1,2-Naphthoquinone as a Poison of Human Type II Topoisomerases

Abstract

1,2-Naphthoquinone, a secondary metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles that displays cytotoxic and genotoxic properties. Because many quinones have been shown to act as topoisomerase II poisons, the effects of this compound on DNA cleavage mediated by human topoisomerase IIα and IIβ were examined. The compound increased levels of double-stranded DNA breaks generated by both enzyme isoforms and did so better than a series of naphthoquinone derivatives. Furthermore, 1,2-naphthoquinone was a more efficacious poison against topoisomerase IIα than IIβ. Topoisomerase II poisons can be classified as interfacial (which interact non-covalently at the enzyme-DNA interface and increase DNA cleavage by blocking ligation) or covalent (which adduct the protein and increase DNA cleavage by closing the N-terminal gate of the enzyme). Therefore, experiments were performed to determine the mechanistic basis for the actions of 1,2-naphthoquinone. In contrast to results with etoposide (an interfacial poison), the activity of 1,2-naphthoquinone against topoisomerase IIα was abrogated in the presence of sulfhydryl and reducing agents. Moreover, the compound inhibited cleavage activity when incubated with the enzyme prior to the addition of DNA and induced virtually no cleavage with the catalytic core of the enzyme. It also induced stable covalent topoisomerase IIα-DNA cleavage complexes and was a partial inhibitor of DNA ligation. Findings were also consistent with 1,2-naphthoquinone acting as covalent poison of topoisomerase IIβ; however, mechanistic studies with this isoform were less conclusive. Whereas the activity of 1,2-naphthoquinone was blocked in the presence of a sulfhydryl reagent, it was much less sensitive to the presence of a reducing agent. Furthermore, the reduced form of 1,2-naphthoquinone, 1,2-dihydroxynaphthalene, displayed high activity against the β isoform. Taken together, results suggest that 1,2-naphthoquinone increases topoisomerase II-mediated double-stranded DNA scission (at least in part) by acting as a covalent poison of the human type II enzymes.

Graphical Abstract

INTRODUCTION

Naphthoquinones are secondary metabolites of naphthalene that are found in multiple species of bacteria, fungi, plants, and mammals.^{1, 2} These compounds serve important physiological roles in the electron transport chain (ubiquinone and plastoquinone) and in vitamin K synthesis (menadione).^{1, 2} Due to their yellow or brown color, naphthoquinones also have been used as pigmentation agents for centuries.¹ In addition, these compounds have been used in a broad range of medicinal applications, including antibiotic, antiviral, antiparasitic, fungicidal, and insecticidal (among others) treatments.¹ Thus, many traditional medicines across the world include naphthoquinone-based components.¹

Despite the medicinal uses of many naphthoquinones, some members of this family have been associated with negative health outcomes.^1–3 For example, 1,2-naphthoquinone is an electrophilic environmental pollutant found in diesel exhaust particles (Figure 1).^3–7 It has been associated with the formation of cataracts and altered pulmonary function in vertebrate animal models.^{3, 8, 9} Furthermore, this compound is cytotoxic and genotoxic, impairs mitochondrial function, and promotes an enhanced inflammatory response.^{3, 10–14}

Figure 1.

Structures of 1,2-naphthoquinone, 1,4-naphthoquinone, and related compounds.

Several quinones and polyphenols (that can be converted to quinones by redox cycling) increase levels of DNA cleavage mediated by human type II topoisomerases.^15–25 Among these compounds, environmental toxins such as 1,4-benzoquinone (leukemogenic benzene metabolite) and polychlorinated biphenyl (PCB) quinones (toxic metabolites of PCBs) are associated with the formation of human cancers.^{17, 18} In contrast, a variety of dietary quinones/polyphenols associated with human health benefits have also been identified as topoisomerase II poisons. Compounds included on this list are (−)-epigallocatechin gallate (EGCG, the active ingredient in green tea), thymoquinone (the active ingredient in Nigella sativa, or black seed), curcumin (the primary olfactory and taste component of turmeric), hydroxytyrosol and oleuropein (antioxidants found in olives), and menadione (vitamin K3).^{16, 19–22} These natural products display chemoprotective, anti-inflammatory, and antioxidant properties.^15–22 Many have been components of traditional and herbal medicines for millennia.^{15, 19–22} It has been suggested that at least some of the toxic and medicinal properties of quinones/polyphenols are mediated through interactions with the human type II topoisomerases.^17–22

