Analysis of potential biomarkers of estrogen-initiated cancer in the urine of Syrian golden hamsters treated with 4-hydroxyestradiol

Rosa Todorovic, Prabu Devanesan, Sheila Higginbotham, John Zhao1,, Michael L. Gross1,, Eleanor G. Rogan and Ercole L. Cavalieri2,

Eppley Institute for Research in Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805,
1 Department of Chemistry, Washington University, One Brookings Drive, St Louis, MO 63130-4899, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Estrone (E1) and 17ß-estradiol (E2) are metabolized to catechol estrogens (CE), which may be oxidized to semiquinones and quinones (CE-Q). CE-Q can react with glutathione (GSH) and DNA, or be reduced to CE. In particular, CE-3,4-Q react with DNA to form depurinating adducts (N7Gua and N3Ade), which are cleaved from DNA to leave behind apurinic sites. We report the determination of 22 estrogen metabolites, conjugates and adducts in the urine of male Syrian golden hamsters treated with 4-hydroxyestradiol (4-OHE2). After initial purification, urine samples were analyzed by HPLC with multichannel electrochemical detection and by capillary HPLC/tandem mass spectrometry. 4-Hydroxyestrogen-2-cysteine [4-OHE1(E2)-2-Cys] and N-acetylcysteine [4-OHE1(E2)-2-NAcCys] conjugates, as well as the methoxy CE, were identified and quantified by HPLC, whereas the 4-OHE1(E2)-1-N7Gua depurinating adducts and 4-OHE1(E2)-2-SG conjugates could only be identified by the mass spectrometry method. Most of the administered 4-OHE2 was metabolically converted to 4-OHE1. Formation of thioether (GSH, Cys and NAcCys) conjugates and depurinating adducts [4-OHE1(E2)-1-N7Gua] indicates that oxidation of 4-CE to CE-3,4-Q and subsequent reaction with GSH and DNA, respectively, do occur. The major conjugates in the urine were 4-OHE1(E2)-2-NAcCys. The oxidative pathway of 4-OHE1(E2) accounted for approximately twice the level of products compared with those from the methylation pathway. The metabolites and methoxy CE were excreted predominantly (>90%) as glucuronides, whereas the thioether conjugates were not further conjugated. These results provide strong evidence that exposure to 4-OHE1(E2) leads to the formation of E1(E2)-3,4-Q and, subsequently, depurinating DNA adducts. This process is a putative tumor initiating event. The estrogen metabolites, conjugates and adducts can be used as biomarkers for detecting enzymatic oxidation of estrogens to reactive electrophilic metabolites and possible susceptibility to estrogen-induced cancer.

Abbreviations: CE, catechol estrogen(s); CE-Q, catechol estrogen quinone(s); COMT, catechol-O-methyltransferase; Cys, cysteine; E1, estrone; E2, 17ß-estradiol; ESI, electrospray ionization; GSH, glutathione; LC/MS, liquid chromatography/mass spectrometry; LC/MS/MS, liquid chromatography/tandem mass spectrometry; NAcCys, N-acetylcysteine; OHE, hydroxyestrogen(s); SG, glutathione moiety.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Estrogens are associated with carcinogenic events in both humans and animals (14). These hormones can play dual roles in the induction of cancer: generation of electrophilic species that can covalently bind to DNA (5,6) to induce oncogenic mutations (7) and stimulation of cell proliferation by receptor-mediated processes (811). Estrogens are mainly metabolized via two main pathways: 16{alpha}-hydroxylation and formation of catechol estrogens (CE) (1214). Epidemiological evidence in women indicates that CE formation may be a greater risk factor for breast cancer than high 16{alpha}-hydroxylation of estrogens (15). We attribute this to DNA damage by CE metabolites, which are in part oxidized to CE quinones (CE-Q) and bind to DNA (Figure 1Go). This hypothesis is also supported by three studies in which increased risk of breast cancer was associated with the low activity polymorphic allele of catechol-O-methyltransferase (COMT) (1618).



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Fig. 1. Scheme showing the formation of metabolites, conjugates and DNA adducts of estrogens.

