Henning F. Bjerregaard

Toxicity of CuO nanoparticles and Cu ions to tight epithelial cells from Xenopus laevis (A6): Effects on proliferation, cell cycle progression and cell death

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.

Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells.

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 <100 nm (Poly) and CuO Micro particles of <5 μm (Micro), at 200 μM, equal to 12.7 mg Cu/L. Poly was significantly more toxic than NP6, Micro and Cu2+ to A6 cells, causing DNA damage, decreased cell viability and levels of reduced glutathione (GSH) and eventually cell death. We show that ROS (Reactive Oxygen Species) generation plays a key role and occurs early in Poly toxicity as Poly-induced DNA damage and cell death could be mitigated by the antioxidant NAC (N-acetyl-cysteine). Here we propose a model of the sequence of events explaining Poly toxicity. Briefly, the events include: cellular uptake, most likely via endocytosis, production of ROS, which cause DNA damage that activates a signaling pathway which eventually leads to cell death, mainly via apoptosis.

Effect of cadmium on active ion transport and cytotoxicity in cultured renal epithelial cells (A6).

A cultured epithelial cell line from toad kidney (A6) was used to study the mechanism by which cadmium (Cd) affects transepithelial resistance (TER) and active transepithelial ion transport measured as short-circuit current (SCC) in vitro. The influence of Cd on cell integrity was investigated by measuring time-dependent TER under controlled conditions and the half-maximal inhibition concentration (IC(50)) 24 hr after exposure to 1 mm CdCl(2). The data suggest that Cd deterioration of cell integrity is stronger when applied to the apical relative to the basolateral solution (IC(50) = 173.9 and 147.8 muM, respectively). Also, the data demonstrate that addition of Cd to the basolateral solution results in a prompt and transient stimulation of the active ion transport from 2.6 +/- 0.4 to 8.7 +/- 1.1 muA/cm(2). Use of the sodium channel blocker amiloride indicate that Na transport is not involved in Cd-stimulated SCC. Substitution of Cl with SO(4)(2-) in the basolateral solution and use of the Cl channel inhibitors, diphenylamine-2-carboxylase (DPC) and niflumic acid indicate strongly that Cd increases Cl secretion in A6 epithelium. Thapsigargin (TG), an intracellular Ca-ATPase blocker, inhibits Cd-stimulated active ion transport indicating that Ca-activated Cl channels are probably involved. Therefore, we suggest that Cd by interaction with the basolateral membrane, become internalized and increase Ca intracellularly. In a dose- and time-dependent way an increase in Ca activates specific Cl channels leading to an increased SCC. Thereafter, the increase in Ca leads to disruption of tight junctions thereby decreasing TER. This may lead to deterioration of cell integrity and perhaps even cell death.

Mechanism of calcium ionophore stimulated Cl secretion from frog skin glands

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. From the present data it is suggested that the calcium ionophore stimulates the Cl and water secretion from frog skin gland not by a direct effect of Ca2+-ions per se, but in an indirect manner by stimulating the prostaglandin synthesis, which probably results in an increase in the cAMP level in the gland cells.

Effect of linear alkylbenzene sulfonate (LAS) on ion transport and intracellular calcium in kidney distal epithelial cells (A6)

Linear alkylbenzene sulfonate (LAS) is found in near-shore environments receiving wastewater from urban treatment plants in a concentration reported to have physiological and toxic effect on aquatic organisms. The aim of this study was to investigate the effect LAS on ion transport and homeostasis in epithelia cells. A6 cells form a polarised epithelium when grown on permeable supports, actively absorb sodium and secrete chloride. Only the addition of LAS (100 microM) to the apical solution of A6 epithelia resulted in an increase in the active ion transport measured as short circuit current (SCC) and transepithelial conductance (G(t)). This increase could not be affected by the sodium channel inhibitor amiloride (100 microM), indicating that LAS stimulated the chloride secretion. Change in the intracellular calcium concentration (Ca(2+))(i) was measured in fura-2 loaded A6 cells, since it known that increase in (Ca(2+))(i) stimulate chloride secretion. LAS induced a concentration-dependent increase in (Ca(2+))(i) from 5 to 200 microM, where the half-maximal stimulating concentration on 100 mM resulted in an increase in (Ca(2+))(i) from 108+/-15 to 570+/-26 nM (n=4; P<0.01). The increase in (Ca(2+))(i) could be blocked by the calcium chelator ethylenebis(5-oxyethylenenitrilo)tetraacetic acid (EGTA), showing that the effect of LAS was due to influx of extracellular calcium. Furthermore, it was shown that the calcium channel inhibitor verapamil (0.2 mM) abolished the LAS induced increase in (Ca(2+))(i) and Gt when applied to the apical solution. However, verapamil has no inhibitory effect on these parameters when the non-ionic detergent Triton X-100 (100 microM) was added to A6 cells. These results indicate that LAS induced a specific activation of calcium channels in the apical membrane of A6 epithelia, leading to increase in (Ca(2+))(i) and thereby increased chloride secretion as a result of stimulation of calcium-dependent chloride channels in the apical membrane. The change in ion homeostasis is thought to be the fundamental reason to the physiological and toxic effects induced by LAS in marine organism.

Effect of Cisplatin on transepithelial resistance and ion transport in the a6 renal epithelial cell line.

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 mum) 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 mum 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/TER(0) (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 mum 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.

Electrophysiological measurements of a toad renal epithelial cell line (A6) as an assay to evaluate cellular toxicity in vitro

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.

Stimulation of active transepithelial sodium transport in isolated frog skin by hydrogen peroxide

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.