Here, we determined that tetrachloroaurate oxidizes M4PO and liposome by screening for metal ions that amplify NTP-induced lipid peroxidation. As a result, we observed that ferric and vanadium ions induced higher lipid peroxidation than tetrachloroaurate (Fig. 1B–D). Ferric ions cause oxidative injury to cells that ultimately causes carcinogenesis in mammals26. Contrastingly, in plasma medicine, iron is used for the therapeutic purpose of killing cancer cells6,12,20,23,24. Vanadium exists in several oxidation states, including V(V), V(IV), V(III), and V(II), among which V(IV) is a major oxidation state in crude oil and clay rock27. Regarding therapeutic application, the insulin-like effects that maintain signal transduction for glucose uptake and enhance lipid metabolism have been investigated extensively27. In addition, vanadium is known to catalyze the generation of superoxide, [V(IV) + O2 ⇌ O2●− + V(V)] and the hydroxyl radical from H2O2 [V(IV) + H2O2 ⇢ V(V) + OH− + ●OH]25,28,29. 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 H2O2; however, these did not dramatically increase lipid peroxidation in our screening.
Tetrachloroaurate-induced oxidation yielded M4PO-X, which is characterized by a triplet signal30. 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 4.0–7.030. Here, we did not detect DMPO-X with 100 µM tetrachloroaurate after Ar gas treatment for 2 min at pH 7.4 (Supplementary Fig. 1). Meanwhile, the characteristic signals of M4PO-X and DMPO-X spin adducts were observed following reaction with chlorine dioxide (ClO2●) at pH 2.0 and that of Ti3+ with potassium chlorate (KClO3)31. These results suggest that tetrachloroaurate-induced oxidation may be associated with ClO2●, especially under acidic conditions. Here, tetrachloroaurate-induced M4PO-X was generated in a dose-dependent manner, decaying after NTP exposure (Fig. 2B). On the other hand, in the presence of amino acids and dipeptide, such as Gly (Fig. 3B), Glu (Fig. 3C), and L-alanine (Ala) with L-glutamine (Gln) (Supplementary Fig. 3A), NTP irradiation did not significantly decay the M4PO-X spin adduct. These results suggest that amino acids may stabilize the M4PO-X spin adduct. The rate constant for the reaction between Au(III) and Gly and Ala was less than 100 M− 1s− 1 32,33. Furthermore, Au(III) reacts with two GSH to form reduced Au(I) and GSSG or GSH sulfonic acid that may disrupt normal biological function by altering the secondary and tertiary structures of a protein34,35. On the other hand, gold nanoparticles generated ●OH under microwave irradiation36 so did gold nanocages in photodynamic therapy37, indicating that ionized gold status is critical for gold-catalyzed oxidation.
Tetrachloroaurate significantly induced lipid peroxidation (Fig. 4). The NTP irradiation overloaded ROS in tetrachloroaurate-induced oxidation (Fig. 4B, 4D, 4F, 4H). The supplementation of GSH and GSSG significantly suppressed tetrachloroaurate-induced elevation of TBARS in a dose-dependent manner (Fig. 4A, 4B, 4G, 4H). In contrast, the TBARS were not suppressed by GSH or GSSG at up to 250 µM after NTP irradiation without tetrachloroaurate, suggesting that ●OH is not scavenged effectively by these compounds. 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 suppress NTP-induced lipid peroxidation (Fig. 4C–F and Supplementary Fig. 3C, D). Furthermore, Gly, Glu, and Ala-Gln were not significantly scavenged ●OH (Supplementary Fig. 2 and Supplementary Fig. 3B). These TBARS assay results are consistent with those of EPR (Fig. 3), indicating that thiol and disulfide bonds attenuated tetrachloroaurate-induced oxidation. Recently, we observed that GSSG reacted with NTP-induced H2O218. Further study may reveal the role of disulfide bonds in ROS scavenging.
Gold exists in several oxidation states from I to V; among these, only Au(I/III) have been used for medication38. 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 hepatocytes39. Recently, Au(I/III) complexes inhibited thioredoxin reductases (TrxRs) with high potency and specificity through covalent interaction with the selenocysteine-containing active site and potently reacted with Cys residues of GSH reductase (GR), GSH peroxidases (GPxs)38,40, and L-histidine residues of ferritin heavy chain32. Furthermore, depending on the chelating structure, Au(III) complexes inhibited growth in invading microbes41 and cellular proliferation in cancer cells42. 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 MYCN-amplified neuroblastoma43, 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.