Functional Food and Health - American Chemical Society

Chapter 27

Downloaded by CALIFORNIA INST OF TECHNOLOGY on November 30, 2016 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch027

DNA Intercalation, Topoisomerase I Inhibition, and Oxidative Reactions of Polyphenols Michael R. Webb, Kyungmi Min, and Susan E. Ebeler Department of Viticulture and Enology, University of California, One Shields Avenue, Davis, CA 95616

Polyphenols occur naturally in many foods and have a range of biological effects. Although the mechanisms for these effects are not clear, inhibition of metabolic enzymes may be one possible mode of action in the cell. Using a gel electrophoresis method validated in our laboratory we evaluated the effects of several polyphenols on the DNA -maintenance enzyme topoisomerase IB (topo I). This enzyme (along with topoisomerase II) regulates supercoiling of chromosomal DNA and plays a pivotal role in chromosome replication, transcription, recombination, segregation, condensation and repair. In our studies, the flavones and flavonols had the greatest intercalating and poisoning activity. On the other hand, no evidence for DNA intercalation or topo I inhibition was observed with the anthocyan(id)ins we evaluated. Experimental conditions, including pH, ionic strength and the presence of reductants, free radical scavengers, and trace metals may all have an effect on the solution properties and reactivities of the polyphenols. These experimental conditions must be carefully considered when relating results of in vitro studies to potential biological effects

in vivo.

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© 2008 American Chemical Society Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


321 Polyphenols are widely distributed in fruits and vegetables and contribute bitterness, astringency, and color to foods and beverages. Numerous studies have shown that diets rich in fruits and vegetables are associated with a decreased risk of many degenerative diseases, including cardiovascular disease and cancer (1-6). The compounds responsible for the health protective effects are not fully known, however, dietary polyphenols have been widely studied for their antiviral, anti-inflammatory, antimutagenic and antiproliferative activity (712). In addition, the antioxidant activity of polyphenols, both in vitro and in vivo is thought to play at least a partial role in the observed biological effects (13-17). However, in general, the mechanisms by which polyphenols confer their health protective effects remain poorly understood. This is due to a number of factors including the dynamic and unstable nature of polyphenols in aqueous, aerobic solutions and difficulties in relating in vitro conditions to those that exist in vivo. In our laboratory we have previously observed that dietary polyphenols in red wine, including the flavonoid, catechin, can delay tumor onset in a transgenic mouse model of neurofibromatosis (9, 18). In our most recent efforts we have focused on understanding how dietary polyphenols can interact with DNA and DNA-maintenance enzymes to influence cellular processes (19-21). The enzymes topoisomerase IB (topo I) and topoisomerase II (topo II) regulate chromosomal DNA supercoiling and are important in chromosome replication, transcription, recombination, segregation, condensation and repair (22). DNA supercoiling or relaxation is regulated by controlling the breakage of the phosphodiester bond for one strand (topo I) or both strands (topo II) of duplex DNA. In our studies we have focused on the effects of polyphenols on the activity of the topo I enzyme. Topo I is thought to regulate relaxation/unwinding of DNA strands through a multi-step process. In the first step, topo I binds at the DNA reaction site. Next, strand scission occurs and a tyrosine residue of the enzyme is covalently attached to the 3' end of the broken strand. Finally, the DNA strand unwinds, the broken strand is re-ligated and the enzyme released. The topo I enzyme can be inhibited at various steps in the process. Catalytic (or relaxation) inhibitors bind to the enzyme active site and prevent reaction with the DNA. Another type of inhibition occurs when the inhibitor prevents religation and/or detachment of the enzyme from the DNA strand. This type of inhibition is referred to as "poisoning" or cleavable complex stabilization and results in DNA strand breakage (also termed "nicking"). There is much interest in the identification of various types of topo I inhibitors due to their potential to have antitumor effects. Finally, topoisomerases are useful for studying the intercalative binding of compounds to DNA. Because intercalators can prevent full relaxation by the topo enzyme, assays designed to study relaxation and supercoiling of DNA in the presence and absence of intercalating compounds can provide valuable information on how exogenous compounds interact with DNA to influence cellular processes. However, in many assays it can be difficult to fully distinguish the effects of inhibition, poisoning, and intercalation unless careful

Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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controls and reference standards are introduced. Therefore, our work in this area has been focused on developing a gel electrophoresis assay that can be used for simultaneous determination of inhibition, poisoning, and intercalation by flavonoids. We subsequently used the assay to compare activity of several structurally related flavonoids. In this chapter we present a brief summary and overview of our studies in this area (19-21).