Type II topoisomerases regulate the topological state of the genetic material in vivo.^26–32 These enzymes control levels of DNA supercoiling and remove knots and tangles from the genome by generating transient double-stranded breaks in the DNA backbone and passing an intact double helix through the DNA gate.^26–32 This double-stranded DNA passage reaction is driven by cycles of ATP binding and hydrolysis.^{26, 30–32} In order to maintain genomic integrity during the DNA cleavage/ligation process, type II topoisomerases form covalent links between their active site tyrosine residues and the newly generated 5’-termini of the cleaved DNA.^{26, 30–32} This covalent topoisomerase II-cleaved DNA complex is known as the cleavage complex.²⁶ Compounds that increase levels of this requisite enzyme intermediate are referred to as topoisomerase II poisons in order to distinguish them from catalytic inhibitors of the enzyme.^{26, 29}

Humans encode two isoforms of topoisomerase II, α and β.^{26, 31–33} Topoisomerase IIα is an essential enzyme that is expressed in proliferating cells.^{26, 28, 31, 34} It is the isoform required for DNA replication and the unlinking of daughter chromosomes in mitosis and meiosis and also appears to play roles in transcription.^{26, 28, 31, 34} Topoisomerase IIβ is not essential at the cellular level but is required for proper neural development in mice.^{26, 28, 31, 33–36} In contrast to the α isoform, topoisomerase IIβ is expressed in all cell types irrespective of proliferation status.^{26, 28, 31, 33–36} Although its cellular functions are not well defined, the β isoform is involved in the transcription of hormonally regulated genes.^{26, 28, 31, 33–36}

Two previous studies characterized interactions between 1,2-naphthoquinone and eukaryotic type II topoisomerases. One examined the effects of the compound on the isolated ATPase domain of human topoisomerase IIα and determined that 1,2-naphthquinone inhibited ATP hydrolysis.³⁷ However, this study did not examine the effects of the compound on DNA scission. The second study reported that 1,2-naphthoquinone increased DNA cleavage mediated by calf thymus topoisomerase II (a mixture of the α and β isoforms) but reported no information other than its potency being higher than that of menadione (vitamin K3).¹⁶

Therefore, to further understand the effects of 1,2-naphthoquinone on the human type II enzymes, we characterized the ability of the compound to act as a topoisomerase II poison. Results indicate that 1,2-naphthoquinone increases levels of double-stranded DNA scission mediated by topoisomerase IIα and IIβ but was more potent and efficacious toward the α isoform. Furthermore, the characteristics of DNA cleavage induced by 1,2-naphthoquinone indicate that the compound acts as a covalent, rather than an interfacial, poison of human topoisomerase IIα. Although mechanistic studies with topoisomerase IIβ were less conclusive, they also suggest that 1,2-naphthoquinone acts (at least in part) as a covalent poison of the β isoform.

EXPERIMENTAL PROCEDURES

Enzymes and Materials.

Recombinant human wild-type topoisomerase IIα and IIβ were expressed in Saccharomyces cerevisiae and purified as described previously.^{38, 39} The catalytic core of human topoisomerase IIα (containing amino acids 431–1193)⁴⁰ was expressed in S. cerevisiae and purified using a Ni²⁺ nitriloacetic acid agarose column (Qiagen, Hilden, Germany) as described previously.^{41, 42} The enzymes were stored at −80 °C as 1.5 or 7.5 mg/mL stocks in 50 mM Tris-HCl (pH 7.7), 0.1 mM EDTA (pH 8.0), 750 mM KCl, and 40% (v/v) glycerol. For all enzymes, the concentration of dithiothreitol (DTT) remaining from purification was <2 μM in final reaction mixtures. Negatively supercoiled pBR322 plasmid DNA was prepared from Escherichia coli using a Plasmid Mega Kit (Qiagen) according to the manufacturer’s protocol. Etoposide, 1,4-benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 5,8-dihydroxy-1,4-naphthoquinone were purchased from Sigma-Aldrich (St. Louis, MO). 1,2-Dihydroxynaphthalene was purchased from TCI (Portland, OR). All compounds except 1,4-benzoquinone were stored at 4 °C as 20 mM stock solutions in 100% DMSO. 1,4-Benzoquinone was stored at −20 °C as a 20 mM stock solution in water. In all cases, the activities of compounds in DMSO or water stock solutions were stable for at least 6 months. ATP, ascorbic acid, and DTT were purchased Sigma-Aldrich. All other chemicals were analytical reagent grade.

DNA Cleavage.

