Membrane Currents in Madin-Darby Bovine Kidney Cells are Enhanced by Exposure to Dioxin

The name ”dioxins” is used to indicate a great family of structurally and chemically related Polychlorinated-Dibenzo-P-Dioxins (PCDDs) and Polychlorinated-Dibenzofurans (PCDFs). They are environmental toxins of current interest [1] as they can bio-accumulate in food chain, due to their lipophilic nature. The most toxic of these compounds is the 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD). This chemical, commonly known as dioxin, can cause a wide range of tissueand species-specific toxic effects, as chloracne, liver damage, disruption of hormone signaling pathways, reproductive and developmental defects [2-4]. Indeed, dioxin can alter brain development, produce cognitive disability, motor dysfunction [5,6], and also induce important alterations in neurodevelopment of human newborns following gestational exposure [7]. Moreover, TCDD may provoke immunosuppression and leads to an increased susceptibility to infectious agents [4,8]. Although the molecular mechanisms for carcinogenicity by dioxin have not been clarified, in 1997 TCDD was classified as a cancer promoter by the International Agency for Research on Cancer [9].


Introduction
The name "dioxins" is used to indicate a great family of structurally and chemically related Polychlorinated-Dibenzo-P-Dioxins (PCDDs) and Polychlorinated-Dibenzofurans (PCDFs). They are environmental toxins of current interest [1] as they can bio-accumulate in food chain, due to their lipophilic nature. The most toxic of these compounds is the 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCDD). This chemical, commonly known as dioxin, can cause a wide range of tissue-and species-specific toxic effects, as chloracne, liver damage, disruption of hormone signaling pathways, reproductive and developmental defects [2][3][4]. Indeed, dioxin can alter brain development, produce cognitive disability, motor dysfunction [5,6], and also induce important alterations in neurodevelopment of human newborns following gestational exposure [7]. Moreover, TCDD may provoke immunosuppression and leads to an increased susceptibility to infectious agents [4,8]. Although the molecular mechanisms for carcinogenicity by dioxin have not been clarified, in 1997 TCDD was classified as a cancer promoter by the International Agency for Research on Cancer [9].
However, data from literature are sometimes contrasting. For example, the effect on apoptosis signaling can result, dependent on the cell contest, either inducing [19,20] and repressing [17].
Very few are the studies that report effects on the membrane electrical activity after TCDD exposure [26]. The membrane elec-trical activity is due to the influx/efflux of ions across ion channels and pumps. It regulates the homeosta-sis of the cell [27] and many fundamental processes like as autophagy [28] and apoptosis [29] that also result to be very sensitive to TCDD [4,[19][20][21][22][23][24][25].
In the present study we used MDBK, a cell line affected by TCDD as defined by our previous studies [4,[19][20][21][22][23][24][25]. By using a patchclamp technique in the whole cell configuration, we have tested the possibility that the effect of this chemical can alter the normal ionic membrane currents. The preliminary results presented herein show that our cell line undergoes to significant variation of the ionic membrane currents when cultured in the presence of TCDD.
Moreover, these variations are largely depending on the increase of the chloride component (Table 1).

Electrophysiological Recordings
Experiments were performed by using an inverted microscope (Nikon, Diaphot 300) mounted with a recording chamber (∼1 ml).

Results
We   Only at membrane values below 0mV the responses remain unchanged (p < 0.05) with all solutions used. The curves in the panel Figure 2, although roughly resembling the same shape of the control ones, always present larger density currents. Also, in this case, for positive membrane values the curve C loses its linearity. In addition, for values ranging from -30mV to +30mV, the curves present a more complex combination of the different responses. The results obtained by the solution C (Na + −free) in fact, significantly (p < 0.05) differ from the total and, similarly, those obtained by solutions B (K + −free), significantly (p < 0.05) differ from D (Na + K + −free).
The differences between the two groups (a) and (b) are stressed in Figure 3, where the comparison between control and treated data is shown for the same solution. As can be seen the differences are always statistically significant (p < 0.05).

Discussion
As shown in our results, the mostly evident effect of TCDD treatment (1pg/ml) on MDBK cells is to increase the total membrane density currents. Several other observations can be made mostly looking at Figure 4. In the control group for the values below 0mV the non-significant difference be-tween the results obtained with the different solutions groups, indicates that the produced current is due to Cl − ions. In fact, the Na + K + free solution (D) has only Cl − ions, and this indicates that other ions do not contribute.
This assumption is also supported by the fact that all curves invert polarity at 0mV which the reverse potential of our solutions couple for is Cl − . The linearity of results obtained with solution D (Figure 3) indicates that this contribution is probably due to an Ohmic (non-voltage-dependent) conductance. Essentially the membrane behaves like a resistor obeying to the Ohm's low and giving a current directly proportional to the voltage level. This holds both for control and treated group.
Another observation by considering the results of solution C (green line in Figures 2 & 3) is that a K + voltage dependent contribution is added for values greater than 0mV both in the control and treated group. In fact, only for the positive voltage steps, the results obtained with this solution significantly diverge from those obtained with solution D. However, the difference between the results obtained with the solutions C and D is significantly larger (p < 0.05) in the control with respect to the treated group, (panel (a) in Figure   4). This could suggest that, following the TCDD treatment, the Cl − component grows more than K + component, which could instead diminish. The curve B (K + −f ree) shows a significant (p < 0.05) difference in the treated group respect the control one ( Figure 2) and, only in the treated group, it significantly diverges (p < 0.05) from results obtained with solution D for values of membrane potential ranging -30mV to 30mV , see Figure 4b. This difference become not significant for values of membrane potential close to Na + equilibrium potential (+80mV for our solutions). The lack of significance while the membrane potential is approaching the Na + reverse potential, indicates that this component could be contributed by Na + ions. However, the extremely positive value of the reverse potential for Na + suggests that a current contributed by this ion should be an inward current but the comparison in Figure 4b shows that the contribution is of an outward current. This finding is not easy to explain. At present stage we can only hypothesize a possible mechanism. TCDD treatment could induce the appearance of a possible Na + current by a possible expression of newly formed Na + channels, not present in the control conditions. These ionic channels should be voltage dependent because this current appears only for values of the membrane potential greater than -30mV which is in the range where normally this kind of channels become active. Moreover, as we have collected data from traces only after 20ms from the stimulus onset, if Na + channels are induced, they should be of the non-inactivating type.  The fact that, the current contributed by N a+ is of unexpectedly of the outward type, could be explained hypothesizing that the Na + current activates a pumping system that pumps out a quantity of current significantly larger than the inward current produced by the ionic channels. Pumps sensitive to local ionic changes of concentrations have been largely described [31] and complex transport systems have been also identified in a cell line similar to ours [32][33][34][35]. However, these results are preliminary and, of course, need of further confirmations to be supported.

Conclusion
By the previous results and discussions, we can summarize the following conclusions: c) The density of K+ current probably changes after TCDD exposure; d) TCDD probably activates a Na+ conductance which we have hypothesized to become an outward current thanks to activation of a (newly or pre-existent) pumping system which produces an efflux of ions larger than the influx one.