 
Estradiol (E2) and estrone (E1) induce kidney tumors in male Syrian golden hamsters (1,2). Of the two catechol metabolites formed, 2-CE and 4-CE, relatively high levels of 4-CE occur in the kidney of hamsters, the site of tumor formation, compared with the liver and other organs in which tumors are not induced (19). In fact, in Syrian golden hamsters, 4-hydroxyestradiol (4-OHE2) induces renal tumors, whereas 2-OHE2 is not carcinogenic (20,21).

If CE are not detoxified by O-methylation catalyzed by COMT, they can be oxidized to reactive quinones by cytochrome P450s and peroxidases (Figure 1Go). CE-Q can be reduced to CE by quinone reductase and/or cytochrome P450 reductase (22,23 and Cavalieri,E.L., Devane-San,P., Bosland,M.C., Badawri,A.F. and Rogan,E.C., in preparation) or conjugated with glutathione (GSH). GSH conjugates of CE-Q (CE-SG) are catabolized via a three-step enzymic hydrolysis of the GSH tripeptide (24). First, the glutamyl moiety is released by {gamma}-glutamyl transpeptidase. Second, the resulting cysteinylglycine conjugate is hydrolyzed by cysteinylglycinase to yield the CE-cysteine (Cys) conjugate, which, in a final step, is acetylated to form the CE-N-acetylcysteine (NAcCys) conjugate.

CE-2,3-Q can also react with DNA to form stable adducts (25,26), which remain in DNA unless repaired, whereas CE-3,4-Q also react with DNA to form predominantly depurinating adducts (CE-1-N7Gua and CE-1-N3Ade) (5,6,27). (In this article we use the term `adduct' for products formed by reaction of CE-Q with DNA and the term `conjugate' for products formed with GSH and its derivatives.) These adducts are lost from DNA by cleavage of the glycosyl bond, leaving behind apurinic sites. GSH conjugates and depurinating DNA adducts have already been identified and quantified in the kidney of male Syrian golden hamsters treated with 4-OHE2 (28).

Based on evidence from our previous studies (5), we proposed that oxidation of 4-CE to CE-3,4-Q and their subsequent reaction with DNA, resulting in the formation of depurinating adducts, is a pathway that initiates cancer. Formation of the endogenous carcinogens CE-3,4-Q is due to the prominent role of the oxidative pathway for 4-CE to their quinones in competition with methylation of 4-CE by COMT. This imbalance can be achieved in male Syrian golden hamsters by implantation of E1, E2 or their 4-hydroxyestrogens (4-OHE), which induce tumors (1,2,20,21).

Because CE are both methylated and oxidized (28), our analytical method must identify and quantify estrogen metabolites, methoxy CE, CE-thioether conjugates (CE-SG, CE-Cys and CE-NAcCys) and CE-DNA depurinating adducts (CE-1-N7Gua) in the urine of male Syrian hamsters treated with 4-OHE2. The thioether conjugates and DNA adducts (Figure 2Go) are produced by enzymatic oxidation of 4-CE to CE-3,4-Q and subsequent reaction with GSH or DNA, respectively. The identity of the compounds produced by both pathways was determined by HPLC with a multichannel electrochemical detector, and then confirmed by mass spectrometry. Their levels in human urine could serve as potential biomarkers for susceptibility to estrogen-induced tumor initiation.




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Fig. 2. Structures of (A) E1, E2 and metabolites, and (B) CE conjugates, CE-Q conjugates and N7Gua adducts.

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
4-OHE1, 4-OHE2, 2-OHE1 and 2-OHE2 were synthesized according to Dwivedy et al. (25). The 4-CE-2-SG, 4-CE-2-Cys and 4-CE-2-NAcCys conjugates were synthesized as previously described (29). Methoxy derivatives of CE and 16{alpha}-OHE1 were purchased from Steraloids (Newport, RI). E1, E2, estriol (16{alpha}-OHE2), ascorbic acid and ß-glucuronidase (G1512), saccharic acid-1,4-lactone (S0375) and estrone sulfamate (E9645) were purchased from Sigma (St Louis, MO). Bond Elut Certify SPE cartridges were purchased from Varian (Palo Alto, CA).

