Knapp J.S., Cabrera W.L. Metabolomics: Metabolites, Metabonomics, and Analytical Technologies

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Metabolomics 237

A useful approach to unraveling and understanding complex biological networks is to

decompose networks into basic functional and structural units [48]. Recent application of

convex analysis to metabolic networks leads to the development of network-based metabolic

pathway analysis and the decomposition of metabolic networks into metabolic extreme

pathways that are true functional units of metabolic systems [48]. Metabolic extreme

pathways are derived from limited knowledge of the metabolic networks, but provide an

integrated predictive description of metabolic networks [48]. Some interesting reports on

usefulness of pathway analysis are presented in Table 1.

D. Pathway Analysis Tool

There are also many pathway analysis tools at present. These tools are very useful.

Details of some important tools will be hereby presented.

1. Pathway Miner [53]

Pathway Miner catalogs genes based on their role in metabolic, cellular and regulatory

pathways [53]. A Fisher exact test is provided as an option to rank pathways [53]. The genes

are mapped onto pathways and gene product association networks are extracted for genes that

co-occur in pathways [53]. Pathway Miner is a freely available web accessible tool at

http://www.biorag.org/pathway.html [53].

2. WholePathwayScope [54]

WholePathwayScope (WPS) is for deriving biological insights from analysis of High

Throughput data [54]. WPS extracts gene lists with shared biological themes through color

cue templates [54]. WPS statistically evaluates global functional category enrichment of gene

lists and pathway-level pattern enrichment of data [54]. WPS incorporates well-known

biological pathways from KEGG (Kyoto Encyclopedia of Genes and Genomes) and Biocarta,

GO (Gene Ontology) terms as well as user-defined pathways or relevant gene clusters or

groups, and explores gene-term relationships within the derived gene-term association

networks (GTANs) [54]. WPS simultaneously compares multiple datasets within biological

contexts either as pathways or as association networks. WPS also integrates Genetic

Association Database and Partial MedGene Database for disease-association information. We

have used this program to analyze and compare microarray and proteomics datasets derived

from a variety of biological systems [54]. The tool is freely available at

http://www.abcc.ncifcrf.gov/wps/wps_index.php [54].+

3. ArrayxPath [55]

ArrayXPath (http://www.snubi.org/software/ArrayXPath/) is a web-based service for

mapping and visualizing microarray gene-expression data for integrated biological pathway

resources using Scalable Vector Graphics [55]. By integrating major bio-databases and

searching pathway resources, ArrayXPath automatically maps different types of identifiers

from microarray probes and pathway elements [55]. When one inputs gene-expression

clusters, ArrayXPath produces a list of the best matching pathways for each cluster [55].

Viroj Wiwanikit 238

4. Genome Expression Pathway Analysis Tool [56]

Genome Expression Pathway Analysis Tool (GEPAT) offers an analysis of gene

expression data under genomic, proteomic and metabolic context [56]. GEPAT offers various

statistical data analysis methods, as hierarchical, k-means and PCA clustering, a linear model

based t-test or chromosomal profile comparison [56]. GEPAT offers no linear work flow, but

allows the usage of any subset of probes and samples as a start for a new data analysis [56].

GEPAT relies on established data analysis packages, offers a modular approach for an easy

extension, and can be run on a computer grid to allow a large number of users [56]. It is freely

available under the LGPL open source license for academic and commercial users at http://

gepat.sourceforge.net [56].

Introduction to Metabolonics

Metabolonics is a science to study cellular metabolic activities. The aim of metabolonics

is to study the complete metabolic response of living things to genetic modifications or

environmental stimuli. The knowledge in this new “omic” science is still limited but carries

great hope in science and medicine.

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In: Metabolomics: Metabolites, Metabonomics… ISBN: 978-1-61668-006-0

Chapter 9

THE ROLE OF SPECIFIC ESTROGEN METABOLITES

IN THE INITIATION OF BREAST AND OTHER

UMAN CANCERS

Eleanor G. Rogan

and Ercole L. Cavalieri

Eppley Institute for Research in Cancer and Allied Diseases,

University of Nebraska Medical Center, 986805 Nebraska Medical Center,

Omaha, NE, USA

Keywords: Breast cancer, cancer initiation, catechol estrogen quinones, depurinating DNA

adducts, estrogens.

