Tetrachloroaurate (III)-induced oxidation increases non-thermal plasma-induced oxidative stress

Abstract Non-thermal plasma (NTP) devices have been explored for medical applications. NTP devices discharge electrons, positive ions, ultraviolet (UV), reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as the hydroxyl radical (•OH), singlet oxygen (1O2), superoxide (O2 •-), hydrogen peroxide (H2O2), ozone, and nitric oxide, at near-physiological temperature. At preclinical stages or in human clinical trials, NTP promotes blood coagulation, eradication of bacterial, viral, and biofilm-related infections, wound healing, and cancer cell death. Here, we observed that ferric, vanadium, and gold(III) ions significantly elevated lipid peroxidation, which was measured by 2-thiobarbituric acid-reactive substances (TBARS) in combination with NTP exposure. Using 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) as a spin probe in electron paramagnetic resonance (EPR), we observed that tetrachloroaurate (III) yielded an M4PO-X spin adduct. Tetrachloroaurate-induced oxidation was attenuated efficiently by reduced (GSH) and oxidized glutathione (GSSG), while glycine (Gly), and L-glutamate (Glu), components of GSH, were ineffective. Furthermore, GSH and GSSG efficiently suppressed tetrachloroaurate-induced lipid peroxidation, while Gly and Glu were ineffective in suppressing TBARS elevation. These results indicate that tetrachloroaurate-induced oxidation is attenuated by GSH as well as GSSG. Further studies are warranted to elucidate the redox reactions between metal ions and biomolecules to advance the clinical application of NTP.


Introduction
The medical application of non-thermal plasma (NTP) originated in the late 1970s and has been improving since the mid-1990s [1]. NTP is composed of electrons, various ions, ultraviolet (UV), and reactive oxygen/nitrogen species (ROS/RNS), such as ozone (O 3 ), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), superoxide (O 2 -), the hydroxyl radical ( OH), and nitric oxide (NO). These molecules initiate blood coagulation, eradication of bacterial, viral, and biofilm-related infections, promotion of inorganic surface optimization in biomaterial, wound healing, and cancer cell death [2][3][4]. Recently, Conformit e Europ eenne (CE)-certified NTP devices have been available for disinfection (bacteria/fungi/viruses), atopic eczema, and wound healing. Furthermore, clinical studies on advanced head and neck squamous cell carcinomas and malignant melanoma have been conducted [5]. However, the biological outcomes of NTP may be affected by the generated levels of ROS/RNS, which depend on the design and operation of NTP sources. Thus, there is a need to optimize conditions to achieve the ideal biological effects.
To overcome the limitation of direct NTP exposure, NTP-activated medium (PAM) [6,7] or NTP-activated Ringer's lactate (PAL) [8][9][10][11][12] have been explored for eradicating cancer cells, and results demonstrated that PAM and PAL effectively kill a wide range of tumors. However, the biological role of ROS/RNS in selective killing is yet to be elucidated. Previously, we have demonstrated that NTP simultaneously generates H 2 O 2 , 1 O 2 , O 2 À , OH, and UV modifications with electron paramagnetic resonance (EPR) or specific fluorescent or luminescent probes [13][14][15][16]. Using this device, selective killing was demonstrated in oral squamous cell carcinoma [17], glioblastoma [3], ovarian adenocarcinoma [18], and malignant mesothelioma [19,20]. Indeed, iron, manganese, copper, lead, cobalt, mercury, beryllium, nickel, cadmium, molybdenum, tin, titanium, vanadium, chromium, aluminum, selenium, and arsenic have been shown to increase oxidative injury [21]. However, the intensity at which NTP-induced oxidative injury is amplified remains to be elucidated. In this study, we screened soluble element ions, which could enhance lipid peroxidation determined using a 2-thiobarbituric acid-reactive substances (TBARS) assay. Using this screening, we determined that ferric, vanadium, and gold (III) ions elevated TBARS, and EPR showed that tetrachloroaurate (HAuCl 4 ) yielded an oxidized spin adduct. These results suggest the utility of metal ions in medical NTP application for clinical settings.

Experimental setting of NTP
The photo and scheme of NTP device are shown in Figure 1. Within the visible plasma plume from the plasma head, high O atom (10 13 cm À3 ) due to dominating electron collision-induced dissociation of oxygen molecules, high electron density (10 15 cm À3 ), and larger amount of OH than NO are detected [1,22]. The NTP device, classified as plasma jet-type, was applied with a sinusoidal alternative current of 9 kV, transformed from a 100-V to 60-Hz commercial power line [13][14][15][16]. The distance from the bottom of the plasma head round window to the top surface of a 96-well plate was fixed as 10 mm. The flow rate of argon gas was set at 2 standard L/min.