Sample collection
Two male Syrian golden hamsters (6 weeks old, Eppley Colony) at each dose were intraperitoneally injected with 1, 2, 4, 8 or 12 µmol 4-OHE2/100 g body weight in 300 µl 9:1 (v/v) trioctanoin/DMSO. The experiment was repeated two additional times, the three sets of data were averaged and standard deviations were calculated. The hamsters were maintained individually for 24 h in metabolism cages. Water was provided ad libitum. To prevent food particles from contaminating the collected urine, the hamsters were removed to regular cages for 2 h each morning while food was provided. Urine was collected for 24 h into vials containing 2.5 ml 50 mM ammonium acetate, pH 3.5, and 4 mg/ml ascorbic acid, kept on dry ice in the dark. After collection, urine samples were stored at –80°C until use.

Solid phase extraction of metabolites, conjugates and adducts
For determination of metabolites, conjugates and adducts, one-third of the volume of the urine sample collected from each hamster was diluted 2-fold with 20 mM ammonium acetate, pH 3.8; the final pH of the sample was adjusted to between 3.5 and 3.8, and the concentration of ascorbic acid to 2 mg/ml. For determination of total (free + conjugated) metabolites, one-third of the volume of urine was diluted with 50 mM ammonium acetate, pH 5, to a total volume of 5 ml, which was incubated with 3000 U ß-glucuronidase (also containing about 200 U sulfatase) for 6 h at 37°C in the presence of 2 mg/ml ascorbic acid. For inhibition studies, 10 µM estrone sulfamate, a sulfatase inhibitor (30), or 50 mM D-saccharic acid-1,4-lactone, a ß-glucuronidase inhibitor (31), was included in mixtures incubated for 6 h at 37°C with added ß-glucuronidase/sulfatase.

The samples in 20 mM ammonium acetate, pH 3.8, were then applied to a Bond Elut Certify Sep-Pak cartridge (130 mg) pre-conditioned with methanol and equilibrated with 20 mM ammonium acetate, pH 3.8. Impurities were washed off with 4 ml buffer, then with 2 ml 20% methanol and 2.5 ml 40% methanol in 50 mM acetic acid. CE metabolites, conjugates and adducts were eluted with 2 ml 50% methanol and 6 ml 60% methanol in 50 mM acetic acid (or 4 ml 60% methanol in 30 mM HCl). To prevent oxidation of CE, it was essential to have ascorbic acid in all aqueous solvents. The collected 50 and 60% methanol fractions were analyzed separately by HPLC and then by capillary liquid chromatography/mass spectrometry (LC/MS), and the amounts of each analyte in the two fractions were added together.

HPLC analysis of metabolites, conjugates and adducts
HPLC analysis was carried out on a reverse phase Luna(2) C18 column (250x4.6 mm, 5 µm; Phenomenex, Torrance, CA) on an HPLC system equipped with dual ESA Model 580 solvent-delivery modules, an ESA Model 540 autosampler and an 8-channel CoulArray electrochemical detector (ESA, Chelmsford, MA). Metabolites, conjugates and adducts of 4-OHE2 were determined and confirmed in at least two different gradient systems. Gradient system 1 consisted of applying a linear gradient starting with 100% mobile phase A [0.1 M ammonium acetate, pH 4.4/acetonitrile/methanol (80:15:5, v/v/v)] to 90% mobile phase B [0.1 M ammonium acetate, pH 4.4/acetonitrile/methanol (30:50:20, v/v/v)] over 50 min at a flow rate of 1 ml/min. The eight electrochemical detector channels were set at 0, 40, 90, 130, 250, 300, 420 and 470 mV with respect to the internal standard electrode. Gradient system 2 consisted of applying a linear gradient starting with 100% mobile phase A [50 mM ammonium acetate, pH 4.4/acetonitrile/methanol (80:10:10)] to 90% mobile phase B [50 mM ammonium acetate, pH 4.4/acetonitrile (30:70)] over 50 min at a flow rate of 1 ml/min. The detector channel potentials were set at 0, 50, 125, 250, 300, 420, 450 and 520 mV. Gradient system 3 consisted of applying a linear gradient starting with 100% mobile phase A [0.1 M ammonium acetate, pH 4.4/acetonitrile/methanol (75:15:10)] to 90% mobile phase B [0.1 M ammonium acetate, pH 4.4/acetonitrile/methanol (60:30:10)] over 45 min at flow rate of 1 ml/min. The detector channel potentials were set at 0, 40, 80, 120, 160, 200, 240 and 280 mV. The first two gradients were used for analysis of metabolites, conjugates and adducts, whereas the third one was used for conjugates and adducts.