DNA cleavage reactions were performed according to the procedure of Fortune and Osheroff.⁴³ Reaction mixtures contained 10 nM pBR322 and 300 nM wild-type topoisomerase IIα, 275 nM wild-type topoisomerase IIβ, or 850 nM topoisomerase IIα catalytic core in a total of 20 μL of cleavage buffer [10 mM Tris-HCl (pH 7.9), 0.1 mM EDTA (pH 8.0), 100 mM KCl, 5 mM MgCl₂, 2.5% (w/v) glycerol]. The concentrations of topoisomerase IIα and IIβ that were used yielded equivalent enzyme activities. DNA cleavage reactions were incubated for 6 min at 37 °C before enzyme-DNA cleavage complexes were trapped by adding 2 μL of 4% SDS followed by 2 μL of 250 mM EDTA (pH 8.0). Proteinase K was added (2 μL of a 0.8 mg/mL solution), and reaction mixtures were incubated for 30 min at 45 °C to digest the enzyme. Samples were mixed with 2 μL of agarose loading buffer [60% (w/v) sucrose, 10 mM Tris-HCl (pH 7.9), 0.5% (w/v) bromophenol blue, and 0.5% (w/v) xylene cyanol FF] and heated for 2 min at 45 °C prior to electrophoresis in 1% agarose in 40 mM Tris-acetate (pH 8.3), and 2 mM EDTA (pH 8.0) containing 0.5 μg/mL ethidium bromide. DNA bands were visualized by ultraviolet light and quantified using an Alpha Innotech digital imaging system. Double-stranded DNA cleavage was monitored by the conversion of supercoiled plasmid DNA to linear molecules.

DNA cleavage reactions were performed in the presence of 0–100 μM 1,2-naphthoquinone, 1,4-naphthoquinone, 1,2-dihydroxynaphthalene, 2-hydroxy-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, etoposide, or 1,4-benzoquinone. Unless stated otherwise, the above compounds were added last to the reaction mixtures. In some cases, reactions contained 1 mM ATP, 2 mM DTT, or 2 mM ascorbic acid. Alternatively, DTT or ascorbic acid were added after the 6 min cleavage reaction, and samples were incubated for an additional 5 min.

Persistence of Topoisomerase II-DNA Cleavage Complexes.

The persistence of topoisomerase IIα-DNA cleavage complexes was determined using the procedure of Gentry et al.⁴⁴ Initial reactions contained 55 nM pBR322, 2.2 μM wild-type topoisomerase IIα (in the absence of compound) or 300 nM wild-type topoisomerase IIα in the presence of 50 μM 1,2-naphthoquinone or 100 μM etoposide in a total of 20 μL of DNA cleavage buffer. In the reaction that contained no compound, 5 mM MgCl₂ in the cleavage buffer was replaced with 5 mM CaCl₂ in order to increase baseline levels of DNA cleavage. To establish a DNA cleavage/ligation equilibrium, reactions were incubated for 6 min at 37 °C and diluted 20-fold with DNA cleavage buffer lacking divalent cation at 37 °C. Samples (20 μL) were removed at times ranging from 0–120 min. DNA cleavage complexes were trapped, and samples were analyzed as discussed under DNA cleavage. Levels of DNA cleavage were set to 100% at time zero, and the persistence of cleavage complexes was determined by the decay of linear reaction product over time.

DNA Ligation.

DNA ligation mediated by human topoisomerase IIα was monitored as described previously.⁴⁵ Initial reactions contained 10 nM pBR322 and 860 nM wild-type topoisomerase IIα (no compound, in order to raise levels of double-stranded breaks) or 300 nM wild-type topoisomerase IIα (with compound). As described for the DNA cleavage assays, DNA cleavage/ligation equilibria were established for 6 min at 37 °C in the absence or presence of 50 μM 1,2-naphthoquinone or 100 μM etoposide. DNA ligation was initiated by shifting samples from 37 to 0 °C, which allowed enzyme-mediated ligation but prevented new rounds of DNA cleavage from occurring. This resulted in a unidirectional sealing of the cleaved DNA. DNA cleavage complexes were trapped at time points ranging from 0–20 s, and samples were analyzed as discussed under DNA cleavage. Linear DNA cleavage product at time zero was set to 100% to allow direct comparison between the different compounds, and DNA ligation was monitored by the loss of linear DNA.

RESULTS

1,2-Naphthoquinone Is a Poison of Human Type II Topoisomerases.

1,2-Naphthoquinone (see Figure 1) is a cytotoxic and genotoxic quinone-based electrophile that is an environmental pollutant found in diesel exhaust particles.^{2–7, 10} Several other quinone-based biologically active compounds enhance topoisomerase II-mediated DNA scission.^15–25 Therefore, the effects of 1,2-naphthoquinone on DNA strand breaks generated by human topoisomerase IIα and IIβ were examined.