Development of a Screening Assay for Determining DNA Intercalation and Topoisomerase I Inhibition and Poisoning The full screening assay has been previously described (19). The assay uses closed, circular plasmid DNA which can supercoil to different degrees, ranging from fully relaxed (Rel), to fully supercoiled (SC) forms (Figure 1). The degree of supercoiling defines distinct forms of the plasmid referred to as topoisomers (R ). The topoisomers are separated using gel electrophoresis and result in distinct bands as the various supercoiled forms migrate through the gel (more supercoiled forms travel further through the gel since their tightly wound and compact shape results in less resistance to migration). Intercalators insert into the DNA strand, alter the extent of supercoiling and change the topoisomer distribution (Figure 1). For example, addition of ethidium bromide (EB), a known intercalator, to fully relaxed plasmid results in increasing degrees of supercoiling as the concentration of EB is increased (Figure 2, Lanes 1-5). Similarly, intercalation by the flavonoid, luteolin is observed in Lanes 14-18 (Figure 2). Reference/controls showing fully relaxed plasmid without (Lane 8) and with (Lanes 9 and 10) addition of topo I are included in the assay for comparison. Additional references/controls using fully supercoiled plasmid without (Lane 11) and with topo I (Lane 12) are also shown. In the absence of topo I, no unwinding of the supercoiled plasmid, which forms as a result of luteolin intercalation, is observed (Lane 13). To observe poisoning activity (which results in formation of 'nicked' DNA strands), the covalently closed, relaxed plasmid must be separated from the nicked form (Nik). This is done by performing a second electrophoresis step. Following the first electrophoresis (to distinguish intercalation and catalytic inhibition activity), the gel is incubated with ethidium bromide (EB), which intercalates into the DNA in the gel causing the relaxed form to supercoil but leaving the nicked form relatively unchanged since, being nicked, it cannot supercoil. In this way the shape of the two forms becomes radically different and the nicked and relaxed forms will then separate from each other rapidly if the electrophoresis is continued. The poisoning activity of the naturally occurring alkaloid, camptothecin, can be observed in Figure 2 (Lanes 6 and 7) as an increase in the intensity of the nicked plasmid bands (Nik). Fully linear-ized plasmid (breakage of both DNA strands) is also observed as a distinct band n



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Negatively Supercoiled DNA

h

I

i

Figure 1. DNA topoisomerase assay showing intercalation of test compounds into plasmid DNA. Topoisomerase relaxes the supercoiled DNA and the test compound (portrayed here as a short, solid black line) inserts into the relaxed DNA). The topoisomerase is inactivated and the test comound is then either extracted and/or removed during gel electrophoresis. The plasmid rewinds to a degree determined by the amount of compound which was intercalated at equilibrium. The degree of supercoiling determines how fast the plasmid migrates in the gel. If no intercalation occurs, the plasmid remains relaxed.



