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Supplement: Promises and Perils of Lycopene/Tomato Supplementation and Cancer Prevention

How Do Intermediate Endpoint Markers Respond to Lycopene in Men with Prostate Cancer or Benign Prostate Hyperplasia?^1,2

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, Chicago, IL 60612

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

KEY WORDS: • lycopene • prostate cancer • benign prostate hyperplasia • humans

	EXPANDED ABSTRACT

TOP EXPANDED ABSTRACT METHODS PRELIMINARY RESULTS LITERATURE CITED

 
Epidemiological studies indicate that a tomato-rich diet is associated with a lower risk of prostate cancer (1). In a study conducted in a cohort of 14,000 Seventh Day Adventist men, the consumption of tomato products (also beans, lentils, and peas) was associated with a lower risk of prostate cancer (2). In addition to retrospective epidemiological studies, several dietary prospective studies have evaluated the relation between consumption of tomatoes and risk of prostate cancer (3). Among the possible tomato constituents, lycopene has been suggested to be responsible for the beneficial effect of tomatoes and tomato-based products in the prevention of prostate cancer (4).

We are carrying out a randomized, placebo-controlled, double-blind clinical investigation (a phase II clinical trial) exploring the effects of lycopene in 120 men with prostate cancer and benign prostate hyperplasia (BPH).⁴ The hypothesis to be tested is that lycopene prevents oxidation in the prostate and therefore might be effective as a prostate cancer chemoprevention agent. Therefore, DNA oxidation and lipid peroxidation are intermediate endpoints that might be measured in a short-term clinical trial of lycopene and prostate cancer chemoprevention. Additional questions that will be addressed include the following: Does lycopene prevent the formation of multiple DNA oxidation products or only certain products in vivo? Does lycopene prevent lipid peroxidation in vivo? Is lycopene a pro-oxidant instead of an anti-oxidant in vivo? Can in vitro experiments predict in vivo effects of lycopene? And finally, do plasma lycopene concentrations reflect prostate levels?

The specific aims of this study are to determine the blood and prostate tissue levels of lycopene in men receiving daily lycopene supplementation and to investigate the correlation between lycopene concentration and measures of oxidative stress such as DNA oxidation and lipid peroxidation. The potential for lycopene supplementation to reduce serum prostate-specific antigen (PSA) and the percentage of free PSA in men with prostate cancer or BPH is also being examined, because a preliminary whole-foods arm of the study suggested that lycopene might reduce total PSA in the blood of men with prostate cancer (5). To complement this clinical trial, in vitro studies are being carried out using human prostate cancer and BPH cell lines treated with lycopene.

	METHODS

TOP EXPANDED ABSTRACT METHODS PRELIMINARY RESULTS LITERATURE CITED

 
Men with elevated PSA levels (>4 µg/L), who were scheduled for prostate biopsy for the diagnosis of either BPH or prostate cancer, were recruited for this lycopene intervention trial. After randomization, each subject received 30 mg/d of lycopene or placebo for 21 d prior to prostate biopsy (Fig. 1). Dietary intakes of carotenoids (including lycopene), vitamin E, and other nutrients at the start of the intervention and during the study were assessed by five 24-h dietary recalls. Blood samples were obtained immediately before and at the end of the intervention. Plasma was prepared from the blood specimens and stored at –80°C until analysis. Extra prostate needle biopsy samples were collected by a pathologist on d 21 of the intervention for chemical analysis in addition to the biopsies for diagnosis of BPH or prostate cancer.

View larger version (30K): [in this window] [in a new window]   FIGURE 1 Design of the placebo-controlled double-blind phase II study of the effects of lycopene on intermediate endpoint markers in men with prostate cancer or BPH. A preliminary whole-foods intervention fifth arm of the study was carried out previously (5) using tomato sauce containing 30 mg lycopene/d.

