Mitigation of copper toxicity by DNA oligomers in green paramecia

Abstract

Impact of transition metals which catalyze the generation of reactive oxygen species (ROS), on activation of cell death signaling in plant cells have been documented to date. Similarly in green paramecia (Paramecium bursaria), an aquatic protozoan species harboring symbiotic green algae in the cytoplasm, toxicities of various metallic ions have been documented. We have recently examined the effects of double-stranded GC-rich DNA fragments with copper-binding nature and ROS removal catalytic activity as novel plant cell-protecting agents, using the suspension-cultured tobacco cells. Here, we show that above DNA oligomers protect the cells of green paramecia from copper-induced cell death, suggesting that the phenomenon firstly observed in tobacco cells is not limited only within higher plants but it could be universally observable in wider range of organisms.

Homeostasis in the levels of reactive oxygen species (ROS) and crosstalk among ROS and other signaling events are crucial to plant cell responses and adaptation to abiotic environmental factors threatening the plants.^1-3 One of key signaling events contributing to crosstalk in plant responses to abiotic stress would be transient increase in cytosolic free calcium ion concentration stimulated by generation of ROS through ROS-dependent activation of calcium channels on the vacuolar and/or plasma membranes.^1,4

Impact of various ions of transition metals, chiefly of copper, on oxidative and calcium signal transductions leading to cell death in plant cells have been documented to date. Among such metals, copper is known to be a phytotoxic metal which reportedly induces a series of biological and chemical reactions in plant cells including the burst of reactive oxygen species production and stimulation of calcium channel opening allowing a transient increase in cytosolic calcium concentrations.^5-8

According to earlier works, ion of copper (chiefly Cu²⁺) was shown to bind and damage the DNA,^9,10 through binding to the guanine (G) and cytosine (C) bases at physiological pH.¹¹ In addition, among 3 of known biologically active forms of DNA, Z-DNA showing a non-Watson-Crick type structure,¹² possesses the highest affinity to binding of Cu²⁺, especially at G bases.¹³ In our recent report,¹⁴ effects of the DNA fragments with Cu-binding motifs as novel plant cell-protecting agents were examined by using suspension cultured transgenic cells of tobacco (Nicotiana tabacum L., cell line BY-2) expressing aequorin, a calcium-responsive luminescent protein, in cytosolic space. Addition of double-stranded GC-rich DNA fragments (especially CGCGCG), prior to the addition of copper ions, effectively blocked both the copper-induced calcium influx and cell death.¹⁴ In addition, the DNA-Cu complex examined was shown to possess a catalytic activity for scavenging of superoxide anion radical, suggesting that DNA-mediated protection of the cells from copper toxicity is largely due to mitigation of oxidative stress.¹⁴

Apart from terrestrial plant models, aquatic protozoan cells including photosynthetic green protozoa such as Euglena gracilis and Paramecium bursaria (known as green paramecia) have been used as models for studying the impacts of polluting chemicals in aquatic ecosystem.^15,16 In case of green paramecia, oxidative stress was shown to be a driving force to develop a symbiotic relationship between Chlorella-like green algae,^17-19 or experimentally introduced cyanobacteria,²⁰ explaining the view that green paramecia survived the oxidative evolutional selection.^21,22

According to Aonuma et al.,²³ green paramecia are sensitive to various metal ions and show cytotoxicity at high dose and loss of galvanotactic swimming capability at low dose. As shown in Figure 1, P. bursaria cells showed sensitivity to Cu²⁺ and the median lethal concentration (LC₅₀) were shown to be around 10 μM.

Figure 1.

Copper-induced cell death in P. bursaria. (A) Control cells. (B) Typical morphological changes in dead cells (observed in the presence of 100 μM Cu²⁺). (C) Effect of Cu²⁺ concentration on induction of cell death in P. bursaria. Bars, SD (n = 4). P. bursaria cultured in a yeast extract-based nutrition mixture (1 EBIOS tablet/L; Asahi food & Healthcare, Tokyo) at the stationary phase was used. Viability tests were carried out on 12-well microplates. Each well on the plates was filled with 0.9 ml of nutrition mixture harboring 100 paramecium cells plus 0.1 ml of CuSO₄ solutions. Then the cells were incubated for 12 h at 23°C under continuous dark condition, and the number of living cells was counted at the end of incubation under a stereomicroscope (SMZ645; Nikon, Tokyo).

Here, we show that GC-rich DNA oligomers protect the cells of green paramecia from copper-induced cell death (Fig. 2). For mitigation of copper toxicity, effects of 5 different sequences of DNA hexamers were examined. Among DNA hexamers GC-rich double stranded sequence (CGCGCG) showed most strong action preventing the toxicity of copper, in a dose-dependent manner (Fig. 2). In contrast, ATATAT, a double-stranded AT-rich model sequence failed to rescue the cells of P. bursaria exposed to copper stress as similar results were already reported for plant cells.¹⁴ The data obtained here imply that the phenomenon of interest firstly observed in our previous work (with tobacco cells) should not be limited only within higher plants but it could be universally applicable in wider range of organisms covering protozoan cells.

Figure 2.

Effects of oligo-DNA sequences on mitigation of copper toxicity in P. bursaria. (A) Five different sequences of DNA hexamers examined. DNA sequences in gray-colored letters indicate the presence of complementary strands. (B) Comparison of the action of 5 different DNA oligomers on mitigation of copper toxicity. (C) Effect of GC-rich double stranded oligo-DNA concentration on mitigation of copper toxicity. Bars, SD (n = 4). Conditions for assays are identical as described for Fig. 1, except addition of DNA oligomers.

Ca²⁺ is one of the key regulatory elements for cellular function and ciliary movements in Paramecium species. At present, our knowledge on the involvement of calcium signaling in copper-induced cell death is largely limited. Although calcium signaling in green paramecium cells is out of our key interest in this work, we included a brief description of the calcium signaling mechanism in green paramecia.

Some Paramecium species possess calcium channels on the ciliary membrane which are voltage-dependently gated and involved in ciliary movements.²⁴ Therefore, most visible event with involvement of calcium signaling in Paramecium species is ciliary reversal which depends on the intracellular increase in Ca²⁺ concentration.²⁵ It has long been known that members of Paramecium species including P. bursaria exhibit galvanotaxis, a directed swimming of cells toward the electrode, induced in response to an applied voltage.^23,26 A pharmacological approach has revealed that T-type calcium channels are involved in galvanotactic movement of P. bursaria involving electrically forced ciliary reversal, as galvanotactic behavior was shown to be sensitive to a variety of Ca²⁺-related inhibitors such as Ca²⁺ chelators, inhibitors of intracellular Ca²⁺ release (such as ruthenium red and neomycin), lanthanide ions (general calcium channel blockers), and inhibitors of T-type calcium channels (such as NNC 55-0396, 1-octanol and Ni²⁺), while inhibitors of L-type calcium channels such as nimodipine, nifedipine, verapamil, diltiazem and Cd²⁺ all failed to block the galvanotactic movement of P. bursaria.²³

Funding

TK was supported by a grant of Regional Innovation Strategy Support Program implemented by Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.