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Persistent perfluorinated organic compounds such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are distributed widely in the global environment including wildlife and human. In this study, we investigated the genotoxicity of PFOS and PFOA using the novel in vivo comet assay developed for Paramecium caudatum. For the comet assay, large nuclei squeezed out of the paramecia with 0.25 M sucrose containing 0.6% Triton X-100 were embedded in a layer of agarose gel placed over the slide glass. N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) and 2-aminoanthracene (2-AA) were successfully used for positive controls. Productions of 8-hydroxydeoxyguanosine (8-OH-dG) and intracellular reactive oxygen species (ROS) were also measured in paramecia. PFOS did not cause DNA damage on any conditions examined. On the other hand, 12 and 24 hr exposure to PFOA (100 µM) increased DNA migration in electrophoresis condition at pH 13, but not at pH 12.1, suggesting that the DNA damage may be alkali labile site (such as apurinic/apyrimidinic (AP) site). Exposure of paramecia to 100 µM PFOA for 1, 3 and 24 hr and to 10 µM PFOA for 24 hr significantly increased intracellular ROS. Under the same condition, however, 8-OH-dG level was not affected by PFOA. The PFOA-induced DNA damage was not abolished by the application of 100 µM GSH which completely inhibited the increase of intracellular ROS. In conclusion, the PFOA-induced in vivo DNA damage was first shown in paramecia, and the DNA damage might not be directly attributable to increase in intracellular ROS.

Eeffects of perfluorooctane sulfonate (PFOS) on maleic dialdehyde (MDA) content, superoxide dismutase (SOD) activity and total antioxidation capability (T-AOC) were compared in mice at different postnatal developmental stages, and concentrations and distributions of PFOS in different tissues were measured simultaneously. The male and female mice at postnatal day (PD) 7, PD 14, PD 21, PD 28 and PD 35 were distributed randomly to dosage group (50 mg/kg body weight) and control group (0 mg/kg body weight). Mice were administered with PFOS by once subcutaneous injection. Subsequently, after 24 hr, MDA content, SOD activity and T-AOC in brain and liver were analyzed. The PFOS concentrations in blood, brain and liver were determined by high-performance liquid chromatography negative electrospray tandem mass spectrometry (LC-MS). PFOS induced degression of the body weights of mice evidently and increase of relative weights of liver. Meanwhile, it depressed the SOD activity and T-AOC in brain and liver. The concentrations and distribution percentages of PFOS in blood, brain and liver of mice were significantly different at various postnatal developmental stages. Achieved results in this study indicate that younger mice pups were more sensitive to PFOS exposure. In addition, significant distinctions in concentrations and distribution percentages of PFOS in various tissues were demonstrated in this study. The gender difference observed was greater in the older mice. Thus it is worth giving attention especially to adverse effects of PFOS on foetus and children.

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca2+ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K+ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca2+ channels, and the hyperpolarizing quinine receptor potential by an activation of K+ channels.

Effects of the external pH on the voltage-dependent Na conductance in the marine dinoflagellate Noctiluca miliaris were examined under voltage-clamp conditions. The depolarizing Na spike (positive spike) in the tentacle regulating potentials became attenuated when the externa! pH was lowered from 8 (normal value) to less than 5. Under voltage-clamp conditions, a depolarization-activated transient inward current corresponding to the positive spike decreased with lowering the external pH to less than 5, and entirely disappeared at pH 3. Threshold for the inward current shifted towards the depolarizing direction while the reversal potential of the current shifted towards the hyperpolarizing direction with lowering the external pH. Rate of increase in the inward current reduced whereas rate of decrease did not change at low pH. Lowering of the external pH did not affect the steady state inactivation of the inward current. It is concluded that the external pH controls appearance of the positive spike ...

