The Metabolic Fate of ortho-Quinones Derived from Catecholamine Metabolites

ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase or by transition metal ions. Neuromelanin, a dark pigment present in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid (DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible spectrophotometry. HPLC analysis after reduction with NaBH₄ or ascorbic acid enabled measurement of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric examination showed that the ortho-quinones initially formed underwent extensive degradation at pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from DOPE-quinone was DOPEG which was produced through the addition of a water molecule to the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc) and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min. This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are converted to quinone methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The unstable quinone methides afford stable alcoholic or carbonyl products.     

The Aldo-Keto Reductases and Polycyclic Aromatic Hydrocarbon Activation

The aldo-keto reductases (AKRs), are monomeric 37 kDa oxidoreductases that catalyze the NADP + -dependent oxidation of PAH trans -dihydrodiol proximate carcinogens to their reactive and redox-active o -quinones. Five human isoforms have been cloned and expressed as purified recombinant enzymes (AKR1C1-AKR1C4 and AKR1A1). Of these the general metabolic enzyme aldehyde reductase (AKR1A1) displays the highest utilization ratio for the oxidation of ( m ) R,R -benzo[ a ]pyrene-7,8-diol (BP-7,8-diol) to benzo[ a ]pyrene-7,8-dione (BP-7,8-dione). AKR1A1 is coexpressed with CYP1A1 and epoxide hydrolase suggesting that it is exposed to its trans -dihydrodiol substrates in situ. PAH o -quinones produced by AKRs are highly electrophilic yielding bimolecular rate constants for the addition of GSH of at least 1.3 2 10 3 M m 1 min m 1 . By reacting with DNA, PAH o -quinones form both stable and depurinating adducts in vitro. By entering into futile redox-cycles the PAH o -quinones can amplify the production of reactive oxygen species and increase 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dGuo) formation. The spectrum of DNA-adducts and generation of prooxidants (reactive oxygen) may explain how PAHs can act as complete carcinogens.     

Front Cover: Similar but Still Different: Which Amino Acid Residues Are Responsible for Varying Activities in Type-III Copper Enzymes? (7/2021)

Type-III copper enzymes like polyphenol oxidases (PPOs) are ubiquitous among organisms and play a significant role in the formation of pigments. PPOs comprise different enzyme groups, including tyrosinases (TYRs) and catechol oxidases (COs). TYRs catalyze the o-hydroxylation of monophenols and the oxidation of o-diphenols to the corresponding o-quinones (EC 1.14.18.1). In contrast, COs only catalyze the oxidation of o-diphenols to the corresponding o-quinones (EC 1.10.3.1). To date (August 2020), 102 PDB entries encompassing 18 different proteins from 16 organisms and several mutants have been reported, identifying key residues for tyrosinase activity. The structural similarity between TYRs and COs, especially within and around the active center, complicates the elucidation of their modes of action on a structural basis. However, mutagenesis studies illuminate residues that influence the two activities and show that crystallography on its own cannot elucidate the enzymatic activity mode. Several amino acid residues around the dicopper active center have been proposed to play an essential role in the two different activities. Herein, we critically review the role of all residues identified so far that putatively affect the two activities of PPOs.     

(−)‐Tarchonanthuslactone: Design of New Analogues,Evaluation of their Antiproliferative Activity on Cancer Cell Lines,and Preliminary Mechanistic Studies

Natural products containing the α,β‐unsaturated δ‐lactone skeleton have been shown to possess a variety of biological activities. The natural product (?)‐tarchonanthuslactone ( 1 ) possessing this privileged scaffold is a popular synthetic target, but its biological activity remains underexplored. Herein, the total syntheses of dihydropyran‐2‐ones modeled on the structure of 1 were undertaken. These compounds were obtained in overall yields of 17–21 % based on the Keck asymmetric allylation reaction and were evaluated in vitro against eight different cultured human tumor cell lines. We further conducted initial investigation into the mechanism of action of selected analogues. Dihydropyran‐2‐one 8 [(S,E)‐(6‐oxo‐3,6‐dihydro‐2H‐pyran‐2‐yl)methyl 3‐(3,4‐dihydroxyphenyl)acrylate], a simplified analogue of (?)‐tarchonanthuslactone ( 1 ) bearing an additional electrophilic site and a catechol system, was the most cytotoxic and selective compound against six of the eight cancer cell lines analyzed, including the pancreatic cancer cell line. Preliminary studies on the mechanism of action of compound 8 on pancreatic cancer demonstrated that apoptotic cell death takes place mediated by an increase in the level of reactive oxygen species. It appears as though compound 8 , possessing two Michael acceptors and a catechol system, may be a promising scaffold for the selective killing of cancer cells, and thus, it deserves further investigation to determine its potential for cancer therapy.     

Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents

Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy.     

Screening of Human Gut Bacterial Culture Collection Identifies Species That Biotransform Quercetin into Metabolites with Anticancer Properties

We previously demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK-dependent and -independent mechanisms. The existing evidence suggests that gut microbiota is capable of flavonoid biotransformation to generate bioactive metabolites including 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3,4,5-trihyroxybenzoic acid (3,4,5-THBA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In this study, we screened 94 human gut bacterial species for their ability to biotransform flavonoid quercetin into different metabolites. We demonstrated that five of these species were able to degrade quercetin including Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthii, Olsenella scatoligenes and Eubacterium eligens. Additional studies showed that B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generates DOPAC. In addition to the differences in the metabolites produced, we also observed that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the antiproliferative effects of 2,4,6-THBA and 3,4-DHBA demonstrated previously, DOPAC also inhibited colony formation ex vivo in the HCT-116 colon cancer cell line. Consistent with this, the bacterial culture supernatant of F. plautii also inhibited colony formation in this cell line. Thus, as F. plautii and B. glycinifermentans generate metabolites possessing antiproliferative activity, we suggest that these strains have the potential to be developed into probiotics to improve human gut health.     

A Metabolic Activation Mechanism of 7 H -Dibenzo[ c,g ]carbazole Via o -Quinone. Part 1: Synthesis of 7 H -Dibenzo[ c,g ]carbazole-3,4-dione and Reactions with Nucleophiles

7 H -Dibenzo[ c,g ]carbazole (DBC) is a potent carcinogen present in the environment. The metabolic activation pathway of DBC has not been completely understood. The formation of the DBC- o -quinone was proposed and the novel DBC-3,4-dione was chemically synthesized from 4-OH- or 3-OH-DBC, using the Fremy's salt at room temperature. The DBC-3,4-dione was found to react readily with model nucleophile, 2-mercaptoethanol, to afford 1-(2'-hydroxyethylthio)-DBC-3,4-dione through 1,4-Michael addition. An adduct of DBC-3,4-dione with adenine was obtained; adducts formed with other DNA bases and nucleosides were partially characterized and are under further analysis. Under the same conditions, DBC-3,4-dione with guanine did not yield an adduct. These results are encouraging for further studies on DNA binding of DBC through 3,4-dione in vitro and in vivo.     

Nitroxide-Derived N-Oxide Phenazines for Noncovalent Spin-Labeling of DNA

Two o-benzoquinone derivatives of isoindoline were synthesized for use as building blocks to incorporate isoindoline nitroxides into different compounds and materials. These o-quinones were condensed with a number of o-phenylenediamines to form isoindoline-phenazines in high yields. Subsequent oxidation gave phenazine-di-N-oxide isoindoline nitroxides that were evaluated for noncovalent and site-directed spin-labeling of duplex DNA and RNA that contained abasic sites. Although only minor binding was observed for RNA, the unsubstituted phenazine-N,N-dioxide tetramethyl isoindoline nitroxide showed high binding affinity and selectivity towards abasic sites in duplex DNA that contained cytosine as the orphan base.     