In general, the peak-height ratios of compounds that oxidize at lower potentials [4-OHE1(E2)-N7Gua, thioether conjugates and CE] were better when the detector was set at the potentials in gradient system 3, whereas compounds that need higher potentials [E1, E2, 16{alpha}-OHE1(E2) and methoxy derivatives of the CE] gave better responses at the potentials in gradient system 2.

The HPLC system was controlled and data were acquired, processed and analyzed using CoulArray software. Analytes were identified by comparison with authentic standards based on their retention time and peak-height ratios between the dominant peak and the peaks in the two adjacent channels. The metabolites, conjugates and adducts were quantified by comparison of peak heights with those of known amounts of standards.

Liquid chromatography/mass spectrometry analysis
To verify the presence and identity of the metabolites, conjugates and DNA adducts, fractions from the solid phase extraction were also determined by capillary reverse-phase HPLC coupled with electrospray ionization (ESI) MS. The HPLC was an Ultra-Plus MicroLC system (Micro Tech, Sunnyvale, CA), supplying a total flow of 4 µl/min to a 20 µl Dyna-Mix Plus mixer. The mobile phase was a binary gradient that was previously described (28). The total column eluant was directed to the mass spectrometer via an electrospray interface.

Electrospray ionization was performed on a Finnigan liquid chromatography quadrupole ion-trap mass spectrometer (LCQ; Finnigan, San Jose, CA). The eluant from the HPLC was sprayed to a heated capillary (200°C), with a spray voltage at 5.7 kV, a nitrogen-sheath-gas flow of 55 ml/min, and an auxiliary gas flow of 10 ml/min. The LCQ was set in the MS/MS mode, in which the molecular ions of each analyte were isolated with a mass window of m/z 1.5. The resonant excitation energy was set at 25% of the maximum (approximately 5 eV), and the scan range was m/z 250–600. The spectra were taken in profile mode, and each spectrum was an average of two `microscans' that were of 1000 ms duration.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analytical methods
Hamster urine contains a wide variety of electrochemically active compounds, requiring extensive pre-purification prior to HPLC analysis. All procedures, except incubation with ß-glucuronidase/sulfatase, were carried out under acidic conditions (pH 3.5–3.8) in the presence of ascorbic acid to minimize oxidation of metabolites, conjugates and adducts. In addition, under acidic conditions many of the urinary proteins were denatured and easily removed by centrifugation. Urine is a highly polar matrix with variable and high salt content, containing phosphates, ureates, metal ions, sugars, proteins, creatinine and many metabolic products. On the other hand, the analytes of interest, CE metabolites, conjugates and DNA adducts, are moderately polar compounds. Although the efficiency of extraction of these compounds was relatively good on non-polar cartridges (tC18, Waters), the purity of the estrogen compounds was better with Bond-Elut Certify II SPE cartridges with moderately non-polar, polar and strong anion exchange properties and best with Certify SPE cartridges with moderately non-polar, polar and strong cation exchange properties. Certify SPE cartridges were used for the studies described in this article.

Recovery of each analyte from control hamster urine spiked with 0.5–0.8 nmol of the 22 estrogen compounds analyzed by HPLC (shown in Figure 3AGo) was approximately 90%. Most impurities passed thorough the cartridge, or were removed by the 40% methanol wash. Compounds of interest were eluted in two fractions with 50 and 60% methanol in 50 mM acetic acid. Some of the compounds were contained in both fractions, for example, 4-OHE1(E2)-2-SG and 4-OHE1(E2)-1-N7Gua. The predominant analytes in the 50% fraction were CE and 4-OHE1(E2)-2-NAcCys, while 4-OHE1(E2)-2-Cys and methoxy-CE were mostly found in the 60% methanol fraction. In all the studies reported in this article, the 50 and 60% methanol fractions were analyzed separately by HPLC and the amounts of each analyte in the two fractions were added together.