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Figure 2. Intercalation activity by ethidium bromide and luteolin using relaxed plasmid DNA. Lanes 7-5. Ethidium bromide (O.I, 0.13, 0.15, 0.17, 0.2 jug/mL) + relaxed DNA; Lanes 6,7(duplicates): Camptothecin + relaxed DNA + 10X topo; Lane 8: Control-relaxed DNA, no topo; Lanes 9,10: Control-relaxed DNA + lOXtopo; Lane 11: Control-supercoiled DNA; Lane 12: Controlsupercoiled DNA + 1 OX topo; Lane 13: Luteolin + supercoiled DNA, no topo; Lanes 14-18: Luteolin (10, 25, 75, 150, 200 pM) + relaxed DNA + topo. Reproduced with permissionfromreference 19. Copyright 2003 Elsevier Inc.

between the nicked and relaxed forms, but no linear plasmid is observed in these samples. Finally, the ability of topo I to relax supercoiled DNA is the basis for the inhibition portion of the assay (see Figure 3, Lane 1 vs Lane 2 containing supercoiled plasmid without and with topo I, respectively). By comparing the amounts of relaxed and supercoiled forms, in the presence of test compounds, catalytic inhibition relaxation activity can be readily observed. If topo I is inhibited it will not be able to relax the supercoiled form and inhibition is indicated by the progressive increase in the amount of supercoiled plasmid still present as inhibitor concentration is increased (Lanes 9-13 show inhibition by luteolin; Lanes 14-17 show inhibition by morin). By comparing these results to those using fully relaxed plasmid (Lanes 4-8), the catalytic inhibition activity can be distinguished from intercalative activity. An additional comparison is also included showing that luteolin alone, when added to fully supercoiled DNA, does not influence the extent of relaxation if no topo I is added (Lane 3).

Effects of Flavonoids on Topo I Poisoning and Inhibition and DNA Intercalation Using the screening assay described above, 34 polyphenols, from a range of flavonoid classes (Figure 4; Table I) were surveyed for their ability to act as topo I poisons, inhibitors, and DNA intercalators (20). Of the compounds studied, the



325

Figure 3. Catalytic relaxation inhibition by luteolin and morin. Lane 1: Control supercoiled DNA; Lane 2: Control supercoiled DNA + 1 OX topo; Lane 3: Luteolin (150 juM) + supercoiled DNA; Lanes 4-8: Luteolin (10, 25, 75, 150, 200 pM) + relaxed DNA + 1 OX topo; Lanes 9-13: Luteolin (0, 25, 50 75, 150 juM) + supercoiled DNA + 2X topo; Lanes 14-17: Morin (50, 75, 100, 150 pM) + supercoiled DNA + 2Xtopo. Reproduced with permission from reference 19. Copyright 2003 Elsevier Inc.

Figure 4. General structure offlavonoid classes studied.


326 flavones and flavonols were the most potent poisons (Figure 4; Table I). These compounds generally showed a dose-response relationship with higher concentrations resulting in increased poisoning activity. The flavones and flavonols were also strong intercalators (Table I), however, there was no clear relationship between poisoning activity and intercalating activity (R = 0.213 when degree of intercalation was plotted against degree of poisoning for compounds showing both activities). There was no or only weak poisoning and intercalation activity for the flavanonols, flavan-3-ols, flavanones, isoflavones, chalcones, and stilbenes studied (Table I). These results indicate that DNA intercalation is not required for stabilization of the topo-I-DNA cleavable complex (i.e., poisoning), although intercalation may be associated with poisoning in some cases. Structure activity determinates of the intercalating activity indicated that the presence of a double bond between the 2 and 3 carbon of the C-ring (Figure 4) results in a sufficiently planar structure to allow intercalaction between adjacent bases of the DNA duplex. For example, no intercalating activity was observed for the flavanonol, taxifolin, which, lacks this double bond, while quercetin, the structural analog containing this double bond, is a strong intercalator (see also Table I). The presence of a free hydroxy at the 4' position of B-ring of the flavones and flavonols also appears necessary for intercalation (Figure 4; Table I). For example, chrysin, which lacks this hydroxyl is unable to intercalate. In this study, inhibition activity could not be accurately assessed due to flavonoid aggregation in solution. Based on these observations, we determined that solution conditions are critical for understanding chemical reactions of the flavonoids. Flavonoids (FH) will ionize (F"), oxidize, and aggregate (FH ) in solution according to the following relationships:


2

n

FH

(precip)

«

(FH)

n

«

•

FH

Functional Food and Health - American Chemical Society

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