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Supplement: International Research Conference on Food, Nutrition, and Cancer

Polyphenols, Inflammatory Response, and Cancer Prevention: Chlorination of Isoflavones by Human Neutrophils^1,2

Tracy D'Alessandro^*, Jeevan Prasain^*,, M. R. Benton, Nigel Botting, Ray Moore, Victor Darley-Usmar, Rakesh Patel^, and Stephen Barnes^*,**,,,3

Departments of ^* Pharmacology & Toxicology and Pathology, ^** Center for Nutrient-Gene Interaction in Cancer Prevention, Comprehensive Cancer Center Mass Spectrometry Shared Facility, and Purdue-UAB Botanicals Center for Age-Related Disease, University of Alabama at Birmingham, Birmingham, AL 35294; and Department of Chemistry, University of St. Andrews, Fife, UK

³ To whom correspondence should be addressed. E-mail: sbarnes{at}uab.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
An important aspect of the risk of cancer is the involvement of the inflammatory response. Currently, antiinflammatory agents are used in chemopreventive strategies. For example, aspirin is recommended for the prevention of colon cancer as well as breast and other cancers. The inflammatory response involves the production of cytokines and proinflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONO₂^-) produced by neutrophils and macrophages, respectively. These oxidants react with phenolic tyrosine residues on proteins to form chloro- and nitrotyrosine. Diets rich in polyphenols (green tea catechins, soy isoflavones) have also been shown to be chemopreventive. The aromatic nature of polyphenols makes them potential targets of HOCl and ONO₂^-. These reactions may create novel pharmacophores at the site of inflammation. Previous studies in the neutrophil-like cell line, differentiated HL-60 cells, demonstrated the formation of chlorinated and nitrated isoflavones. In this study we have examined whether similar reactions occur in freshly isolated human neutrophils. After induction of a respiratory burst with a phorbol ester, isoflavones and their metabolites were identified by liquid chromatography-tandem mass spectrometry and then quantitatively measured by LC-mass spectrometry using multiple-reaction ion monitoring. The data obtained indicate that both chlorinated and nitrated genistein are formed by human neutrophils. The extent of chlorination of genistein was markedly increased by the phorbol ester whereas the low level of nitration of genistein was constitutive and unaffected. These data imply a potential role for modified forms of genistein that would be produced in the inflammatory environment in and around a tumor.

KEY WORDS: • polyphenols • genistein • neutrophils • chlorination • nitration

The importance of inflammation as a component of the development of cancer has become increasingly clear. Recruitment of inflammatory cells into the tissue sites of tumors leads to pro- and antitumor growth effects. The activation status as well as environmental factors influence the inflammatory cell's diverse functions, such as angiogenesis, neoplastic cell mitogenesis, antigen presentation, matrix degradation, and cytotoxicity (1). Indeed, the medical profession has become so convinced of the role of antiinflammatory agents that a daily dose (one tablet) of aspirin is strongly recommended as a way to prevent colon cancer as well as heart disease.

The immune response is a complex system of soluble agents and cells whose principal purpose is to protect the human body from infectious agents, chemical and physical damage, and tumors (2). A feature of aging is that the immune response declines, thereby increasing the risk of infection as well as potentially allowing tumor cells to escape immune surveillance. Inflammatory cells have specialized biochemical pathways that can locally generate reactive compounds that are used to attack foreign cells. Typically, inflammatory cells convert oxidants such as singlet oxygen, superoxide anion, and hydrogen peroxide into more highly reactive compounds, such as hydroxyl radicals (OH^·), hypochlorous acid (HOCl)⁴, hypobromous acid (HOBr), and peroxynitrite (ONO₂^-) (2). These compounds react with DNA, lipids, and proteins, resulting in the disruption of the target's structure as well as its function. Although these compounds are reactive, their copious amounts (locally in the millimolar range) result in their diffusion away from the inflammatory focus into the local normal environment. This causes collateral damage to otherwise uninvolved cells and may contribute to lesion formation, thereby creating a separate disease process. Investigators have proposed that this is a significant event in the evolution of atherosclerosis.