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Fig. 3. Multichannel electrochemical response from HPLC of (A) standard mixture of estrogens, estrogen metabolites, estrogen conjugates and estrogen-DNA adducts, (B) 60% methanol fraction from hamster urine, and (C) 60% methanol fraction from hamster urine treated with ß-glucuronidase/sulfatase. The compounds analyzed are, in order of elution, 4-OHE2-2-SG, 4-OHE2-1-N7Gua, 4-OHE1-2-SG, 4-OHE2-2-Cys, 4-OHE1-1-N7Gua, 16{alpha}-OHE2, 4-OHE1-2-Cys, 4-OHE2-2-NAcCys, 4-OHE1-2-NAcCys, 16{alpha}-OHE1, 4-OHE2, 2-OHE2, 2-OHE1, 4-OHE1, E2, 4-OCH3E2, 2-OCH3E2, E1, 4-OCH3E1, 2-OH-3-OCH3E2, 2-OCH3E1 and 2-OH-3-OCH3E1.

 
For most of the 22 compounds analyzed, the best purity, retention time and peak-height ratio correlations were obtained by using gradient 1 (Figure 3AGo). Most of the analytes were detectable at picomole levels per injection. The presence and identity of conjugates and adducts were confirmed by LC/MS.

The strategy we used in the liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis was to monitor as a function of time the product-ion mass spectrum of the [M + H]+ of the precursor over approximately two thirds of the mass range, as per the capabilities of the ion-trap mass spectrometer. The product-ion spectra that were obtained during the elution time of the analyte, as determined from reference compounds, were averaged and compared with those of the reference compounds. We generally found that the product-ion spectra were more complicated than those of the references, owing to co-elution of compounds with [M + H]+ ions that have m/z values with a mass unit of the analyte. This co-elution is a consequence of the complexity of the urine samples even though we used extensive clean-up. We considered a detection to be validated if the three to five most abundant product ions were seen in the averaged spectrum obtained over the elution profile of the various analytes. We did not quantify the adducts by LC/MS/MS.

CE metabolites, conjugates and adducts detected in the urine
When hamsters were intraperitoneally injected with 4-OHE2 at doses of 1–12 µmol/100 g body weight, various estrogen metabolites, conjugates and DNA adducts were detected in the urine within 24 h. Solvent-treated animals excreted only small amounts of E1, E2 and CE, but no CE-3,4-Q-derived products were detected (Table IGo). The levels of CE metabolites and conjugates observed in the urine of hamsters treated with 2 or 4 µmol 4-OHE2/100 g body weight are presented in Table IGo. In addition to the expected metabolites, conjugates and adducts, E2, 2-OHE2 and 2-OCH3E2 were also formed, and their levels increased with higher doses of 4-OHE2 used to treat the hamsters (Table IGo).


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Table I. Estrogen metabolites and conjugates in the urine of hamsters treated with 4-OHE2
 
The separation of estrogen derivatives present in the 60% methanol fraction of urine from hamsters treated with 4 µmol 4-OHE2 is shown in Figure 3BGo as an example of the profiles obtained with the various fractions. Most of the administered 4-OHE2 was converted by 17ß-estradiol dehydrogenase to 4-OHE1 (Table IGo). The amounts of the metabolites and conjugates detected after incubation of urine samples with ß-glucuronidase/sulfatase are also presented (Table IGo, Figure 3CGo). The latter amounts represent the sum of free metabolites plus their conjugates with glucuronic and/or sulfuric acid. Most of the CE and their methoxy derivatives were further conjugated. To determine the nature of this conjugation, aliquots of urine from hamsters treated with 4 µmol 4-OHE2 were incubated for 6 h at 37°C with ß-glucuronidase/sulfatase plus saccharic acid lactone, an inhibitor of ß-glucuronidase (31), or estrone sulfamate, an inhibitor of sulfatase (30) (Table IIGo). The ß-glucuronidase inhibitor eliminated virtually all of the effect of added ß-glucuronidase/sulfatase, whereas the sulfatase inhibitor had no effect. As can be concluded from the results in Table IIGo, the further conjugation of E2, CE and 4-OCH3E1(E2) was by glucuronidation.