As seen in Figures 2 and 3, 1,2-naphthoquinone is an efficacious human topoisomerase II poison. The compound generated primarily double-stranded DNA breaks and enhanced levels of enzyme-mediated cleavage more than an order of magnitude compared to baseline values. At 50 μM 1,2-naphthoquinone, the compound induced ~25% and ~14% double-stranded DNA cleavage mediated by human topoisomerase IIα and IIβ, respectively. By comparison, the same concentration of etoposide induced only ~7% and ~3.5% DNA scission by the two enzyme isoforms.

Figure 2.

1,2-naphthoquinone and 1,4-naphthoquinone enhance DNA cleavage mediated by human topoisomerase II⍺ and IIβ. The graphs show the effects of 1,2-naphthoquinone (maroon) and 1,4-naphthoquinone (green) on double-stranded DNA cleavage mediated by human topoisomerase II⍺ (hTII⍺, left) and IIβ (hTIIβ, right). Results for etoposide (blue) are displayed for comparison. Error bars represent standard deviations (SDs) for at least three independent experiments. Representative gels for DNA cleavage assays with 1,2-naphthoquinone and the respective enzymes are shown above the graphs. Negatively supercoiled DNA (DNA) is shown as a control. The mobilities of negatively supercoiled DNA (form I, FI), nicked circular DNA (form II, FII), and linear DNA (form III, FIII) are indicated.

Figure 3.

Effects of naphthoquinone derivatives on double-stranded DNA cleavage mediated by human topoisomerase II⍺ (hTII⍺, top panel) and IIβ (hTIIβ, bottom panel). Results are shown for 50 or 100 μM 1,2-naphthoquinone (1,2-NQ, maroon); 1,4-naphthoquinone (1,4-NQ, green); 2-hydroxy-1,4-naphthoquinone (2-hydroxy-1,4-NQ, purple); 5-hydroxy-1,4-naphthoquinone (5-hydroxy-1,4-NQ, orange); and 5,8-dihydroxy-1,4-naphthoquinone (5,8-dihydroxy-1,4-NQ, red). Etoposide (blue) is shown for comparison. Error bars represent SDs for at least three independent experiments.

To assess the effects of carbonyl position and hydroxyl group inclusion on the naphthoquinone ring system, the abilities of 1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 5,8-dihydroxy-1,4-naphthoquinone (see Figure 1) to induce enzyme-mediated double-stranded DNA cleavage were examined (Figures 2 and 3). 1,4-naphthoquinone forms the backbone of Vitamin K and the other compounds are used as dyes.^{1, 2, 16} Of these naphthoquinone derivatives, only 1,4-naphthoquinone displayed appreciable activity. 1,4-naphthoquinone induced less DNA cleavage than the 1,2-isomer and displayed a different preference for the two isoforms of topoisomerase II. Whereas 1,2-naphthoquinone was twice as efficacious against topoisomerase IIα compared to IIβ, 1,4-naphthoquinone was at least two times more active against the β isoform. The reasons underlying these different preferences are unknown. Therefore, additional studies were carried out to further characterize the effects of 1,2-naphthoquinone on topoisomerase IIα and IIβ.

To ensure that DNA cleavage induced by 1,2-naphthoquinone was mediated by the human type II topoisomerases, a series of control experiments was carried out (Figure 4). First, the compound failed to induce DNA cleavage in the absence of enzyme. Second, linear and nicked DNA reaction products did not enter the gel unless samples were treated with Proteinase K. This result is consistent with a covalent protein-DNA linkage. Third, the addition of EDTA (which chelates the required active site divalent cations)⁴⁶ prior to trapping the cleavage complex with SDS reversed double-stranded DNA cleavage. Similarly, treatment of reaction mixtures with NaCl (which diminishes enzyme-DNA binding) prior to trapping the cleavage complex also lowered levels of DNA scission. Reversibility of DNA cleavage is a hallmark of the type II enzymes. It is notable that greater salt reversibility was observed with topoisomerase IIβ, which is consistent with previous reports that cleavage complexes formed with the β isoform are less stable than those generated by topoisomerase IIα.^{47, 48}

Figure 4.