Antiinflammatory agents fall into two broad categories—those that inhibit the biosynthesis of prostanoids [nonsteroidal antiinflammatory drugs (NSAID) such as aspirin] and those that alter the production and the action of proinflammatory oxidants (antioxidants). The activity of aspirin, although now made by chemical synthesis, was originally discovered 2000 years ago from a natural plant source and is a cyclooxygenase (COX) inhibitor. Other pharmacologically derived COX inhibitors to prevent colon cancer have been developed because of the relatively low efficacy of NSAID (50% reduction in incidence and mortality) and their wide spectrum of side effects, some of which are fatal (3). Examples of these new COX inhibitors are the NO-NSAID, which are traditional NSAID linked to an NO-releasing group via a chemical spacer. The addition of the NO-releasing group is believed to reduce gastric toxicity seen with traditional NSAID (3).

Many epidemiologic studies and preclinical laboratory experiments have suggested roles of dietary antioxidants in the prevention of cancer. Fruits and vegetables contain large amounts of these antioxidants, and higher intakes of fruits and vegetables are the subject of federal dietary recommendations. However, the reluctance of the public to make a change in diet has led to an industry-based focus on dietary supplements to deliver these compounds, where either foods and edible plants enriched in these compounds are added to the regular diet or the compounds are extracted (or synthesized as occurred for aspirin) to create a pill form. This latter step may have unforeseen consequences in that the original food may have had more than one bioactive compound or a compound-food matrix effect may exist.

Conventional thinking about antioxidants has been that they react with either free radicals or the chemically reactive species generated by inflammatory cells. This has caused concern for some investigators because it appears to imply that the normal function of the oxidant reactive species in terminating invading cells or viruses would be compromised. However, the antioxidants may have a role in preventing the collateral damage associated with the chemically reactive species. This may occur at the perimeter of the inflammatory cells, not in the termination zone in the phagosome. Of course, at very high doses of the antioxidants, a progressive decrease in tissue oxidants may occur and unwanted effects on immune function could result. This emphasizes the importance of using foods and not pills in the context of preventing chronic disease risk. Therapeutic drugs may have greater bioactivity but they are rarely used chronically because of their substantial side effects.

An alternative role for antioxidants may occur as a result of their reaction with the proinflammatory oxidants. The products should not be considered as being biologically neutral and may have properties of locally produced, and hence active, novel pharmacophores. In our research program at the University of Alabama at Birmingham, we are interested in the properties and mechanisms of action of several types of polyphenols that are present in foods and plants whose consumption is associated with lowered risk of breast and prostate cancer as well as several other chronic diseases. In this article we describe our investigations of the reactions between certain polyphenols and reactive chemical species (HOCl and ONO₂^-). We (4) previously showed that human leukemia HL-60 cells differentiated with dimethylsulfoxide (DMSO) (a renewable source and model of human neutrophils) and induced to have a respiratory burst rapidly convert isoflavones to chlorinated and nitrated products. In this study we investigate whether freshly isolated human neutrophils carry out similar reactions.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials

Isoflavones were obtained both from biological sources and chemical suppliers, as described elsewhere (5). Chlorinated isoflavones standards were chemically synthesized using selected chlorinated starting materials (6). The 3'-chlorinated genistein derivative was synthesized from commercially available 3-chloro-4-hydroxyphenylacetic acid. They were fully characterized by ¹H and ¹³C nuclear magnetic resonance spectroscopy and their purity was established by reverse-phase HPLC, using acetonitrile-water as solvent.

DMSO and sodium nitrite (NaNO₂) were purchased from Fisher (Fair Lawn, NJ). RPMI 1640, fetal bovine serum (FBS), and ACK Lysis Buffer were purchased from Mediatech, Inc. (Herdon, VA). Histopaque-1077, Histopaque-1119, and phorbol 12-myristate 13-actetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, MO). Krebs-Henseleit buffer (K-H; 118.0 mmol NaCl/L, 27.2 mmol NaHCO₃/L, 4.8 mmol KCl/L, 1.75 mmol CaCl₂/L, 1.0 mmol KH₂PO₄/L, 1.2 mmol MgSO₄/L, 11.1 mmol glucose/L, pH 7.4) was used for incubations with HL-60 cells and human polymorphonuclear cells (PMN).