GeromiRs Are Downregulated in the Tumor Microenvironment during Colon Cancer Colonization of the Liver in a Murine Metastasis Model

Cancer is a phenomenon broadly related to ageing in various ways such as cell cycle deregulation, metabolic defects or telomerases dysfunction as principal processes. Although the tumor cell is the main actor in cancer progression, it is not the only element of the disease. Cells and the matrix surrounding the tumor, called the tumor microenvironment (TME), play key roles in cancer progression. Phenotypic changes of the TME are indispensable for disease progression and a few of these transformations are produced by epigenetic changes including miRNA dysregulation. In this study, we found that a specific group of miRNAs in the liver TME produced by colon cancer called geromiRs, which are miRNAs related to the ageing process, are significantly downregulated. The three principal cell types involved in the liver TME, namely, liver sinusoidal endothelial cells, hepatic stellate (Ito) cells and Kupffer cells, were isolated from a murine hepatic metastasis model, and the miRNA and gene expression profiles were studied. From the 115 geromiRs and their associated hallmarks of aging, which we compiled from the literature, 75 were represented in the used microarrays, 26 out of them were downregulated in the TME cells during colon cancer colonization of the liver, and none of them were upregulated. The histone modification hallmark of the downregulated geromiRs is significantly enriched with the geromiRs miR-15a, miR-16, miR-26a, miR-29a, miR-29b and miR-29c. We built a network of all of the geromiRs downregulated in the TME cells and their gene targets from the MirTarBase database, and we analyzed the expression of these geromiR gene targets in the TME. We found that Cercam and Spsb4, identified as prognostic markers in a few cancer types, are associated with downregulated geromiRs and are upregulated in the TME cells.     

Mass Spectral Analysis of Unstable N7-Aralkyl DNA Adducts Resulting from Reaction of 7-Sulfooxymethyl-12-methylbenz[ a ]anthracene (SMBA) with DNA and Deoxynucleotides

The unified hypothesis for PAH activation predicts that SMBA plays a role in the metabolic activation and carcinogenicity of 7,12-dimethylbenz[ a ]anthracene (DMBA). SMBA and closely related aralkylating agents are derived from 7-hydroxymethyl-12-methylbenz[ a ]anthracene (HMBA), a direct metabolite of DMBA, and react with bases in nucleic acids. This occurs by generation of a benzylic carbonium ion owing to the fact that sulfate is a good leaving group. Previous characterization of reaction products with deoxynucleotide-3'-monophosphates detected stable adducts as primarily resulting from reaction with adenine at the N6- and guanine at the N2-amino groups, respectively. Pyrimidine adducts were also found; however, examination of SMBA-reacted DNA confirmed that the purine bases were the major targets for reaction. We now report evidence for the formation of unstable DNA adducts, most likely by reaction with purine N-7 positions. Treatment of SMBA-reacted DNA with formic acid at 70°C for 60 min and examination of acidified reaction products with electrospray-positive mode mass spectrometry (ESI + MS) disclosed m/z 390 and 406 products corresponding to 12-methylbenz[ a ]anthracene-7-methylene-N7-adenine or -guanine, respectively. Release of these adducts is expected to be accompanied by generation of apurinic sites in DNA structure. Examination of alternative leaving groups on the aralkylating agent suggest the following relative reactivities with deoxyguanosine or deoxyadenosine: chloro > sulfooxy > acetoxy > iodo. It is our expectation that development of these analytical methodologies will enable us to assign a role for the participation of aralkylating agents in PAH carcinogenicity.     

Studies on the Synthesis and Activity of Three Tripalladium Complexes Containing Planaramine Ligands

The present study deals with the synthesis, characterization and activity against human cancer cell lines: A2780, A2780^cisR and A2780^ZD0473R of three tripalladium complexes, MH3, MH4 and MH5, that each have two planaramine ligands bound to the central metal ion. Cellular uptake levels, extent of DNA binding, and nature of interaction with salmon sperm and pBR322 plasmid DNA were determined for each complex. Palladium compounds are much more reactive than their corresponding platinum derivatives, which makes them therapeutically inactive but toxic. However, the results of the present study suggest that significant antitumour activity can be introduced in palladium complexes by lessening their reactivity by the introduction of sterically hindered ligands such as 2‐hydroxypyridine, 3‐hydroxypyridine and 4‐hydroxypyridine. When bound to the central palladium ion, 4‐hydroxypyridine appears to be more activating than 2‐hydroxypyridine and 3‐hydroxypyridine, suggesting that noncovalent interactions, such as hydrogen bonding, may also be key determinants of antitumour activity in addition to the steric effect. While cisplatin binds with DNA to form intrastrand GG adducts that causes local bending of a DNA strand, these planaramine‐derived palladium complexes are expected to bind with DNA and form a number of long‐range interstrand GG adducts that would cause a global change in DNA conformation, provided the tripalladium cations in MH3, MH4 and MH5 persist under physiological conditions.     