DNA cleavage induced by 1,2-naphthoquinone is mediated by mediated by human topoisomerase II⍺ and IIβ. Results with topoisomerase IIα (hTII⍺) are shown at left and topoisomerase IIβ (hTIIβ) at right. The bar graph shows double-stranded DNA cleavage from reactions that contained negatively supercoiled DNA alone (DNA), DNA with 50 μM or 100 μM 1,2-naphthoquinone (for reactions with hTII⍺ and hTIIβ, respectively) in the absence of enzyme (1,2-NQ), topoisomerase IIα or IIβ with DNA in the absence of 1,2-NQ (hTIIα, hTIIβ), or complete reactions that were stopped with SDS prior to the addition of EDTA and Proteinase K (SDS). In other reactions, Proteinase K was omitted (No ProK) or reactions were treated with 22 mM EDTA (EDTA) or 500 mM NaCl (NaCl) for 5 min prior to the addition of SDS and Proteinase K. Error bars represent SDs for at least three independent experiments. A representative gel is shown at the top and DNA positions are as indicated in Figure 2.

As an additional control, time courses of 1,2-naphthquinone-induced DNA cleavage against topoisomerase IIα and IIβ were performed (Figure 5). For both enzyme isoforms, levels of DNA cleavage plateaued within the duration of the 6 min cleavage assay and were followed by a DNA cleavage/ligation equilibrium. These hyperbolic time courses are consistent with topoisomerase II-mediated DNA cleavage. Taken together, control experiments provide strong evidence that the enhanced DNA scission observed in the presence of 1,2-naphthoquinone is mediated by the human type II enzymes.

Figure 5.

Establishment of DNA cleavage/ligation equilibria induced by 1,2-naphthoquinone with human topoisomerase II⍺ or IIβ. The graph displays the time course of DNA cleavage mediated by human topoisomerase II⍺ (hTII⍺, filled circles, left) and IIβ (hTIIβ, open circles, right) in the presence of 50 μM or 100 μM 1,2-naphthoquinone (1,2-NQ), respectively. Error bars represent SDs for at least three independent experiments.

Effects of ATP on the Enhancement of Double-stranded DNA Cleavage Induced by 1,2-Naphthoquinone.

Type II topoisomerases do not require ATP for DNA cleavage or ligation (although the high energy cofactor raises baseline levels of DNA scission).^{49, 50} However, ATP is required for the enzyme to carry out its complete double-stranded DNA passage reaction.^{49, 50} Furthermore, a previous study provided evidence that 1,2-naphthoquinone interacts at the ATP binding site of topoisomerase IIα and inhibits ATP hydrolysis (at least in the isolated ATPase domain).³⁷ Therefore, the effects of the high energy cofactor on 1,2-naphthoquinone-induced DNA cleavage were assessed (note that the other cleavage assays shown in this paper did not contain ATP). As seen in Figure 6, similar levels of DNA cleavage were observed in the absence or presence of ATP for both topoisomerase IIα and IIβ. Therefore, 1,2-naphthoquinone poisons the human type II topoisomerases under conditions that support the complete catalytic reaction of the enzymes.

Figure 6.

Effects of ATP on the enhancement of double-stranded DNA cleavage induced by 1,2-naphthoquinone with human topoisomerase II⍺ or IIβ. Results with topoisomerase IIα (hTII⍺, filled circles) are shown at left and topoisomerase IIβ (hTIIβ, open circles) at right. The graphs show the concentration dependence of 1,2-naphthoquinone-induced DNA cleavage in the absence (-ATP, maroon) or presence (+ATP, grey) of 1 mM ATP. Error bars represent SDs for at least three independent experiments.

Covalent vs Interfacial Topoisomerase II Poisons.

Topoisomerase II poisons can be classified as being either interfacial or covalent.^{26, 29, 51–53} Interfacial poisons include anticancer drugs such as etoposide, amsacrine, and mitoxantrone and bind non-covalently at the interface between the enzyme and its DNA substrate.^{26, 29, 51, 52, 54} These compounds interact with both the protein and the DNA in the cleavage complex and block ligation by inserting between the 3’-hydroxyl and 5’-phosphate of the cleaved double helix.^{26, 29, 51, 52, 54} As a result, most interfacial poisons are strong inhibitors of topoisomerase II-mediated DNA ligation.^{26, 29, 51, 52, 54, 55}

In contrast, covalent topoisomerase II poisons, which include a variety of quinone-, polyphenol-, and isothiocyanate-containing compounds,^{15–19, 21–25} form protein-adducts via an acylation reaction at cysteine (and potentially other) residues that are outside of the active site of type II topoisomerases.^{26, 56–58} Our understanding of the mechanism by which covalent poisons enhance enzyme-mediated DNA cleavage is less detailed than that described for interfacial poisons. However, covalent poisons appear to act (at least in part) by closing the N-terminal protein gate of topoisomerase II.^{18, 59} Because of their covalent attachment to the enzyme, covalent poisons can induce long-lived (i.e. highly persistent) DNA cleavage/ligation equilibria.⁴⁷ However, because these compounds do not act by intercalating between the cleaved DNA ends at the scissile bond, they often display a lesser ability to inhibit ligation compared to interfacial poisons that physically block the rejoining of the DNA termini.^{21, 22, 60}