Detection of OH with EPR spin trapping
About 100 mM M4PO or 2 mM DMPO in 50 mM phosphate buffer (pH 7.4) was directly exposed to NTP for 2 min in the presence of tetrachloroaurate (1-100 mM) or a vehicle in 96-well plates (375 mL; Thermo Fisher Scientific, Waltham, MA). When evaluating the antioxidative compounds, Chelex-treated water (vehicle) or an antioxidant at the indicated concentration was irradiated simultaneously in the same well. Each sample was immediately transferred to a disposable flat quartz cell (Radical Research, Tokyo, Japan), as previously described [13][14][15][16]. The EPR settings were as follows: microwave power, 4 mW; frequency, 9. . When the signal was over-ranged, the amplitude was decreased to optimize the setting.
The effect of metal ions on lipid peroxidation using TBARS PC was dispersed at 2.55 mM in phosphate buffer (50 mM, pH 7.4) in Chelex-treated milli-Q water, as previously described [16]. The PC suspension was prepared with extensive vortexing. The samples were then divided into three groups (untreated, argon gas 2 min, and NTP 2 min) and transferred into 96-well clear-bottom plates containing designated metal ions (100 mM) or an appropriate vehicle. To assess the antioxidative activity of each compound against tetrachloroaurate-induced oxidation, the compounds were added to the PC suspensions and transferred into 96-well plates containing a vehicle or tetrachloroaurate (10, 50, or 100 mM). TBARS were determined according to the manufacturer's instructions. In brief, samples were incubated with a chromogenic reagent consisting of 2-thiobarbituric acid (TBA) and acid reagent for 20 min at room temperature. The fluorometric assay was subsequently performed using Powerscan4 or Cytation5 (Biotek, Winooski, VT), with malondialdehyde (MDA) as a standard.

Statistical analysis
Statistical analyses were performed using a paired t-test in GraphPad Prism version 7 (Graphpad Software, La Jolla, CA). Differences were considered significant when p<.05. The data were expressed as the mean ± SEM (n ¼ 3-6) unless otherwise specified.

Results
Determination of PC-derived TBARS after NTP exposure for 2 min in the presence of ions In the screening system, we observed that NTP irradiation significantly elevated TBARS in several ions and identified ferric, vanadium, and gold (III) ions as candidates that amplify NTP-induced oxidative stress ( Figure 2(A)). In the first step of screening, many samples were irradiated simultaneously that causes autooxidation and may mask the significant NTP-induced lipid peroxidations in the presence of ferric, vanadium, and gold (III) ions ( Figure 2(A)). In line with this autooxidation, the tendency of TBARS to increase in Ar gastreated samples than in untreated samples may be caused by spraying Ar gas in the ambient air. To clarify the overload of NTP-induced ROS in metal-mediated lipid peroxidation, a limited number of experimental samples were measured simultaneously. In these experiments, NTP significantly elevated TBARS in ferric ( Figure 2(B)), vanadium ( Figure 2(C)), and gold (III) ions  ( Figure 2(D)) relative to Ar gas treatment. Notably, the metal ions elevated TBARS in a dose-dependent manner ( Figure 2(B-D)), indicating that metal-mediated auto-oxidation is an important factor in the present experimental setting.
Tetrachloroaurate (HAuCl 4 ) yields an oxidized spin adduct (M4PO-X) Exposure to NTP for 2 min without tetrachloroaurate generated M4PO-OH and M4PO-H spin adducts ( Figure  3(A)), as previously observed [14,16]. With an increase in tetrachloroaurate, NTP-induced M4PO-OH decreased (Figure 3(A)). This tendency was also observed in DMPO-H and DMPO-OH in the presence of tetrachloroaurate (Supplementary Figure 1). On the other hand, a triplet signal was observed from 5 mM tetrachloroaurate in the Ar gas treatment (Figure 2(B)). M4PO-X production was elevated in accordance with the concentration of tetrachloroaurate in the NTP and Ar gas treatments ( Figure 3(B)). The formation of M4PO-X was significantly higher in Ar gas than in NTP, indicating that NTP decayed M4PO-X as observed in other spin probes [15]. DMPO-X was not detected (Supplementary Figure 1).