Analysis of chlorinated genistein

HL-60 cell differentiation was induced with 1.3% (v/v) DMSO-supplemented 10% FBS/RPMI-1640 media for 7 d with media refreshment on d 4. Cell medium was exchanged with K-H buffer, and cells were suspended at 1 x 10⁶ cells/mL. Cells were activated with PMA (10 µmol/L) in the presence of genistein (10 µmol/L) and NaNO₂ (50 µmol/L) for 60 min at 37 °C. Each sample was treated with catalase (5 U/mL) to stop the respiratory burst by scavenging any remaining H₂O₂ and placed on ice for 10 min. The cells were centrifuged for 5 min at 800 x g at 4 °C and the supernatant was removed and extracted as follows: the cell supernatant (950 µL) was added to diethyl ether (2 mL). The samples were vortex mixed and centrifuged at 2000 x g, whereupon the ethereal top layer was removed. The same steps were repeated until a total volume of 6 mL of ether was added. Ether layers were combined and dried to evaporation under air. Before analysis by liquid chromatography-multiple reaction ion monitoring-mass spectrometry (LC-MRM-MS), 80% methanol (100 µL) was added to redissolve the dried residues.

Human PMN were isolated from freshly drawn human blood from volunteers using Histopaque according to manufacturer's instructions. This study was approved by the University of Alabama at Birmingham Institutional Review Board for Human Use. Contaminating red blood cells were lysed with ACK lysis buffer for 5 min at room temperature. The cells were centrifuged at 800 x g for 5 min at room temperature. The buffer was aspirated leaving the PMN pellet. The PMN were suspended in K-H at 1 x 10⁶ cells/mL. Genistein (10 µmol/L) was added to 1 mL of cell suspension. Cells were activated with PMA (10 µmol/L) and NaNO₂ (50 µmol/L) and incubated for 60 min at 37 °C. Each sample was treated with catalase (5 U/mL) and placed on ice for 10 min. The cells were centrifuged for 5 min at 800 x g at 4 °C and the supernatant was removed and extracted in the same manner as described above.

HPLC analysis of differentiated HL-60 reaction products

Analyses of differentiated HL-60 reaction products were performed on a HP1100 series HPLC module with diode array detector. Reaction products were separated by reversed-phase HPLC as previously described (7).

Mass spectrometry analysis of reaction products

Reaction mixtures were separated by HPLC using a 10 cm x 4.6 mm i.d., C-8 Aquapore reverse-phase column preequilibrated with 10 mmol/L ammonium acetate (NH₄OAc). The mobile phase composition was 6 min isocratically with 40% acetonitrile in 10 mmol/L NH₄OAc at 1 mL/min flow rate. The column eluant was passed into the Ionspray^TM ionization interface operating in the negative ion mode and passed into a PE-Sciex (Concord, Ontario, Canada) API III triple quadrupole mass spectrometer. The voltage on the Ionspray^TM interface needle was -4900 V and the orifice potential was set at -70 V. Negative ion mass spectra were recorded over an m/z range of 20–400. Selected [M-H]^- molecular ions were analyzed by collision-induced dissociation mass spectrometry with 100% argon as the collision gas, and the product ion mass spectra were recorded. To obtain quantitative data, specific parent ion–product ion combinations were used in LC-MRM-MS analysis (4). Daidzein was added as the internal standard for experiments with genistein. Data were analyzed using software provided by the manufacturer on Macintosh Quadra 950 and PowerPC 9500 computers (Apple Computers, Cupertino, CA). A series of samples prepared with several known concentrations of the varying genistein and a single concentration of the internal standard were analyzed to generate an area response-concentration curve. These typically gave correlation coefficients of 0.98 for a five-point curve.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Previously, when genistein was reacted with either HOCl or ONO₂^-, the products were analyzed by ¹H nuclear magnetic resonance spectroscopy to determine the positions of chlorination and nitration (7). Chlorination took place in positions 6, 8, and 3', whereas the nitrated product was found only in the 3' position (Fig. 1). HPLC-diode array analysis detected chlorinated and nitrated forms of genistein in differentiated HL-60 cell medium after the addition of genistein to culture medium in the presence or absence of PMA and NaNO₂ (Fig. 2). In the presence of genistein without PMA activation, chlorinated and nitrated genistein were not detected (Fig. 2A). However, in the presence of genistein with PMA activation, monochlorogenistein was detected (Fig. 2B) with peak enhancement upon the addition of NaNO₂ (Fig. 2C). Nitrated genistein was not detectable by HPLC analysis. At this time, it is not known whether genistein was nitrated under these conditions because of the decreased sensitivity of HPLC analysis compared with LC-MRM-MS analysis.