Introduction

Various types of evidence have implicated estrogens in the etiology of human breast

cancer [1-8]. They are generally thought to cause proliferation of breast epithelial cells

through estrogen receptor-mediated processes [4]. Rapidly proliferating cells are susceptible

to genetic errors during DNA replication, which, if uncorrected, can ultimately lead to

malignancy. While receptor-mediated processes may play an important role in the

development and growth of tumors, accumulating evidence suggests that specific oxidative

metabolites of estrogens, if formed, can be endogenous ultimate carcinogens that react with

DNA to cause the mutations leading to initiation of cancer [6-9]. Thus, estrogen metabolites,

specifically catechol estrogen-3,4-quinones, are hypothesized to be endogenous initiators of

breast, prostate and other human cancers.

Several lines of evidence, including metabolism and carcinogenicity studies by Liehr and

coworkers, led to the recognition that the 4-hydroxylated estrogens play a major role in the

genotoxic properties of estrogens [1-3]. We have hypothesized that the estrogens estrone (E

)

and estradiol (E

) initiate breast and other human cancers by reaction of their electrophilic

E-mail address: egrogan@unmc.edu. Tel: 402-559-4095, Fax: 402-559-8068. (Corresponding author)

Eleanor G. Rogan and Ercole L. Cavalieri 244

metabolites, catechol estrogen-3,4-quinones [E

)-3,4-Q], with DNA to form depurinating

adducts [5-8]. These adducts generate apurinic sites leading to mutations that may initiate

breast, prostate and other human cancers [6-9].

The estrogens, E

and E

, are obtained via aromatization of 4-androstene-3,17-dione and

testosterone, respectively, catalyzed by cytochrome P450(CYP)19, aromatase (Figure 1). E

and E

, which are biochemically interconvertible by the enzyme 17β-estradiol dehydrogenase,

are metabolized to the 2-catechol estrogens, 2-OHE

), and 4-OHE

), predominantly

catalyzed by the activating enzymes CYP1A1 [10] and 1B1 [10-13], respectively, in

extrahepatic tissues. The estrogens are also metabolized, to a lesser extent, to 16α-hydroxy

derivatives (not shown). The catechol estrogens are further easily oxidized to the catechol

estrogen quinones, E

)-2,3-Q and E

)-3,4-Q (Figure 1) by metal ions, peroxidases and

cytochrome P450. In general, the catechol estrogens are inactivated by conjugating reactions,

such as glucuronidation and sulfation. A common pathway of inactivation in extrahepatic

tissues, however, occurs by O-methylation catalyzed by the ubiquitous catechol-O-

methyltransferase (COMT) [14]. If formation of E

or E

is excessive, due to overexpression

of aromatase and/or the presence of excess sulfatase that converts the stored E

sulfate to E

increased formation of catechol estrogens is expected. In particular, the presence and/or

induction of CYP1B1 and other 4-hydroxylases could render the 4-OHE

), which are

usually minor metabolites, as the major metabolites [15-17]. Thus, conjugation of 4-

OHE

) via methylation in extrahepatic tissues might become insufficient, and competitive

catalytic oxidation of 4-OHE

) to E

)-3,4-Q could occur. (Figure 1)

Protection at the quinone level can occur by conjugation of E

)-Q with glutathione

(GSH, Figure 1). A second inactivating process for E

)-Q is their reduction to catechol

estrogens by quinone reductase. If these two inactivating processes are not effective, E

)-Q

may react with DNA to form stable and depurinating adducts [5-8,18-20]. Imbalances in

estrogen homeostasis [17,20], that is the equilibrium between activating and protective

enzymes with the scope of avoiding formation of catechol estrogen semiquinones and

quinones, could lead to initiation of cancer by estrogens.

Catechol Estrogen Quinones as Mutagens Initiating Breast,

Prostate and Other Human Cancers

Experiments on estrogen metabolism [17,19-22], formation of DNA adducts [5-8],

carcinogenicity [23-25], and mutagenicity [9,26,27] provide a basis for the hypothesis that

reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with

DNA can generate the critical mutations initiating breast, prostate and other cancers [7-9,28].

Imbalance in Estrogen Homeostasis

Estrogen metabolism involves a balance between activating and deactivating (protective)

pathways. There are several factors that can unbalance estrogen homeostasis, that is, the

equilibrium between activating and deactivating pathways, to limit formation and/or reaction

of the endogenous carcinogenic E

)-Q with DNA. The first imbalancing factor could be

excessive synthesis of E

by high expression of aromatase (CYP19) in target tissues [29-31]

The Role of Specific Estrogen Metabolites… 245

and/or the presence of sulfatase that excessively converts stored E

sulfate to E

[32,33]. A

striking result of in situ production of E

in human breast tissue is the similar levels of E

breast tissue in pre-menopausal and post-menopausal women, even though plasma levels are

10-50 fold higher in pre-menopausal than post-menopausal women [34]. Both aromatase and

sulfatase contribute to in situ estrogen production [32,33].