Due to the mechanism of action of covalent topoisomerase II poisons, there are a number of additional hallmarks that distinguish compounds in this class from interfacial poisons. First, because the oxidation state of covalent poisons is critical for their adduction chemistry, reducing agents, such as ascorbic acid, prevent their activity against topoisomerase II.⁶¹ Similarly, sulfhydryl reagents, such as DTT, adduct to covalent poisons and inactivate them.^{17, 19–23, 60, 61} In contrast, as interfacial poisons are not dependent on redox chemistry for their activity, they are generally unaffected by reducing or sulfhydryl reagents.^{17, 19, 60} Second, because covalent poisons close the N-terminal gate of topoisomerase II, they block plasmid DNA from binding to the enzyme.^{18, 57, 59} As such, when covalent poisons are incubated with topoisomerase II prior to the addition of DNA, they inhibit, rather than enhance, DNA cleavage.^{17–22, 57, 59–62} Interfacial poisons, which act at the active site of the enzyme, induce DNA scission regardless of the order of addition.^{16, 18, 51, 60} Finally, because of the reliance of covalent poisons on the N-terminal domain of topoisomerase II, these compounds are unable to induce DNA cleavage with the catalytic core of topoisomerase IIα (which lacks both the N-terminal and C-terminal domains of the protein).^{61, 63} Due to their interactions at the active site of the enzyme, interfacial poisons maintain their ability to induce scission with the truncated protein.^{61, 63}

1,2-Naphthoquinone Is a Covalent Poison of Human Topoisomerase IIα.

As a first step toward determining the mechanism by which 1,2-naphthoquinone poisons human topoisomerase IIα, the effects of sulfhydryl and reducing agents on the activity of the compound were determined (Figure 7). Similar to the effects of the prototypical covalent poison 1,4-benzoquinone on DNA cleavage mediated by human topoisomerase IIα, the activity of 1,2-naphthoquinone was abrogated when DTT or ascorbic acid were incubated with the compound prior to inclusion of the quinone in reaction mixtures. It is notable that the effects of DTT on topoisomerase II poisons were observed only when the sulfhydryl reagent reacted with the free compound; once protein adducts were formed, they were not reversed by the addition of DTT.^{17, 19–23, 60, 61} Similar to the results seen with 1,4-benzoquinone, the addition of DTT following the establishment of cleavage complexes did not affect the induction of DNA scission by 1,2-naphthoquinone (Figure 7). In contrast, DTT and ascorbic acid had little effect on DNA cleavage induced by the interfacial poison etoposide.

Figure 7.

Effects of sulfhydryl and reducing agents on topoisomerase IIα-mediated double-stranded DNA cleavage induced by 1,2-naphthoquinone. Topoisomerase IIα-DNA cleavage reactions were performed in the absence of sulfhydryl/reducing agents (open bars), in the presence of 2 mM DTT (stippled bars) or 2 mM ascorbic acid (AA, diagonal bars) added prior to the start of the 6 min DNA cleavage reaction, or 2 mM DTT added after the 6 min cleavage reaction (filled bars). Results are shown for reactions that contained no compound (hTII⍺, black), 50 μM 1,2-naphthoquinone (1,2-NQ, maroon), 50 μM 1,2-dihydroxynaphthalene (1,2-Dihydroxy-NP, gold), 25 μM 1,4-benzoquinone (BQ, teal), or 100 μM etoposide (blue). 1,4-Benzoquinone and etoposide were included as controls for the effects of the sulfhydryl and reducing agents on a covalent and interfacial topoisomerase II poison, respectively. Error bars represent SDs for at least three independent experiments.

Generally, the reduction of quinone-based covalent poisons to their corresponding hydroxyl forms results in a loss of activity.^{17, 64} Surprisingly, this was not the case with 1,2-naphthoquinone, as 1,2-dihydroxynaphthalene (Figure 1) displayed high activity against topoisomerase IIα (Figure 7). However, the activity of the dihydroxy compound was abrogated when DTT or ascorbic acid were added prior to incubation with the enzyme. Moreover, the majority of the activity was retained when 1,2-dihydroxynaphthalene was incubated with DTT after the establishment of DNA cleavage complexes. Because 1,2-dihydroxynaphthalene displays the hallmarks of a covalent poison, the most likely explanation for these results is that the compound readily undergoes redox cycling to the reactive quinone form. This has been reported previously for polyphenolic bioflavonoids and catechins.^{19, 24, 25}

Consistent with the characteristics of a covalent poison, 1,2-naphthoquinone induced topoisomerase IIα to form long-lived cleavage complexes (Figure 8, left). In the absence of the quinone, cleavage complexes rapidly dissociated following dilution into buffer that lacked divalent metal ion, with a half-life of less than 5 s. In contrast, cleavage complexes formed in the presence of 1,2-naphthoquinone persisted for at least two hours with no observed decrease in DNA scission. Moreover, 1,2-naphthoquinone inhibited ligation but to a much lesser extent than the interfacial poison etoposide (Figure 8, right).