Essential to the success of these clinical and in vitro studies are highly sensitive and selective assays that were developed specifically for these investigations. These included assays for the quantitative analysis of lycopene, DNA oxidation products, and lipid peroxidation products, as well as all incorporated HPLC-tandem MS (LC-MS-MS). The first assay was developed for the measurement of multiple DNA oxidation products, including 8-oxo-deoxyguanosine (8-oxo-dG), 8-oxo-deoxyadenosine (8-oxo-dA), thymidine glycol, and 5-hydroxymethyluridine (HMDU) (7). Sample preparation for the analysis of these oxidized deoxynucleosides included mild and rapid enzymatic hydrolysis of DNA followed by ultrafiltration to remove the enzymes. No derivatization was required. Stable isotopically labeled [¹⁵N and ¹³C] deoxynucleosides were synthesized for use as surrogate standards.

Next, a new LC-MS-MS atmospheric pressure chemical ionization (APCI) assay was developed for the quantitative analysis of the lipid peroxidation product malondialdehyde. This assay is based on the popular HPLC-UV absorbance TBARS assay, in which 1 equivalent of malondialdehyde reacts with 2 molecules of thiobarbituric acid. In our approach, 1,3-diethyl-2-thiobarbituric acid is reacted with malondialdehyde and then detected using LC-MS-MS. An important advantage of using MS-MS over UV or fluorescence detection is the elimination of potential interference from a number of substances, including proteins, sucrose, and urea, all of which might react with 1,3-diethyl-2-thiobarbituric acid and produce signals unrelated to lipid peroxidation.

Finally, a new LC-MS-MS assay using negative-ion APCI was developed for the measurement of lycopene in plasma and prostate tissue needle biopsies (8). After precipitation of proteins from plasma, hexane extraction was carried out immediately prior to analysis using LC-MS-MS. Tissue samples were homogenized and saponified prior to extraction. Echinenone was used as an internal standard to correct for the losses during sample preparation. Lycopene cis- and trans- isomers were also measured using a modification of this assay (9).

	PRELIMINARY RESULTS

TOP EXPANDED ABSTRACT METHODS PRELIMINARY RESULTS LITERATURE CITED

 
One of the challenges of measuring lycopene in human plasma and tissues is distinguishing it from potentially interfering substances even when chromatographic separation is used. Mass selective detection of the molecular ion of lycopene at m/z 536 helps to distinguish it from most other coeluting substances. However, there is still potential interference from the isomeric dietary carotenoids - and ß-carotene. Our solution to the selective detection of lycopene was to use HPLC with tandem MS and selected reaction monitoring (Fig. 2). First, the negative molecular ion of lycopene at m/z 536 was formed using APCI, selected during the first stage of mass spectrometry, and then fragmented using collision-induced dissociation to form an abundant and unique fragment ion of m/z 467. Finally, the fragment ion of m/z 467, formed by elimination of a terminal isoprene group, was selected and detected using a second stage of mass spectrometry. Because - and ß-carotene have terminal rings instead of an isoprene group, they cannot form this fragment ion of m/z 467. As a result, lycopene could be detected selectively in the presence of - and ß-carotene using LC-MS-MS (8). Furthermore, this assay was highly selective and enabled us to measure lycopene levels in prostate biopsies weighing only 2–6 mg (wet wt). The limit of quantitation of this assay, defined as signal to noise 10:1, was 23 fmol lycopene.

View larger version (14K): [in this window] [in a new window]   FIGURE 2 Negative-ion APCI product ion tandem mass spectrum of the lycopene molecular ion radical of m/z 536.43. Collision-induced dissociation was used with argon collision gas at 30 eV in a hybrid quadrupole time-of-flight MS. Note the abundant product ion of m/z 467 corresponding to the elimination of an isoprene group. Because isomeric - and ß-carotene do not fragment via this pathway, LC-MS-MS was used to detect lycopene selectivity based on selected reaction monitoring of the fragmentation pathway m/z 536 to 467.

	ACKNOWLEDGMENTS

 
Marlos Viana is the biostatician for this study, the clinical study manager is Richard Morrissy, and the study dietitian is Phyllis Bowen. The principal investigator thanks the graduate students, postdoctoral fellows, and staff who contributed to the analytical methods development, the cell culture studies, and the analysis of the clinical specimens, including C. Duncan, D. Zhu, L. Yuan, N. Pajkovic, H. Xiong, X. Xu, Y. Wang, C. Gu, Y. Hua, L. Chen, M. Stacewicz-Sapuntzakis, and S. Wainhaus.