Genistein-Inhibited Cancer Stem Cell-Like Properties and Reduced Chemoresistance of Gastric Cancer

Genistein, the predominant isoflavone found in soy products, has exerted its anticarcinogenic effect in many different tumor types in vitro and in vivo. Accumulating evidence in recent years has strongly indicated the existence of cancer stem cells in gastric cancer. Here, we showed that low doses of genistein (15 μM), extracted from Millettia nitida Benth var hirsutissima Z Wei, inhibit tumor cell self-renewal in two types of gastric cancer cells by colony formation assay and tumor sphere formation assay. Treatment of gastric cancer cells with genistein reduced its chemoresistance to 5-Fu (fluorouracil) and ciplatin. Further results indicated that the reduced chemoresistance may be associated with the inhibition of ABCG2 expression and ERK 1/2 activity. Furthermore, genistein reduced tumor mass in the xenograft model. Together, genistein inhibited gastric cancer stem cell-like properties and reduced its chemoresistance. Our results provide a further rationale and experimental basis for using the genistein to improve treatment of patients with gastric cancer.     

Deuterated Curcuminoids: Synthesis,Structures, Computational/Docking and Comparative Cell Viability Assays against Colorectal Cancer

A series of deuterated curcuminoids (CUR) were synthesized, bearing two to six OCD₃ groups, in some cases in combination with methoxy groups, and in others together with fluorine or chlorine atoms. A model ring-deuterated hexamethoxy-CUR–BF₂ and its corresponding CUR compound were also synthesized from a 2,4,6-trimethoxybenzaldehyde-3,5-d₂ precursor. As with their protio analogues, the deuterated compounds were found to remain exclusively in the enolic form. The antiproliferative activities of these compounds were studied by in vitro bioassays against a panel of 60 cancer cell lines, and more specifically in human colorectal cancer (CRC) cells (HCT116, HT29, DLD-1, RKO, SW837, and Caco2) and in normal colon cells (CCD841CoN). The deuterated CUR–BF₂ adducts exhibited better overall growth inhibition by NCI-60 assay, while for other CUR–BF₂ adducts the non-deuterated analogues were more cytotoxic. Results of the more focused comparative cell viability assays followed the same trend, but with some variation depending on cell lines. The CUR–BF₂ adducts exhibited significantly higher cytotoxicity than CURs. Structural studies (X-ray and DFT) and computational molecular docking calculations comparing their inhibitory efficacy with those of known anticancer agents used in chemotherapy are also reported.     

Clinical Perspectives of ERCC1 in Bladder Cancer

ERCC1 is a key regulator of nucleotide excision repair (NER) pathway that repairs bulky DNA adducts, including intrastrand DNA adducts and interstrand crosslinks (ICLs). Overexpression of ERCC1 has been linked to increased DNA repair capacity and platinum resistance in solid tumors. Multiple single nucleotide polymorphisms (SNPs) have been detected in ERCC1 gene that may affect ERCC1 protein expression. Platinum-based treatment remains the cornerstone of urothelial cancer treatment. Given the expanding application of neoadjuvant and adjuvant chemotherapy in locally advanced bladder cancer, there is an emerging need for biomarkers that could distinguish potential responders to cisplatin treatment. Extensive research has been done regarding the prognostic and predictive role of ERCC1 gene expression and polymorphisms in bladder cancer. Moreover, novel compounds have been recently developed to target ERCC1 protein function in order to maximize sensitivity to cisplatin. We aim to review all the existing literature regarding the role of the ERCC1 gene in bladder cancer and address future perspectives for its clinical application.     

The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.     