View larger version (12K): [in this window] [in a new window]   FIGURE 1  Structure of genistein and the sites of chlorination and nitration (modified from reference 6).

View larger version (19K): [in this window] [in a new window]   FIGURE 2  Detection of chlorinated derivatives of genistein using differentiated HL-60 cells. HPLC chromatograms of differentiated HL-60 cell media reaction products in the presence and absence of genistein (10 µmol/L), PMA (10 µmol/L), and NaNO2 (50 µmol/L). (A) Unreacted genistein was detected (retention peak at 9.174 min). (B, C) Unreacted genistein (retention peak at 9.176 min) and monochlorogenistein (retention peak at 10.398) were detected after PMA-induced stimulation in the absence or presence of NaNO2, respectively. Nitrated genistein was not detectable.

View larger version (27K): [in this window] [in a new window]   FIGURE 3  Detection of chlorinated and nitrated derivatives of genistein human polymorphonuclear cells (PMN) with and without NaNO2. Liquid chromatography-multiple reaction ion monitoring-mass spectrometry (LC-MRM-MS) ion chromatograms of human PMN media reaction products in the presence/absence genistein (10 µmol/L), PMA (10 µmol/L), and NaNO2 (50 µmol/L). (A) Unreacted genistein was detected (m/z 269/133, molecular and product ions). (B and C) Mono- and dichlorogenistein were detected after PMA-induced stimulation, respectively. (D) Nitrated genistein was detected in the presence of NaNO2 with or without PMA-induced stimulation.

We previously demonstrated that chlorination of isoflavones results in isomers with substitution at the 3', 6, and 8 positions (Fig. 1). Figure 4 shows the fragmentation patterns for both authentic compounds and those synthesized de novo. Figure 4 shows the MS-MS fragmentation patterns of cell extracts of genistein that had been incubated with PMA-activated PMN for 60 min. Panel A shows the MS/MS spectrum of monochlorogenistein extracted from activated PMN cell medium. The presence of the m/z 133 ion suggests that the chlorine resides in the A ring of genistein. Panel B shows the MS/MS spectrum of dichlorogenistein extracted from the same medium as the monochlorogenistein. The presence of chlorine in the molecule is also corroborated by the m/z 35 ion. A previous study in our laboratory gave a detailed description of the structural information obtained from the MS/MS spectrum of monochloro- and dichlorogenistein (8).

View larger version (14K): [in this window] [in a new window]   FIGURE 4  MS/MS fragmentation of mono- and dichlorogenistein. (A) Monochlorogenistein fragmented into the dominant m/z 133, 211, and 35 product ions. (B) Dichlorogenistein fragmented into the dominant m/z 255 and 209 product ions leaving a small m/z 35 product ion.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genistein also underwent some 3'-nitration of genistein. This was dependent on the addition of nitrite to the incubation medium. However, there was no enhancement of nitration as a result of stimulation with phorbol ester.

These data have important implications for cancer prevention because it has been suggested that tumors are wounds that will not heal (1). The presence of macrophages has been seen in carcinomas of the breast, cervix, and bladder and there have been conflicting reports for prostate, lung, and brain tumors (9). These macrophages are referred to as tumor-associated macrophages (TAM). TAM are recruited to tumor sites by the tumor cells through the release of chemokines monocyte chemotactic protein-1, macrophage colony stimulating factor, and vascular endothelial growth factor (9). It is the recruitment of TAM and their subsequent further production of chemokines and reactive oxygen and nitrogen species that create a heterogeneous microenvironment around the tumor. One study suggested that colorectal adenocarcinoma cell lines were capable of suppressing production of reactive oxygen intermediates by human phenotypes (10). However, the measurements were detecting superoxide production, which can be a measurement of superoxide or one of its metabolites, and did not clarify which reactive oxygen intermediates were being affected. Interestingly, genistein has been noted to decrease the production and release of vascular endothelial growth factor, which is a potent stimulator of angiogenesis (11). Further studies need to be performed looking into genistein's effect on tumor microenvironment. We have shown that genistein reacts with reactive species generated by human neutrophils to form chlorinated and nitrated products; therefore, genistein could be directly affecting the tumor microenvironment by altering its oxidant status. One possibility could be that there is an indirect effect of the native, chlorinated, or nitrated species on chemokine production and release and TAM recruitment. This would affect the tumor's ability to grow and spread because the antitumorigenic properties of TAM would no longer be suppressed.

