Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells

doi:10.1016/0891-5849(92)90099-3

Free Radical Biology and Medicine

Volume 12, Issue 6, 1992, Pages 457-469

https://doi.org/10.1016/0891-5849(92)90099-3 Get rights and content

Abstract

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent 1,10-phenanthroline (OP), the 1:05 complex of 1,10-phenanthroline and CuCl₂ [(OP)₂Cu], and CuCl₂ inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10⁵ cells/mL, respectively. (OP)₂Cu at 10 nmol/10⁵ cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl₂-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)₂Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)₂Cu and CuCl₂. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands blind copper added as (OP)₂Cu or CuCl₂ and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)₂Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H₂O₂ in both processes. In contrast, superoxide dimutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)₂Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye did not inhibit DNA strand scission produced by (OP)₂Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)₂Cu-treated cells. Growth inhibition produced by OP or (OP)₂Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

References (50)

A.C. Mello Filho et al.
In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction
Biochim. Biophys. Acta
(1984)
A.C. de Mello Filho et al.
Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species
Biochim. Biophys. Acta
(1985)
A.L. Feig et al.
Mutagenicity of the nuclease activity of 1,10-phenanthroline-copper ion
Biochem. Biophys. Res. Commun.
(1988)
A. Shulman et al.
Action of 1,10-phenanthroline transition metal chelates on P388 mouse lymphocytic leukaemic cells
Chem.-Biol. Interact.
(1977)
S. Lyman et al.
Activity of bleomycin in iron- and copper-deficient cells
Biochem. Pharmacol.
(1989)
R.W. Byrnes et al.
Inhibition of bleomycin-induced DNA strand scission by 1,10-phenanthroline
Biochem. Pharmacol.
(1991)
L.A. Saryan et al.
Interaction of 2-formylpyridine thiosemicarbazonato copper(II) with Ehrlich ascites tumor cells
Biochem. Pharmacol.
(1981)
D.S. Sigman et al.
Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline · cuprous complex
J. Biol. Chem.
(1979)
J.M.C. Gutteridge et al.
The role of superoxide and hydroxyl radicals in the degradation of DNA and deoxyribose induced by a copper-phenanthroline complex
Biochem. Pharmacol.
(1982)
L.M. Pope et al.
Products of DNA cleavage by the 1,10-phenanthroline-copper complex: Inhibitors of Escherichia coli DNA polymerase I
J. Biol. Chem.
(1982)

S. Inouye

Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid

FEBS

(1984)

O. Cantoni et al.

Hydrogen peroxide insult in cultured mammalian cells: Relationships between DNA single-strand breakage, poly(ADP-ribose) metabolism and cell killing

Biochim. Biophys. Acta

(1989)

D.J. McConkey et al.

Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress

Toxicol. Lett.

(1988)

S. Goldstein et al.

The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from the toxicity of O₂₋

J. Free Rad. Biol. & Med.

(1986)

P. Nicotera et al.

Inhibition of hepatocyte plasma membrane Ca²⁺-ATPase activity by menadione metabolism and its restoration by thiols

FEBS Lett.

(1985)

A.C. Mello Filho et al.

Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron

Biochem. J.

(1984)

I. Allan et al.

Structural damage of lymphocyte nuclei by H₂O₂ or radical production

Br. J. Cancer

(1988)

J.A. Imlay et al.

Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro

Science

(1988)

J.B. Mitchell et al.

Biologically active metal-independent superoxide dismutase mimics

Biochemistry

(1990)

J. Aronovitch et al.

In vivo degradation of bacterial DNA by H₂O₂ and o-phenanthroline

E. Lickl et al.

Intracellular oxidative cleavage of DNA in Escherichia coli by the copper-1,10-phenanthroline complex

Free Rad. Res. Communs.

(1989)

D. Sigman

Nuclease activity of 1,10-phenanthroline-copper ion

Acc. Chem. Res.

(1986)

C. Krishnamurti et al.

Effects of ethylenediaminetetraacetic acid and 1,10-phenanthroline on cell proliferation and DNA synthesis of Ehrlich ascites cells

Cancer Res.

(1980)

K.A. Reich et al.

Cleavage of DNA by the 1,10-phenanthroline-copper ion complex. Superoxide mediates the reaction dependent on NADH and hydrogen and hydrogen peroxide

J. Am. Chem. Soc.

(1981)

R.W. Byrnes et al.