The Primary Role of Apurinic Sites in Tumor Initiation

The profiles of DNA adducts determined for benzo[a]pyrene (BP), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P) reveal that a majority of adducts are released from DNA by depurination. Papillomas were induced in mouse skin by several PAH, and mutations in the c-Harvey-ras oncogene were determined to investigate the relationship between DNA adducts and ras oncogene mutations. The pattern of mutations induced by each PAH correlated with the profile of depurinating adducts. DB[a,l]P and DMBA formed predominantly depurinating adenine adducts (78% and 79%, respectively) and consistently induced a CAA → CTA transversion in codon 61 of ras. In contrast, BP produced both guanine (46%) and adenine (25%) depurinating adducts and induced a GGC → GTC mutation in codon 13 of c-H-ras in 54% of tumors and a CAA → CTA mutation in codon 61 in 15% of tumors. These results support the hypothesis that mis-replication of unrepaired apurinic sites generated by loss of PAH-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.     

Chemical Inhibitors and microRNAs (miRNA) Targeting the Mammalian Target of Rapamycin (mTOR) Pathway: Potential for Novel Anticancer Therapeutics

The mammalian target of rapamycin (mTOR) is a critical regulator of many fundamental features in response to upstream cellular signals, such as growth factors, energy, stress and nutrients, controlling cell growth, proliferation and metabolism through two complexes, mTORC1 and mTORC2. Dysregulation of mTOR signalling often occurs in a variety of human malignant diseases making it a crucial and validated target in the treatment of cancer. Tumour cells have shown high susceptibility to mTOR inhibitors. Rapamycin and its derivatives (rapalogs) have been tested in clinical trials in several tumour types and found to be effective as anticancer agents in patients with advanced cancers. To block mTOR function, they form a complex with FKBP12 and then bind the FRB domain of mTOR. Furthermore, a new generation of mTOR inhibitors targeting ATP-binding in the catalytic site of mTOR showed potent and more selective inhibition. More recently, microRNAs (miRNA) have emerged as modulators of biological pathways that are essential in cancer initiation, development and progression. Evidence collected to date shows that miRNAs may function as tumour suppressors or oncogenes in several human neoplasms. The mTOR pathway is a promising target by miRNAs for anticancer therapy. Extensive studies have indicated that regulation of the mTOR pathway by miRNAs plays a major role in cancer progression, indicating a novel way to investigate the tumorigenesis and therapy of cancer. Here, we summarize current findings of the role of mTOR inhibitors and miRNAs in carcinogenesis through targeting mTOR signalling pathways and determine their potential as novel anti-cancer therapeutics.     

Effect of conformation of the arylamine-DNA adduct on the sensitivity of [Ru(phen)2(dppz)]2 + complex

DNA damage by endogenous/exogenous chemicals and ionizing radiation results in the formation of abasic sites, alkylation products, oxidative lesions and covalent-DNA adducts. The covalent DNA adducts adopt different conformations that might lead to various types of mutations. In the present study, we investigated the interaction of different Ru(II) polypyridyl complex, [Ru(phen)₂(dppz)]^2 + (phen: 1,10-phenanthroline; dppz: dipyrido[3,2-a:2′,3′-c]phenazine) with oligonucleotide sequences containing either [N-(2′-deoxyguanosin-8-yl)-2-aminofluorene] (AF-dG) or [N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene] (AAF-dG) adduct. The modification of the oligonucleotides with the arylamines was characterized by UV–visible and MALDI-TOF spectroscopy. The results of thermal denaturation studies of oligonucleotide with Ru(II) complex indicate that Ru(II) complex stabilize the arylamine adducts better than control oligonucleotide. The integrated luminescence intensity of the Ru(II) complex was increased two-fold in AAF-dG adduct compared to control while a reverse trend was observed with AF-dG adduct. The addition of quencher enhanced the luminescence ratio between AF-modified and control duplex by 1.5 fold compared to 3.6 to 6-fold in AAF-dG adduct. The results from this study demonstrate the role of conformational heterogeneity of the arylamine-dG adduct in the binding of the Ru(II) complex and also provides us insight on the development of new probes.