	FOOTNOTES

 
¹ Presented as part of a symposium, "International Research Conference on Food, Nutrition, and Cancer," given by the American Institute for Cancer Research and the World Cancer Research Fund International in Washington, D.C., July 17–18, 2003. This conference was supported by Balchem Corporation; BASF Aktiengesellschaft; California Dried Plum Board; The Campbell Soup Company; Danisco USA, Inc.; Hill's Pet Nutrition, Inc.; IP-6 International, Inc.; Mead Johnson Nutritionals; Roche Vitamins, Inc.; Ross Products Division; Abbot Laboratories; and The Solae Company. Guest editors for this symposium were Helen A. Norman and Ritva R. Butrum.

² Support was from a National Institutes of Health grant to the Purdue-UAB Botanicals Center for Age-Related Disease from the NIH Office of Dietary Supplements and the National Center for Complementary and Alternative Medicine (P50 AT-00477). The mass spectrometer was purchased by funds from an NIH instrumentation grant (S10RR06487) and from the University of Alabama at Birmingham. Operation of the UAB Mass Spectrometry Shared Facility has been supported in part by a National Cancer Institute Core Research Support Grant (P30 CA13148-31) to the University of Alabama at Birmingham Comprehensive Cancer Center.

⁴ Abbreviations used: COX, cyclooxygenase; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; HOCl, hypochlorous acid; K-H, Krebs-Henseleit; LC-MRM-MS, liquid chromatography-multiple reaction ion monitoring-mass spectrometry; NSAID, nonsteroidal anti-inflammatory drugs; ONO₂^-, peroxynitrite; PMA, phorbol 12-myristate 13-actetate; PMN, polymorphonuclear cells; TAM, tumor-associated macrophages.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Leek, R. D. & Harris, A. L. J. (2002) Tumor-associated macrophages in breast cancer. J. Mammary Gland Biol. Neoplasia 7: 177–189.[Medline]

2. Stvrtinova, V., Jakubovsky, J. & Hulin, I. Inflammation and fever. Academic Electronic Press. Available at http://nic.sav.sk/logos/books/scientific/z.html (accessed June 23, 2003).

3. Rigas, B. & Williams, J. L. (2002) NO-releasing NSAIDs and colon cancer chemoprevention: a promising novel approach (Review). Int. J. Oncol. 20: 885–890.[Medline]

4. Boersma, B. J., D'Alessandro, T. L., Benton, M. R., Kirk, M., Wilson, L. S., Prasain, J., Botting, N. P., Barnes, S., Darley-Usmar, V. M. & Patel, R. P. (2003) Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties. Free Radic. Biol. Med., in press.

5. Peterson, G. & Barnes, S. (1991) Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem. Biophys. Res. Commun. 179: 661–667.[Medline]

6. Whalley, J. L., Oldfield, M. F. & Botting, N. P. (2000) Synthesis of [4-¹³C]-isoflavonoid phytoestrogens. Tetrahedron 56: 455–460.

7. Boersma, B. J., Patel, R. P., Kirk, M., Jackson, P. L., Muccio, D., Darley-Usmar, V. M. & Barnes, S. (1999) Chlorination and nitration of soy isoflavones. Arch. Biochem. Biophys. 368: 265–275.[Medline]

8. Prasain, J., Patel, R. P., Kirk, M., Wilson, L., Botting, N., Darley-Usmar, V. M. & Barnes, S. (2003) Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites. J. Mass Spectrom. 38: 764–771.[Medline]

9. Bingle, L., Brown, N. J. & Lewis, C. E. (2002) The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J. Pathol. 196: 254–265.[Medline]

10. Siegert, A., Denkert, C., Leclere, A. & Hauptmann, S. (1999) Suppression of the reactive oxygen intermediates production of human macrophages by colorectal adenocarcinoma cell lines. Immunology 98: 551–556.[Medline]

11. Shao, Z., Wu, J. & Shen, Z. (2000) Genistein exerts multiple suppressive effects on human breast carcinoma cells. Chinese J. Oncol. 22: 362–365.

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