GSH and GSSG significantly attenuate tetrachloroaurate (HAuCl 4 )-induced lipid peroxidation
NTP yielded red insoluble particles on the surface of PC suspension in the well with 100 mM tetrachloroaurate, while Ar gas did not generate any visible particle (data not shown). In the Ar gas and NTP treatments, 100 and 250 mM GSH significantly suppressed elevation of TBARS and formation of insoluble particle in the presence of 100 mM tetrachloroaurate ( Figure 5(A,B)). GSH (25,50, 100, and 250 mM) significantly suppressed lipid peroxidation in the presence of 10 and 50 mM tetrachloroaurate, while there was no significant suppression of lipid peroxidation without tetrachloroaurate ( Figure 5(A,B)). In the Ar gas and NTP treatments, Gly was ineffective in suppressing lipid peroxidation in the presence of 10, 50, and 100 mM tetrachloroaurate and in its absence ( Figure 5(C,D)). Similar observations were made with Glu under the same conditions ( Figure  5(E,F)). In the Ar gas treatment, 100 and 250 mM GSSG significantly suppressed lipid peroxidation in the presence of 10, 50, and 100 mM tetrachloroaurate ( Figure  5(G)), whereas in the NTP treatment, 250 mM GSSG significantly suppressed lipid peroxidation under similar conditions. Furthermore, 100 mM GSSG significantly suppressed lipid peroxidation in the presence of 10 mM tetrachloroaurate ( Figure 5(H)). The 100 mM tetrachloroaurate with NTP irradiation yielded red insoluble particles was attenuated by 250 mM GSSG, while 10 mM tetrachloroaurate did not yield red insoluble particles (data not shown), suggesting that NTP-induced lipid peroxides formed complexes with Au(III). There was no significant suppression of lipid peroxidation without tetrachloroaurate in all of the examined compounds in this study ( Figure 5), consistent with the EPR results (Supplementary Figure 2) and previous reports [14,15].