A second critical factor leading to imbalances in estrogen homeostasis might be high

levels of 4-OHE

) due to high expression of CYP1B1, which metabolizes E

predominantly to form 4-OHE

[10,12,35]. This could result in relatively large amounts of 4-

OHE

) that, in turn, can lead to more extensive oxidation to the carcinogenic E

)-3,4-Q.

A third factor could be a lack or a low level of activity of the protective COMT enzyme. If

this enzyme is insufficient, 4-OHE

) will not be effectively methylated in extrahepatic

tissues, but will be oxidized to the ultimate carcinogenic metabolites E

)-3,4-Q. A fourth

factor could be a low level of GSH and/or low levels of quinone oxidoreductase and/or CYP

reductase, which could leave available higher levels of E

)-3,4-Q that may react with

DNA.

Imbalances in estrogen homeostasis have been observed in laboratory animals and in

breast tissue from women with breast cancer:

The Kidney of Syrian Golden Hamsters

The hamster provides an excellent model for studying estrogen homeostasis because

implantation of E

or E

in male Syrian golden hamsters induces 100% of renal carcinomas,

but does not induce liver tumors [36]. Therefore, comparison of the profile of estrogen

metabolites, conjugates and DNA adducts in the two organs, after treatment of hamsters with

, should provide information on the relative imbalance in estrogen homeostasis in the two

tissues [20]. In the liver, more O-methylation of 2-OHE

) was observed, whereas more

formation of E

)-Q was detected in the kidney. These results suggest greater oxidation of

catechol estrogens to E

)-Q and less protective methylation of 2-OHE

) in the kidney.

When normal levels of GSH were depleted before hamsters were treated with E

, very low

levels of catechol estrogens and methoxy catechol estrogens were observed in the kidney

compared to the liver, suggesting little protective reduction of E

)-Q to catechol estrogens

in the kidney. More importantly, the 4-OHE

)-1-N7Gua depurinating adduct arising from

reaction of E

)-3,4-Q with DNA was detected in the kidney, but not in the liver [20].

These results suggest that tumor initiation in the kidney occurs because of poor

methylation of catechol estrogens, rendering more likely competitive oxidation of catechol

estrogens to E

)-Q, as well as poor quinone reductase activity to remove the E

)-Q.

These two effects produce a large amount of E

)-Q, which can react with the nucleophilic

groups of DNA.

The Mammary Gland of ERKO/Wnt-1 Mice

Mammary tumors develop in female estrogen receptor-α knock-out (ERKO)/Wnt-1 mice

despite their lack of functional estrogen receptor-α [37]. Extracts of hyperplastic mammary

tissue and mammary tumors from these mice were analyzed by HPLC interfaced with an

Eleanor G. Rogan and Ercole L. Cavalieri 246

electrochemical detector [21]. Picomole amounts of the 4-catechol estrogens were detected,

but their methoxy conjugates were not. Neither the 2-catechol estrogens nor 2-methoxy

catechol estrogens were detected. 4-OHE

)-GSH conjugates or their hydrolytic products

(conjugates of cysteine and N-acetylcysteine) were detected in picomole amounts in both

tumors and hyperplastic mammary tissue, demonstrating the formation of E

)-3,4-Q.

These preliminary findings indicate that estrogen homeostasis is unbalanced in the mammary

tissue, in that the normally minor 4-catechol estrogen metabolites were detected in the

mammary tissue, but not the normally predominant 2-catechol estrogens. Furthermore,

methylation of catechol estrogens was not detected, whereas formation of 4-OHE

)-GSH

conjugates was. These results are consistent with the hypothesis that mammary tumor

development is primarily initiated by metabolism of estrogens to E

)-3,4-Q, which may

react with DNA to induce oncogenic mutations.

The Prostate of Noble Rats

Estrogen metabolites and conjugates were analyzed in the ventral and anterior lobes of

the rat prostate, which are not susceptible to estrogen-induced carcinogenesis, and in the

susceptible dorsolateral and periurethral prostate of rats treated with 4-OHE

or E

-3,4-Q

[22]. The analyses revealed that the areas of the prostate susceptible to induction of

carcinomas have less protection by COMT, quinone reductase and GSH, thereby favoring

reaction of E

)-3,4-Q with DNA.

Figure 1. Formation, metabolism, conjugation and DNA adducts of estrogens.