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Table II. Effect of ß-glucuronidase and arylsulfatase inhibitors on the amount of estrogen metabolites and conjugates in the urine of hamsters treated with 4 µmol 4-OHE2
 
In contrast to CE and their methyl conjugates, the thioether conjugates were not further conjugated (Table IGo). 4-OHE1(E2)-2-Cys and 4-OHE1(E2)-2-NAcCys were the products of mercapturic acid biosynthesis from conjugates of E1(E2)-3,4-Q with GSH. The final products of this biosynthesis pathway, namely, NAcCys conjugates, were the most abundant, whereas the levels of the 4-OHE1(E2)-2-SG conjugates were low and could not be reliably detected by HPLC with electrochemical detection. GSH conjugates were identified by LC/MS/MS (Figure 4Go). The agreement of the product-ion spectrum of the unknown and that of the reference is among the best of the validations that were made by mass spectrometry in this study. Not only are most of the major ions seen in both spectra, but also their abundances are comparable. The spectrum of the reference is very similar to one we published a few years ago in an article describing the mass spectrometry fragmentation of this conjugate and some of its isomers. An explanation of the fragmentation is presented in that article (32).



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Fig. 4. Comparison of the product-ion mass spectrum from LC/MS/MS of the reference 4-OHE1-2-SG with that of a material eluting at the proper retention time in the analysis of a sample of hamster urine.

 
DNA depurinating adducts 4-OHE1(E2)-1-N7Gua and 4-OHE1(E2)-1-N3Ade were not observed by HPLC with electrochemical detection. The 4-OHE1(E2)-1-N7Gua adducts were, however, detected and identified by LC/MS (Figure 5Go shows a typical detection) after treatment of hamsters with 4 or 8 µmol 4-OHE2/100g body weight. The agreement between the product-ion spectrum of the reference and the unknown is typical. Most of the important product ions are seen in both spectra, but the relative abundances do not agree as well as those in Figure 4Go, owing to the lower number of ions that comprise the spectrum of the unknown (note the single-ion spikes along the base line). The N3Ade adducts were not detected by either HPLC or MS.



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Fig. 5. Comparison of the product-ion mass spectrum from LC/MS/MS of the reference 4-OHE1-1-N7Gua with that of a material eluting at the proper retention time in the analysis of a sample of urine from hamsters treated with 4 µmol 4-OHE2/100g body weight.

 
We previously published the product-ion spectra of the E1 adduct taken of fast-atom bombardment-produced ions with a tandem four-sector mass spectrometer (26) and of MALDI-produced ions in the post-source decay mode of a time-of-flight mass spectrometer (5). The spectrum of the reference reported here has many of the same features, but is not identical with those previously reported spectra. Two differences, the ions of m/z 349 and 375, are characteristic of the low-energy collisional activation of the ion trap, and they are likely to be formed by charge-driven losses from the six-membered ring of the purine base. The presence of these ions is consistent with the substitution at N-7 of Gua. Missing in these ion-trap spectra are some products from charge-remote processes, which cannot be induced by the lower energy collisions of the ion trap.

Overall, when hamsters were treated with the 2 or 4 µmol dose, about twice as much quinone conjugates were formed as methoxy derivatives (Table IGo). At the 2 µmol dose, Cys and NAcCys conjugates were formed at a minimum level of 10.6 nmol, compared with 5.7 nmol methoxy CE (both free and glucuronidated). At 4 µmol, 16.7 nmol thioether conjugates were detected, whereas only 9.8 nmol methoxy CE were observed. Far more NAcCys conjugates were always detected than Cys conjugates.

The higher level of 4-OHE1(E2)-2-NAcCys conjugates compared with Cys conjugates was observed at all dose levels (Figure 6Go). The 4-OHE1(E2)-2-NAcCys were determined to be the predominant conjugates identified in hamster urine and were present at nanomole levels. Depending on the dose of 4-OHE2, between 1.2 and 20 nmol 4-OHE1-2-NAcCys and 0.4 and 50 nmol 4-OHE2-2-NAcCys per 100 g body weight were detected after 24 h. Lower doses of 4-OHE2 (1–2 µmol/100 g body weight) favored formation of 4-OHE1 conjugates, whereas higher doses (over 4 µmol/100 g) favored formation of 4-OHE2 conjugates (Figure 6Go).