Figure 8.

Effects of 1,2-naphthoquinone on the persistence and ligation of DNA cleavage complexes generated by human topoisomerase IIα. Left: Persistence of topoisomerase IIα-DNA cleavage complexes formed in the presence of 1,2-naphthoquinone. DNA cleavage/ligation equilibria were established in the absence (hTIIα, black) or presence of 50 μM 1,2-naphthoquinone (1,2-NQ, maroon). Persistence assays were initiated by diluting reaction mixtures 20-fold into buffer that lacked divalent cations, and the stability of cleavage complexes was monitored by the loss of double-stranded DNA breaks over time. DNA cleavage at time zero was set to 100%. Error bars represent SDs for at least three independent experiments. Right: Inhibition of topoisomerase IIα-mediated DNA ligation by 1,2-naphthoquinone. DNA cleavage/ligation equilibria were established in the absence (hTIIα, black) or presence of 50 μM 1,2-naphthoquinone (1,2-NQ, maroon) and ligation was initiated by transferring assays from 37 °C to 0 °C. Results with 100 μM etoposide (blue) are shown for comparison. Double-stranded DNA cleavage levels prior to the induction of ligation were set to 100%. Error bars represent SDs for at least three independent experiments.

As further evidence that 1,2-naphthoquinone is a covalent poison of topoisomerase IIα, incubation of 50 μM compound with topoisomerase IIα prior to the addition of DNA resulted in a rapid loss of enzyme activity (t_1/2 ~80 s) (Figure 9). This exposure completely blocked the ability of the enzyme to cleave DNA within 5 min. In a control experiment that lacked the quinone, topoisomerase IIα maintained its DNA cleavage activity when incubated in the absence of DNA over the same 5 min period (Figure 9, inset).

Figure 9.

1,2-Naphthoquinone inactivates human topoisomerase IIα when incubated with the enzyme prior to the addition of DNA. Double-stranded DNA scission was monitored in the presence of 50 μM 1,2-naphthoquinone (maroon). Times indicate the length of incubation of the naphthoquinone with topoisomerase IIα prior to the addition of DNA. DNA cleavage levels were calculated relative to those induced when 1,2-naphthoquinone and the enzyme were not incubated prior to reaction initiation. The inset shows a control experiment (hTII⍺, open black bars) in which the enzyme (in the absence of compound) was incubated for 5 min at 37 °C prior to the addition of DNA. Error bars represent SDs for at least three independent experiments.

Finally, the ability of 1,2-naphthoquinone to induce DNA scission was severely impeded when the catalytic core of human topoisomerase IIα (which lacks both its N- and C-terminal domains) was used in place of the full-length enzyme (Figure 10). Whereas etoposide was still able to induce DNA cleavage with the catalytic core (albeit at lower levels than with the wild-type enzyme), virtually no DNA cleavage was seen in the presence of the quinone. Taken together, the above experiments provide strong evidence that 1,2-naphthoquinone is a covalent poison of human topoisomerase IIα.

Figure 10.

1,2-Naphthoquinone induces minimal DNA cleavage mediated by the catalytic core of human topoisomerase IIα. The graph compares double-stranded DNA cleavage levels for wild-type (WT) topoisomerase IIα and the catalytic core (CC) of the enzyme (containing residues 431–1193) in the absence (hTII⍺, open black bars) or presence of 50 μM 1,2-naphthoquinone (1,2-NQ, maroon bars). Results with 100 μM etoposide (blue bars) are shown for comparison. Error bars represent SDs for at least three independent experiments.

1,2-Naphthoquinone Displays Characteristics of a Covalent Poison Against Human Topoisomerase IIβ.