	FOOTNOTES

 
¹ Presented as part of the conference "Promises and Perils of Lycopene/Tomato Supplementation and Cancer Prevention," held February 17–18, 2005, in Bethesda, Maryland. This conference was sponsored by the Division of Cancer Prevention (DCP), Division of Cancer Epidemiology and Genetics (DCEG), Center for Cancer Research (CCR), National Cancer Institute, National Institutes of Health (NIH), Department of Health and Human Services (DHHS); Office of Dietary Supplements (ODS), NIH, DHHS; and the Agricultural Research Services (ARS), United States Department of Agriculture (USDA). Guest editors for the supplement publication were Cindy D. Davis, National Cancer Institute, NIH; Johanna Dwyer, Office of Dietary Supplements, NIH; and Beverly A. Clevidence, Agriculture Research Service, USDA.

² Supported by Grants R01 CA70771 and R01 CA101052. Support for the whole foods intervention fifth arm of the study was provided in part by Hunt Wesson. The clinical studies were carried out in collaboration with the General Clinical Research Center of the University of Illinois Medical Center, supported by NIH Grant M01 RR13987.

⁴ Abbreviations used: 8-oxo-dA, 8-oxo-deoxyadenosine; 8-oxo-dG, 8-oxo-deoxyguanosine; APCI, atmospheric pressure chemical ionization; BPH, benign prostate hyperplasia; HMDU, 5-hydroxymethyluridine; PSA, prostate-specific antigen.

	LITERATURE CITED

TOP EXPANDED ABSTRACT METHODS PRELIMINARY RESULTS LITERATURE CITED

 

1. Giovannucci, E. (1999) Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J. Natl. Cancer Inst. 91:317-331.[Abstract/Free Full Text]

2. Mills, P. K., Beeson, W. L., Phillips, R. L. & Fraser, G. E. (1989) Cohort study of diet, lifestyle, and prostate cancer in Adventist men. Cancer 64:598-604.[Medline]

3. Giovannucci, E. (2002) A review of epidemiologic studies of tomatoes, lycopene, and prostate cancer. Exp. Biol. Med. (Maywood) 227:852-859.[Abstract/Free Full Text]

4. Fleshner, N. E. & Klotz, L. H. (1998) Diet, androgens, oxidative stress and prostate cancer susceptibility. Cancer Metast. Rev. 17:325-330.[Medline]

5. Chen, L., Stacewicz-Sapuntzakis, M., Duncan, C., Sharifi, R., Ghosh, L., van Breemen, R. B., Ashton, D. & Bowen, P. E. (2001) Oxidative DNA damage in prostate cancer patients consuming tomato sauce-based entrees as a whole-food intervention. J. Natl. Cancer Inst. 93:1872-1879.[Abstract/Free Full Text]

6. Xu, X., Wang, Y., Constantinou, A. I., Stacewicz-Sapuntzakis, M., Bowen, P. E. & van Breemen, R. B. (1999) Solubilization and stabilization of carotenoids using micelles: delivery of lycopene to cells in culture. Lipids 34:1031-1036.[Medline]

7. Hua, Y., Wainhaus, S. B., Yan, Y., Shen, L., Xiong, Y., Xu, X., Zhang, F., Bolton, J. L. & van Breemen, R. B. (2001) Comparison of negative and positive ion electrospray tandem mass spectrometry for the liquid chromatography tandem mass spectrometry analysis of oxidized deoxynucleosides. J. Am. Soc. Mass Spectrom. 12:80-87.[Medline]

8. Fang, L., Pajkovic, N., Wang, Y., Gu, C. & van Breemen, R. B. (2003) Quantitative analysis of lycopene isomers in human plasma using high performance liquid chromatography-tandem mass spectrometry. Anal. Chem. 75:812-817.[Medline]

9. van Breemen, R. B., Xu, X., Viana, M. A., Chen, L., Stacewicz-Sapuntzakis, M., Duncan, C., Bowen, P. E. & Sharifi, R. (2002) Liquid chromatography-mass spectrometry of cis- and all-trans-lycopene in human serum and prostate tissue after dietary supplementation with tomato sauce. J. Agric. Food Chem. 50:2214-2219.[Medline]

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