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Fig. 6. Levels of Cys and NAcCys conjugates in the urine of hamsters in response to treatment with increasing doses of 4-OHE2. The 50 and 60% methanol fractions from the Certify SPE cartridge were analyzed separately using gradient system 1 and the amounts of each analyte in the two fractions were added together. Error bars indicate the standard deviations.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The oxidation of 4-hydroxyestrogens (4-OHE) to their CE-3,4-Q is a critical pathway that may lead to estrogen-initiated cancer (5,33). Treatment of hamsters with 4-OHE2 produces estrogenic imbalance in the kidney (28). This can also be observed in the urine, as determined by the formation of GSH conjugates and depurinating N7Gua adducts of CE-3,4-Q. After an initial purification of the urine to remove a variety of metabolic products, 22 compounds, including estrogen metabolites, methoxy CE, GSH conjugates and N7Gua adducts, could be identified.

Our results show that most of the administered 4-OHE2 was metabolically transformed to 4-OHE1 (Table IGo). In addition to CE, we observed COMT-catalyzed formation of methoxy CE, which is a measure of protection at the CE level, and thioether conjugates, which is a measure of protection at the CE-Q level. Formation of thioether (GSH, Cys and NAcCys) conjugates and depurinating adducts [4-OHE1(E2)-1-N7Gua] indicates that oxidation of 4-CE to CE-3,4-Q and subsequent reaction with GSH and DNA, respectively, have occurred. These conjugates and adducts can serve as biomarkers of CE oxidation.

The results obtained show that after treatment of hamsters with 2 or 4 µmol 4-OHE2/100 g body weight (Table IGo), the thioether conjugates, representing the oxidative pathway, are present at approximately twice the levels of the products of the methylation pathway. A similar ratio was also observed at the 8 and 12 µmol dose levels (data not shown).

CE and methoxy conjugates were further conjugated by glucuronidation (Tables I and IIGoGo), whereas the thioether conjugates were not (Table IGo). Among the thioether conjugates, the CE-SG were barely detected by HPLC, but were readily identified by LC/MS. The most abundant of the thioether conjugates were the CE-NAcCys.

The low amount of urinary CE-SG is caused by the extensive catabolism of these products to their Cys and NAcCys conjugates, as is consistent with the mercapturic biosynthesis pathway (24). In fact, the level of one enzyme involved in this pathway, {gamma}-glutamyl transpeptidase, is significantly elevated in the urine of hamsters treated with E2 (34).

In the hamsters treated with 4-OHE2 at various doses, E2, 2-OHE2 and 2-OCH3E2 were unexpectedly identified (Table IGo). Their amounts in the urine increased with larger doses of 4-OHE2 administered to the hamsters. These puzzling results may be explained by the ability of 4-OHE2 to induce aromatase, which converts testosterone to E2 (Figure 1Go) (35). Incidentally, aromatase was also found to act as a 2-hydroxylase (36), which can account for the presence of 2-OHE2 and 2-OCH3E2 in the urine.

The 4-OHE1(E2)-1-N7Gua adducts identified by LC/MS/MS clearly indicate that the CE-3,4-Q are endogenous genotoxic electrophilic metabolites that may lead to tumor initiation.

Conclusions
We have developed a sensitive method to identify estrogen metabolites, conjugates and DNA adducts in the urine of male Syrian golden hamsters. After treatment of hamsters with 4-OHE2, the predominant conjugates were found to be 4-OHE1(E2)-2-NAcCys. Formation of these conjugates provides strong evidence that persistent exposure to 4-OHE can lead to formation of the electrophilic CE-3,4-Q, which may then form adducts with DNA, a putative tumor-initiating event. The estrogen metabolites, CE conjugates and adducts identified in urine should be useful as biomarkers for measuring possible susceptibility to estrogen-induced cancer.


    Notes
 
2 To whom correspondence should be addressed Email: ecavalie{at}unmc.edu Back


    Acknowledgments
 
This research was supported by US Public Health Service grants P01 CA49210 and R01 CA49917 from the National Cancer Institute. Core support at the Eppley Institute is provided by grant P30 CA36727 from the National Cancer Institute. The Washington University Mass Spectrometry Laboratory is supported by grant P41 RR00954 from the National Center for Research Resources of the National Institutes of Health. We thank Dr A.F.Badawi for valuable comments and assistance in preparing this article.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 12, 2000; revised February 9, 2001; accepted February 22, 2001.