As observed with topoisomerase IIα (see Figure 7), DTT abolished the ability of 1,2-naphthoquinone to induce topoisomerase IIβ-mediated DNA scission when incubated with the quinone prior to cleavage assays but had no effect on the activity of the compound when added after cleavage complexes were established (Figure 11). This result establishes that 1,2-naphthoquinone can act as a covalent poison of topoisomerase IIβ. However, further experiments suggest that the compound displays mixed activity against the β isoform. Although DTT, which should adduct the quinone, undercut the activity of the compound, ascorbic acid, which should reduce and maintain the compound in its hydroxyl form, had virtually no effect on DNA cleavage. Indeed, levels of DNA scission induced by 100 μM 1,2-naphthoquinone in the presence of 2 mM ascorbic acid were ~90% of those observed in the absence of reducing agent. This result suggests that the hydroxyl form of 1,2-naphthoquinone potentially acts as an interfacial poison of topoisomerase IIβ. Consistent with this hypothesis, 1,2-dihydroxynaphthalene displayed high activity against the β isoform and exhibited responses to the sulfhydryl and reducing agents that were similar to those seen with the quinone.

Figure 11.

Effects of sulfhydryl and reducing agents on topoisomerase IIβ-mediated double-stranded DNA cleavage induced by 1,2-naphthoquinone. Topoisomerase IIβ-DNA cleavage reactions were performed in the absence of sulfhydryl/reducing agents (open bars), in the presence of 2 mM DTT (stippled bars) or 2 mM ascorbic acid (AA, diagonal bars) added prior to the start of the 6 min DNA cleavage reaction, or 2 mM DTT added after the 6 min cleavage reaction (filled bars). Results are shown for reactions that contained no compound (hTIIβ, black), 50 μM 1,2-naphthoquinone (1,2-NQ, maroon), 50 μM 1,2-dihydroxynaphthalene (1,2-Dihydroxy-NP, gold), or 25 μM 1,4-benzoquinone (BQ, teal). 1,4-Benzoquinone was included as a control for the effects of the sulfhydryl and reducing agents on a covalent topoisomerase II poison. Error bars represent SDs for at least three independent experiments.

DISCUSSION

1,2-Naphthoquinone is an environmental pollutant that has been linked to a variety of health issues in mammalian species and exhibits cytotoxic and genotoxic properties.^{2–7, 10} Many environmental and dietary quinones and polyphenols have been shown to act as topoisomerase II poisons.^{15–22, 24, 58} Several of these environmental quinones/polyphenols have been linked to the formation of human cancers; however, many of the dietary topoisomerase II poisons display chemopreventative properties and have been used extensively in traditional and indigenous medicines to treat a variety of conditions.^{1, 17–22, 58} Because of the rich history linking quinones and type II topoisomerases, the effects of 1,2-naphthoquinone on DNA cleavage mediated by human topoisomerase IIα and IIβ were examined.

Results indicate that 1,2-naphthoquinone is a topoisomerase II poison with preferential activity against the α isoform. Furthermore, although the quinone acts as a covalent poison against both enzyme isoforms, our experiments suggest that the dihydroxy form of 1,2-naphthoquinone (or potentially the parent quinone) may also act an interfacial poison of topoisomerase IIβ.

In general, topoisomerase II poisons that induce the most stable cleavage complexes are the most damaging to cells.⁴⁷ As determined by persistence assays, 1,2-naphthoquinone-induced DNA cleavage complexes, at least with topoisomerase IIα, are long-lived and show no degradation over a 2-hour time course. Taken together, the above findings suggest that the type II enzymes may be able to mediate some of the effects of the quinone on the human genome.

A previous study demonstrated that 1,2-naphthoquinone inhibited the ability of the isolated N-terminal domain of human topoisomerase IIα to hydrolyze ATP.³⁷ The relationships between the inhibition of ATPase activity and the enhancement of DNA cleavage by 1,2-naphthoquinone are not known and are likely to be complex. Two possibilities exist: a two-site model and a one-site model. In the two-site model, there are two distinct binding sites for the quinone, one that interferes with ATP interactions and another that stabilizes the closed form of the N-terminal clamp (leading to enhanced cleavage). In support of this model, etoposide, a well-characterized interfacial poison, appears to have two binding sites on topoisomerase II: one in the DNA cleavage/ligation active site that stabilizes cleavage complexes and the other in the ATPase domain.^{23, 54, 65–67} In the one-site model, the same adduction of the quinone that stabilizes the closed N-terminal domain also alters the ability of the enzyme to interact with ATP. It is likely that considerably more detailed structural and enzymological studies will be necessary to deconvolute the actions of 1,2-naphthoquinone in light of these two models.

In summary, 1,2-naphthoquinone acts as a covalent poison against topoisomerase IIα and IIβ. Our findings suggest that some of the genotoxicity and cytotoxicity associated with 1,2-naphthoquinone may be attributed to its ability to induce DNA double-stranded breaks generated by the human type II enzymes.