Persistent perfluorinated organic compounds such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were distributed widely in the global. PFOS (15 microM or higher) caused backward swimming of paramecia. The Triton-extracted paramecia, where the membrane was disrupted and the externally applied chemicals are freely accessible to the ciliary apparatus, showed forward swimming up to 0.1 microM Ca2+ in the medium and backward swimming at about 0.2 microM and higher. PFOS (0.1 mM) did not change the relationship between the swimming directions and free Ca2+ concentrations. Effects of various surfactants including PFOS and PFOA on the swimming direction of paramecia were compared with the hemolysis of mouse erythrocytes as an indicator of surfactant activities. The hemolysis did not correlate with their swimming behavior. PFOS caused triphasic membrane potential changes both in the wild-type paramecia and caudatum non-reversal (CNR) mutants, the latter is defective in voltage-gated Ca2+ channels. An action potential of the wild-type specimen was induced at lower current intensity when PFOS was present in the medium. Voltage-clamp study indicated that PFOS had no effect on the depolarization-induced Ca2+ influx responsible for the action potential. The membrane potential responses obtained were similar to those obtained by the application of some bitter substances such as quinine that activate chemoreceptors of paramecia. Since the CNR specimens did not exhibit PFOS-induced backward swimming at concentrations examined, the backward swimming is attributable to the influx of Ca2+ into the cilia through voltage-gated Ca2+ channels. The Ca2+ channels are most probably activated by the depolarizing receptor potentials resulted from the PFOS-induced activation of chemoreceptors.

Noctiluca miliaris, a marine dinoflagellate, exhibits spontaneous flexion of its tentacle. The movement of the tentacle is always accompanied by perturbations of membrane potential, termed the ‘tentacle regulating potentials’ (TRPs) (Eckert and Sibaoka, 1967). Though the waveform of the TRPs is liable to variation, it consists of four successive basic components: (1) a Na+-dependent depolarizing (positive) spike, (2) a depolarizing plateau potential, (3) a Cl−-dependent hyperpolarizing (negative) spike, and (4) a long-lasting hyperpolarization (Eckert and Sibaoka, 1967; Oami et al. 1988). A slow flexion of the tentacle is seen during the plateau, and the flexion is suddenly accelerated when the negative spike occurs. The tentacle then extends during the long-lasting hyperpolarization. There is no correlation between the positive spike and the tentacular movement (Hisada, 1957; Oami et al. 1988).

In Paramecium , ciliary reversal is coupled with voltage-gated Ca 2+ channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC , with a malfunction of the Ca 2+ channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca 2+ -binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca 2+ channel activity of cnrC . A Pccentrin1p mutant gene lacking Ca 2+ sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca 2+ -dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca 2+ -dependently regulates the Ca 2+ channel activity in vivo .

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.

Voltage-gated Ca 2+ channels play a critical role in controlling Ca 2+ entry in various cells. Ciliary reversal in Paramecium depends on the Ca 2+ influx through voltage-gated Ca 2+ channels on the ciliary membrane. One of the voltage-gated Ca 2+ channel mutants in Paramecium caudatum , cnrC , neither produces Ca 2+ action potentials nor responds to any depolarizing stimuli. Here, we report that the cnrC + gene product is P. caudatum centrin (Pccentrin1p), a member of the Ca 2+ -binding EF-hand protein superfamily. The Pccentrin1p gene of cnrC was found to contain a single-base deletion, a mutation that caused the loss of the fourth EF-hand of Pccentrin1p. Moreover, the wild-type Ca 2+ channel function was impaired by Pccentrin1p gene silencing, leading to the loss of current-evoked Ca 2+ action potentials and stimulated ciliary reversal. These results demonstrate that Pccentrin1p is indispensable for the activity of the voltage-gated Ca 2+ channels that control ciliary reversal in Paramecium .

Membrane potential responses and tentacle movement of the marine dinoflagellate Noctiluca miliaris were recorded simultaneously and their time relationships were examined. The food-gathering tentacle of Noctiluca exhibited slow extension-flexion movements in association with the spontaneously recurring membrane potential responses termed the tentacle regulating potentials (TRPs). The flexion of the tentacle began during the slow depolarization of the TRPs. The rate of the flexion increased after the hyperpolarizing (negative) spike following the slow depolarization. The tentacle then extended slowly during the hyperpolarized level of the TRPs. A TRPs-associated flexion did not occur when the external Ca(2+) ions were removed. On the contrary, the tentacle showed conspicuous flexion (coiling) when the external Ca(2+) concentration was raised. In association with the stimulus-evoked action potential, which triggers bioluminescent flash (flash-triggering action potential; FTP), the tentacle coiled quickly. The FTP-associated coiling took place even in the Ca(2+)-deprived condition. The coupling mechanisms of the TRPs-associated and FTP-associated tentacle movements were compared, and their biological significance was discussed.