Discussion
Here, we screened for metal ions that amplify NTPinduced lipid peroxidation (Figure 2(A)). As a result, we observed that ferric and vanadium ions induced higher lipid peroxidation than tetrachloroaurate ( Figure  2(B-D)). Ferric ions cause oxidative injury to cells that ultimately causes carcinogenesis in mammals [23]. Contrastingly, in plasma medicine, iron is used for the therapeutic purpose of killing cancer cells [9,17,19,20,24]. Vanadium exists in several oxidation states from II to V, among which V(IV) is a major oxidation state in crude oil and clay rock [25]. V solution, which was employed in this study, contains several oxidation states, which are not specified by the supplier; thus, V(II), V(III), or V(IV) may cause oxidative stress synergistically. Regarding therapeutic application, the insulin-like effects that maintain signal transduction for glucose uptake and enhance lipid metabolism have been investigated extensively [25]. In addition, vanadium is known to catalyze the generation of super- [21,26,27]. Further studies on other vanadium salts are needed to explore the utility of vanadium with NTP application. Other transition elements have been reported to catalyze the "Fenton reaction" to generate OH from H 2 O 2 ; however, these did not dramatically increase lipid peroxidation in our screening.   Gold exists in several oxidation states from I to V; among these, only Au(I/III) have been used for medication to kill the bacteria since ancient times and their use can be back more than 2000 years [28,29]. In general, Au(III) complexes exhibit lower stability than Au(I) complexes due to high hydrolysis rates and significant reduction potentials [28]. Au(III) shares similar chemical properties and preference for square-planar coordination geometries with Pt(II) and cisplatin [(H 3 N) 2 PtCl 2 ] that raise the possibility for chemotherapeutic compound. Conversely, the binding of Au(III) complexes to DNA is reversed easily and affected by gold-bound ligands with lower ability than Pt(II); thus, Au(I/III) complexes-induced inhibition of DNA synthesis is triggered by interaction with the sulfhydryl groups of DNA polymerases and modification with the DNA topological state that is vital for DNA topoisomerase I and II [30]. Indeed, 1 mM tetrachloroaurate decays supercoiled form of plasmid (manuscript in preparation). Further study may reveal the association between tetrachloroaurate and NTP-induced DNA damage.
Following the longstanding use of gold drugs for the treatment of rheumatoid arthritis (RA), the epidemiological analyses in RA patients revealed that recipients of gold drugs develop lower malignancy rate, suggesting the promising role of gold drugs in cancer therapy [30]. Here, in addition to TBARS assay, tetrachloroaurate-induced oxidation yielded M4PO-X, which is characterized by a triplet signal [31]. This M4PO-X spin adduct reached a plateau at pH 4.0-6.0 and decreased to 40% at pH 7.0, finally becoming undetectable at pH 9.0. DMPO-X, which reacted 7-fold faster than M4PO-X, was also yielded by tetrachloroaurate at pH 4.0, while relative intensity of the EPR signal for DMPO-X was 30-40% lower than that for M4PO-X at pH 4.0-7.0 [31]. Here, we did not detect DMPO-X with 100 mM tetrachloroaurate after Ar gas treatment for 2 min at pH 7.4 (Supplementary Figure 1). Meanwhile, the characteristic signals of M4PO-X and DMPO-X spin adducts were observed following reaction with chlorine dioxide (ClO 2 ) at pH 2.0 and that of Ti 3þ with potassium chlorate (KClO 3 ) [32], suggesting the tetrachloroaurateinduced oxidation may be similar with ClO 2 . Here, tetrachloroaurate-induced M4PO-X was generated in a dose-dependent manner, decaying after NTP exposure (Figure 3(B)). On the other hand, in the presence of amino acids and dipeptide, such as Gly (Figure 4(B)), Glu (Figure 4(C)), and L-alanine (Ala) with L-glutamine (Gln) (Supplementary Figure 3(A)), NTP irradiation did not significantly decay the M4PO-X spin adduct. These results suggest that amino acids may stabilize the M4PO-X spin adduct. While the rate constant for the reaction between Au(III) and Gly and Ala was less than 100 M À1 s À1 [33,34], Au(III) reacts with two GSH to form reduced Au(I) and GSSG or further oxidized form of GSH sulfonic acid (GSO 3 H) that may disrupt normal biological function by altering the secondary and tertiary structures of a protein [35,36]. On the other hand, gold nanoparticles generated OH under microwave irradiation [37] so did gold nanocages in photodynamic therapy [38], indicating that ionized gold status is critical for gold-catalyzed oxidation. Tetrachloroaurate significantly induced lipid peroxidation (Figures 2(A,D) and 5). The supplementation of GSH and GSSG significantly suppressed tetrachloroaurate-induced elevation of TBARS in a dose-dependent manner ( Figure 5(A,B,G,H)). In contrast, the TBARS were not suppressed by GSH or GSSG at up to 250 mM after NTP irradiation without tetrachloroaurate, suggesting that OH is not scavenged effectively by these compounds. GSSG significantly suppressed M4PO-X faster than GSH (Figure 4(A,D)), while GSSG did not suppress tetrachloroaurate-induced TBARS as well as GSH ( Figure  5(A,B,G,H)). Taken together, the reactivity of tetrachloroaurate is presumed to be the order of PC, GSH > GSSG > M4PO. Meanwhile, Gly and Glu, which are constituents of GSH, as well as Ala-Gln, did not significantly scavenged OH ( Supplementary Figures 2 and  3(B)). Furthermore, Gly, Glu, and Ala-Gln did not suppress NTP-induced and tetrachloroaurate-induced lipid peroxidation ( Figure 5(C-F) and Supplementary Figure  3(C,D)). These TBARS assay results are consistent with those of EPR (Figure 4), indicating that thiol and disulfide bonds attenuated tetrachloroaurate-induced oxidation. Recently, we observed that GSSG reacted with NTP-induced H 2 O 2 [15] that is consistent with the formation of S-oxidized GSH (GSOH, GSO 2 H and GSO 3 H) with NTP irradiation [39]. Further study may reveal the role of disulfide bonds in ROS scavenging.
Among the gold complexes as a therapeutic drug and its derivative, triethylphosphine gold (I) chloride, an analog of auranofin, induced electron-dense deposition within mitochondria, lipid peroxidation, and depletion of GSH and NADPH in rat primary hepatocytes [40]. Recently, Au(I/III) complexes inhibited thioredoxin reductases (TrxRs) with high potency and specificity through covalent interaction with the selenocysteinecontaining active site and potently reacted with Cys residues of GSH reductase (GR), GSH peroxidases (GPxs) [28,29], and L-histidine residues of ferritin heavy chain [33]. In line with this oxidative injury, administration of Au(III) meso-tetraarylporphyrin that is stable in physiological condition significantly prolonged the survival of orthotopic rat hepatocellular carcinoma xenograft model by inducing necrosis and apoptosis in cancer cells, but not in normal hepatocytes, indicating tolerable dosage of Au(III) complex is effective as a chemotherapeutic compound [41]. Furthermore, depending on the chelating structure, Au(III) complexes inhibited growth in invading microbes [42] and cellular proliferation in wide range of cancer cells [43]. These results indicate that Au(I/III) trigger metal-induced oxidative stress and simultaneously disrupt the antioxidative defense system (TrxRs, GR, and GPxs). Moreover, auranofin initiated ferroptosis, which is induced by GPx4 inactivation and suppressed by iron chelating, in MYCNamplified neuroblastoma [44], suggesting the presence of an interaction between iron and gold-induced oxidative stress.
In conclusion, we observed that NTP-induced ROS and tetrachloroaurate-induced oxidation synergistically increase lipid peroxidation, which is attenuated by GSH and GSSG. Thus, combining NTP with metal ions, based on an understanding of redox mechanisms, could expand the possibility of therapeutic applications.

Disclosure statement
No potential conflict of interest was disclosed.

Funding
This work was supported by JSPS KAKENHI 21K06968 to Y.O; and JSPS KAKENHI 17H04064, 19K05462 and 20H05502 to S.T.