-induced increase in SCC was not accompanied by an increase in prostaglandin E2 release from the skins as observed by basolateral addition of TFP. When the apical Na concentration in fast-flow experiments was changed from 0 to 50 mM, the SCC increased promptly and then reclined. The presence of TFP in the apical solution abolished this recline. The apparent inhibition constant for amiloride changed in the presence of TFP from 1.42 +/- 0.12 microM to 0.38 +/- 0.05 microM (n = II) and TFP abolished the inhibition of SCC caused by high apical Na concentrations. These observations indicate that TFP acts by abolishing the Na self-inhibition of the Na channels. Calmidazolium and chlorpromazine stimulated the SCC to the same degree and in the same concentration range as TFP, suggesting that the effect of TFP was not mediated by the Ca2+-calmodulin complex.