The membrane potential responses of Paramecium caudatum to the external application of bitter substances were examined by employing conventional electrophysiological techniques. Mutant cells defective in voltage-gated Ca2+ channels were used to record the potential responses in the absence of contamination by Ca2+ action potentials. The cells produced a transient depolarization followed by a transient hyperpolarization in response to a rapid whole-cell application of chloroquine, strychnine nitrate or brucine. Of these chemicals, chloroquine was the most potent. Cells produced a simple depolarization in response to a localized application of test chemicals to the anterior region, whereas they produced a transient hyperpolarization in response to an application to the posterior region. Membrane potential responses to an application of chloroquine declined with repeated application. The presence of chloroquine in the external bathing solution strongly inhibited the membrane potential responses to an application of brucine or strychnine. However, the presence of chloroquine did not affect the membrane potential responses to an application of quinine. It is suggested that chloroquine, strychnine and brucine share a common component of their transduction pathways, but that the transduction pathway for quinine is different.

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni2+-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca2+ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.

Effects of the external Ca2+ concentration on the depolarization-induced transient inward Na+ current responsible for the Na+ spike in the dinoflagellate Noctiluca miliaris were examined. The peak value and the duration of the Na+ current increased when lowering the external Ca2+ concentration. The threshold potential level for activation and the reversal potential level of the current were not affected by the external Ca2+ concentration. The inactivation took place even in a solution containing EGTA with very low (

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellate Noctiluca miliaris were examined under voltage-clamp conditions. Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of −80 mV to a potential level more positive than −50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na+ concentration was lowered. The transient inward current is assumed to be responsible for the Na+-dependent positive spike in non-clamped specimens of Noctiluca. Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of −20 mV to a potential level more negative than −30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl− concentration was lowered. The transient outward current is responsible for the Cl−-dependent negative spike in non-clamped specimens of Noctiluca.

The intracellular conditions required for contraction in the food-gathering tentacle of a marine dinoflagellate Noctiluca miliaris were examined in the isolated tentacle, treated with Triton X-100. The tentacle flexed to its full extent when the pH of the reactivation medium was lowered to 4·0, and extended when it was raised again to its original value. Adenosine triphosphate (ATP) was not needed for the pH-dependent flexion–extension. The flexion was also produced by Ca2+, but in a concentration range more than a thousand times higher than the effective H+ concentration range for producing the flexion. It is concluded that movement of the tentacle in a live specimen of Noctiluca is controlled by membrane potential-regulated cytoplasmic pH.

Membrane potential responses that regulate movement of the food-gathering tentacle ofNoctiluca miliaris (tentacle regulating potentials, TRPs) were examined electrophysiologically under various ionic conditions. These spontaneous TRPs were modified by changing the external ionic conditions. Positive spike appeared as external Ca2+ concentration was lowered. The peak of the spike became more positive with increasing external Na+ concentration. The spike could be evoked by injecting a depolarizing current when the membrane was hyperpolarized. The positive spike is assumed to be caused by regenerative activation of depolarization-sensitive Na channels. The peak of the negative spike, reported by previous workers, became more negative with increasing external Cl− concentration. The spike was evoked by injecting a hyperpolarizing current when the membrane was depolarized. The negative spike is assumed to be caused by regenerative activation of hyperpolarization-sensitive Cl− channels. The waveforms and amplitudes of the TRPs recorded from the nucleus were identical to those recorded from the flotation vacuole. This suggests that the TRPs are generated on the membrane facing the external solution. Possible roles of the TRPs in the control of tentacle movement are discussed.