Oxidative stress induced by a copper-thioasemicarbazone complex

Biochemistry

(1990)

Cited by (23)

TiO<inf>4</inf>N<inf>2</inf> complexes formed with 1,10-phenanthroline ligands containing a donor-acceptor hydrogen bond site: Synthesis, cytotoxicity and docking experiments
2022, Inorganica Chimica Acta
Citation Excerpt :
The X-ray diffraction data for [Ti(1)2(A)], [Ti(1)2(F)] were collected at and 173 K on a Bruker SMART CCD diffractometer with MoKα radiation (λ = 0.71073 Å). The diffraction data were corrected for absorption using the SADABS program [36]. The structures were solved using SHELXS97 [37] and refined by full matrix least-squares on F2 using SHELXL-2014 [38] in the anisotropic approximation for all non-hydrogen atoms.
The synthesis of five 1,10-phenanthroline-based ligands (ligands A-E), where a pyridinone group is grafted onto a 1,10-phenanthroline backbone through various linkers is described. From these compounds and ligand F (a 2,2′-bipyridine backbone fused with a NH-CO motif), two monomeric octahedral complexes formulated as [Ti(1)₂(A)] and [Ti(1)₂(F)] (1 is 6,6′-diphenyl-2,2′-biphenolato) are obtained. The X-ray crystal structures attested the formation of hydrogen bonded supramolecular dimers in the solid state for both complexes. Colorimetric cell viability assays (MTT) performed on human gastric adenocarcinoma cells (AGS) have permitted to determine IC₅₀ around 20 μM for phenanthrolines A-C whereas compound F was non-toxic. Owing their insolubility, the cytotoxic activities of the two Ti-complexes were not measured. Docking experiments conducted with [Ti(1)₂(A)] and [Ti(1)₂(F)] evidenced major clashes within the MST2 kinase binding pocket while the free ligands have a shape fitting with the size of this enzyme pocket.
In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii
2021, Bioorganic and Medicinal Chemistry
Citation Excerpt :
Apart from sequestering metals, which could affect a wide range of proteins, the resulting 1,10-Phen-metal complexes are highly active and play an important role in the activity of this compound.53,54 Phen derivatives, as a free ligand or in metal complexes, have strong hydrophobicity, planarity and rigidity, enabling them to interact with DNA through intercalation or as groove binding agents, as cleaving agents or may induce oxidative modifications.55–57 Treatment of cancer cells with 1,10-phenanthroline-5,6-dione and its copper derivatives was shown to inhibit DNA synthesis in neoplastic human cell lines.58
Toxoplasma gondii is an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline to new derivatives improved the anti-T. gondii activity 3.4 fold. The most active derivative presented ED₅₀ in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.
The cytotoxicity of some phenanthroline-based antimicrobial copper(II) and ruthenium(II) complexes
2018, Journal of Inorganic Biochemistry
Citation Excerpt :
Although cytotoxicity is a key consideration of compounds with potential antibiotic or disinfectant application, low genotoxicity is desired. Bis-1,10-phenanthroline copper(II) complexes have been observed to undergo reduction to copper(I) and induce single-strand DNA breaks in mouse abdominal tumour cells [15]. The DNA binding capacity of antimicrobial ternary complexes of phen and dach ligands noted in previous studies may contribute to their target cell mode of action, potentially via speciation to bis-phen complexes, however may also induce mutagenesis in human cells [5].
The in vitro cytotoxic properties of antimicrobial copper(II) complexes with 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) or 4,7-dipyridyl-1,10-phenanthroline (DIP) ligands and ruthenium(II) complexes coordinated with TMP or 2,9-dimethyl-1,10-phenanthroline ligands were investigated. Both copper(II) complexes were found to have similar inhibitory concentrations (IC₅₀ ~ 2–2.5 μM). Their cytotoxicity was found to be necrotic, associated with cytoplasmic vacuolisation, rounding, detachment and lack of apoptosis-associated DNA fragmentation, in comparison to the apoptotic effects of cisplatin which demonstrate adherent cell enlargement or detachment, membrane blebbing and condensation. Antimicrobial ruthenium(II) complexes demonstrated a lower renal cytotoxicity than copper(II) complexes or cisplatin (IC₅₀ > 60 μM). [Cu(DIP)(dach)](ClO₄)₂ and [Cu(TMP)(dach)](ClO₄)₂ (where dach = 1,2-diaminocyclohexane) induced dihydroethidium-sensitive ROS and the cytotoxicity of both TMP and DIP coordinated copper(II) complexes was mitigated by catalase, highlighting a role of H₂O₂ generation in their mode of action. The cytotoxicity of either copper(II) complex was not affected by coincubation with organic cation transporter (OCT) inhibitors cimetidine or disopyramide, in contrast to cisplatin, suggesting a non-OCT dependent mode of uptake for the copper(II) complexes in human cells. Coincubation with copper sulfate reduced the cytotoxicity of [Cu(TMP)(dach)](ClO₄)₂ (3-6 ×). The TMP complex induced a greater degree of G2/M accumulation and micronuclei generation than the DIP complex, possibly attributable to its greater DNA binding affinity. These results highlight the potentially low genotoxicity of copper(II) complexes coordinated with TMP or DIP and polypyridyl ruthenium(II) complexes as potential antimicrobial agents.
Radical-induced DNA damage by cytotoxic square-planar copper(II) complexes incorporating o-phthalate and 1,10-phenanthroline or 2,2′-dipyridyl
2012, Free Radical Biology and Medicine
Citation Excerpt :
As a result of the link between copper(I)- and copper(II)-containing molecules and the generation and detoxification of free radicals, there has been a sustained interest in the rational design and therapeutic evaluation of small molecules containing redox-active metal cations [19–27]. In particular, the design of copper(II) anticancer chemotherapeutics capable of targeting DNA represents both an attractive and a multifaceted prospect [28–39]. In comparison to platinum(II) chemotherapeutic agents (Fig. 1), whose coordination chemistry is dominated through the trans effect, and with DNA toxicity being mediated directly through Pt(II)–guanine substitution kinetics [40–42], copper(II) compounds are structurally influenced through the Jahn–Teller effect and induce DNA toxicity both indirectly through free radical modification of 2-deoxyribose or base moieties [43] and directly through Cu(II)–guanine/adenine binding [44,45].
DNA-targeting copper(II) reagents have emerged as suitable drug candidates owing to the clinical success of the copper-activated, natural chemotherapeutic drug bleomycin. This agent and the synthetic chemical nuclease copper(II) bis-1,10-phenanthroline represent important templates for inorganic drug design owing to their ability to initiate free radical DNA scission. Herein, we report the synthesis and biological properties of 1:1:1 square-planar copper(II) complexes incorporating the dicarboxylate o-phthalate and 1,10-phenanthroline (1) or 2,2′-dipyridyl (2) ligands. Their broad-spectrum chemotherapeutic potential has been assessed at 24- and 96-h intervals, along with that of the clinical agent cisplatin, using breast (MCF-7), prostate (DU145), colon (HT29), and intrinsically cisplatin-resistant ovarian (SK-OV-3) human cancer cells. 1 represents a potent cytotoxic agent with IC₅₀ values ranging from 5.6 to 3.4 μM across all cell lines, including SK-OV-3. The production of endogenous reactive oxygen species within SK-OV-3 cancer cells was monitored using the fluorophore 2′,7′-dichlorodihydrofluorescin diacetate, and results indicate a concentration-dependent propensity toward ROS generation by 1 and 2 that mirrors their antitumoral behavior. DNA interaction studies, using fluorescence and viscosity measurements, were conducted in tandem with the DNA-targeting drugs actinomycin D and pentamidine, using calf thymus DNA, poly[d(A-T)₂], and poly[d(G–C)₂], with intercalation of 1 and 2 at the minor groove appearing to be the likely interaction mode. DNA cleavage reactions using superhelical plasmid DNA, in the presence of exogenous reductant, l-ascorbic acid, revealed excellent agreement between double-stranded DNA scission capability and antitumoral IC₅₀ concentration. The presence of double-strand DNA breaks (DSBs) was confirmed within SK-OV-3 cancer cells using immunodetection of γ–H2AX foci by confocal microscopy and flow cytometry, with complex 1 quantitatively producing superior numbers of DSBs compared with complex 2. Superoxide dismutase and catalase mimetic activity assays were conducted, and these activities are related to the ability of both complexes to cleave DNA through free radical generation.
Mechanism of the synergistic cytotoxicity between pentachlorophenol and copper-1,10-phenanthroline complex: The formation of a lipophilic ternary complex
2000, Chemico-Biological Interactions
Citation Excerpt :
It has been shown that Cu(II)(OP)2 complex could non-covalently bind to DNA [14–16]. In the presence of biological reducing agents, both single and double strand breaks could be produced through a site-specific mechanism, both in vitro [14–16] and in vivo systems [17–21]. The ineffective protection of SOD and catalase by their catalytic functions, and the ineffectiveness of DMSO as a hydroxyl radical scavenger, also point to the site-specific nature of cellular damage by the PCP/Cu(II)(OP)2 system.
When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1,10-phenanthroline)cupric complex, Cu(II)(OP)₂, were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation. Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes. The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells. The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.
Inhibition of hydroperoxide-induced DNA single-strand breakage by 1,10- phenanthroline in HL-60 cells: Implications for iron speciation
1996, Archives of Biochemistry and Biophysics
The speciation of intracellular iron which reacts with hydroperoxides to cause DNA damage was investigated in HL-60 cells using 1,10-phenanthroline (OP) to compete with ligands for iron. HL-60 cells were treated with various concentrations of OP for 30 min at 37°C followed by 30 min treatment with H₂O₂ort-butylhydroperoxide (tert-BuOOH) at 4°C. Single-strand breaks (SSBs) were measured by alkaline elution. OP (5 μM) completely inhibited production of SSBs bytert-BuOOH. This contrasts with results for H₂O₂, for which it was not possible to inhibit all SSBs even at OP concentrations up to 60 μM. Induction of SSBs bytert-BuOOH decreased according to a SSB:OP ratio of 1:3.28 ± 0.23 (± 1 SE). This is consistent with inhibition achieved by occupation of all coordination sites on iron by OP, such as by formation of Fe(OP)₃. In contrast, after subtraction of the noninhibitable fraction, SSBs induced by H₂O₂decreased according to a 1:1.53 ± 0.26 ratio. This suggests that inhibition of H₂O₂-induced SSBs is partially or wholly achieved by formation of different structures than Fe(OP)₃.Tert-BuOOH did not cause SSBs in nuclei isolated from HL-60 cells. However, H₂O₂induced SSBs which were inhibited 45 ± 8% (±1 SD) by dimethyl sulfoxide. Analysis of the concentration dependence of inhibition by dimethyl sulfoxide indicated that the H₂O₂-reactive iron in nuclei possessed an average distance of 4.8 nm from DNA. This compares with a previously obtained distance in whole cells of 6.9 nm, for which 5 μMOP pretreatments decreased to 4.8 nm. These data indicate thattert-BuOOH generates DNA-damaging radicals through reaction with a subset of the different cell iron species which react with H₂O₂.

View all citing articles on Scopus

View full text

Original ContributionInteractions of 1,10-phenanthroline and its copper complex with Ehrlich cells

Abstract

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Chem.-Biol. Interact.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

FEBS

Biochim. Biophys. Acta

Toxicol. Lett.

J. Free Rad. Biol. & Med.

FEBS Lett.

Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron

Biochem. J.

Structural damage of lymphocyte nuclei by H2O2 or radical production

Br. J. Cancer

Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro

Science

Biologically active metal-independent superoxide dismutase mimics

Biochemistry

In vivo degradation of bacterial DNA by H2O2 and o-phenanthroline

Intracellular oxidative cleavage of DNA in Escherichia coli by the copper-1,10-phenanthroline complex

Free Rad. Res. Communs.

Nuclease activity of 1,10-phenanthroline-copper ion

Acc. Chem. Res.

Effects of ethylenediaminetetraacetic acid and 1,10-phenanthroline on cell proliferation and DNA synthesis of Ehrlich ascites cells

Cancer Res.

Cleavage of DNA by the 1,10-phenanthroline-copper ion complex. Superoxide mediates the reaction dependent on NADH and hydrogen and hydrogen peroxide

J. Am. Chem. Soc.

Oxidative stress induced by a copper-thioasemicarbazone complex

Biochemistry

Original Contribution
Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells

Structural damage of lymphocyte nuclei by H₂O₂ or radical production

In vivo degradation of bacterial DNA by H₂O₂ and o-phenanthroline