Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian golden hamsters treated with 4-hydroxyestradiol: potential biomarkers for estrogen-initiated cancer

Prabu Devanesan1, Rosa Todorovic1, Jiang Zhao2, Michael L. Gross2, Eleanor G. Rogan1 and Ercole L. Cavalieri1,,3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Formation of depurinating adducts by reaction of catechol estrogen-3,4-quinones with DNA was proposed to be a tumor initiating event by estrogens [E.L.Cavalieri et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10937–10942]. Under estrogenic imbalance, oxidation of catechol estrogens to quinones may compete with their detoxification by protective enzymes. The quinones formed can be detoxified by reaction with glutathione (GSH) or can covalently bind to DNA. To provide more support for this hypothesis, we developed a method to identify and quantify GSH, cysteine (Cys) and N-acetylCys conjugates of 4-hydroxyestrogens (4-OHE) in the kidneys of male Syrian hamsters treated with 4-hydroxyestradiol (4-OHE2) by intraperitoneal injection. The highest level of conjugates was observed 1 h after treatment, and almost none was detected after 24 h. Dose–response studies indicated conjugate formation after treatment with 0.5 µmol of 4-OHE2/100 g body weight, and formation increased up to a treatment level of 12 µmol/100 g body weight. GSH, Cys and N-acetylCys conjugates of 4-OHE were identified in the picomole range by high-performance liquid chromatography (HPLC) with multichannel electrochemical detection and confirmed by HPLC/tandem mass spectrometry. Treatment of tissue homogenates with ß-glucuronidase/sulfatase at 37°C for 6 h before extraction resulted in a 12- to 20-fold increase in Cys conjugates from picomole to nanomole levels. Similar enhancement was observed by just incubating the tissue at 37°C for 6 h. Evidence for the 4-OHE-1-N7Gua depurinating adducts was obtained by mass spectrometry. We conclude that GSH and Cys conjugates of the 4-OHE and the 4-OHE-N7Gua adducts can be utilized as biomarkers to detect estrogenic imbalance and potential susceptibility to tumor initiation.

Abbreviations: CE, catechol estrogen(s); CE-Q, catechol estrogen-3,4-quinone(s); Cys, cysteine; E1, estrone; E2, 17ß-estradiol; ESI, electrospray ionization; GSH, glutathione; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; NAcCys, N-acetylcysteine; OHE, hydroxyestrogen(s); -SG, glutathione moiety.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prolonged exposure to high estrogen levels in women has been linked to increased incidence of breast cancer (13). According to the standard paradigm, estrogens, through receptor-mediated processes, affect the rate of cell proliferation, leading to genetic errors that result in a malignant phenotype (13). Specific metabolites of estrogens, namely catechol estrogen-3,4-quinones (CE-3,4-Q), can react with DNA, however, to produce depurinating adducts that lead to apurinic sites (4,5). [In this article we use the term `adduct' for products formed by reaction of catechol estrogen quinones (CE-Q) with DNA and the term `conjugate' for products formed with glutathione (GSH) and its derivatives.] Such apurinic sites can result in mutations that initiate tumors (6). Evidence that estrogens can indeed initiate tumors is provided by their carcinogenicity in animal models, particularly the Syrian golden hamster, in which kidney tumors are induced without formation of `spontaneous' tumors in untreated animals (710).

The estrogens estrone (E1) and 17ß-estradiol (E2) are biochemically interconvertible by the enzyme 17ß-estradiol dehydrogenase (Figure 1Go). E1 and E2 are metabolized via two major pathways: formation of catechol estrogens (CE) and 16{alpha}-hydroxylation (1113). The CE formed are the 2- and 4-hydroxylated estrogens (Figure 1Go). The 4-hydroxyestrogens (4-OHE) induce kidney tumors in male Syrian golden hamsters, whereas the 2-OHE are not carcinogenic (14,15).



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Fig. 1. Metabolic pathway of oxidation of 4-CE and formation of GSH conjugates and depurinating DNA adducts. SQ, semiquinones.

 
In general, the CE are inactivated, especially in the liver, by conjugation reactions such as glucuronidation and sulfation (11). The most common and ubiquitous pathway of inactivation, however, occurs by O-methylation, catalyzed by catechol-O-methyltransferase (11,16). If these conjugating pathways are insufficient and/or ineffective, the competitive oxidation pathway of CE forms their semiquinones and quinones, which conjugate with GSH. If this inactivating pathway is also insufficient and/or ineffective, the CE-Q may react with DNA to form adducts. For example, if 4-OHE1(E2) are not conjugated, they can be oxidized to E1(E2)-3,4-Q, which react with GSH to form conjugates or with DNA to form the depurinating adducts 4-OHE1(E2)-1-N7Gua and 4-OHE1(E2)-1-N3Ade (Figure 1Go) (4,5).

The GSH conjugates of E1(E2)-3,4-Q are catabolized via a three-step enzymic hydrolysis of the GSH tripeptide (Figure 2Go) (17). First, the glutamyl moiety is released by {gamma}-glutamyl transpeptidase. The resulting cysteinylglycine conjugate is hydrolyzed by cysteinyl glycinase to yield the CE-cysteine (Cys) conjugate, which, in a final step is acetylated to form the CE-N-acetylcysteine (NAcCys) conjugate.



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Fig. 2. GSH conjugation with the electrophilic compound RX, followed by mercapturic acid biosynthesis to yield the NAcCys conjugate as the final product.

 
Formation of the endogenous carcinogens CE-3,4-Q is presumably due to disruption of estrogen homeostasis, namely the balance between activating and deactivating (protective) enzymes. This imbalance can be achieved in male Syrian golden hamsters by implantation of E1, E2 or their 4-OHE that induce tumors (9,10,14,15).

Several studies have been conducted to determine estrogens in urine, plasma, human uterus and endometrial tissue (1823). The methods used in these studies required derivatization and/or the use of radiochemicals, or were set up to detect hydroxylated metabolites or methylated or sulfated conjugates. Formation of CE–GSH conjugates in the bile ducts of hamsters was also reported (24). The studies conducted thus far have tended to focus on thioether conjugates of 2-OHE and not those of 4-OHE.

Utilizing the well-investigated male Syrian golden hamster kidney model, we show in this article that upon treatment with 4-OHE2, the estrogen homeostasis of these animals is unbalanced, as evidenced by formation of thioether conjugates and DNA adducts of CE-3,4-Q. We also report a new, sensitive electrochemical method of detecting conjugates and DNA adducts of CE for use as potential biomarkers of estrogenic imbalance and potential susceptibility to tumor initiation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Male Syrian golden hamsters (6 weeks old) were from the Eppley Colony. 4-OHE2 was synthesized according to Dwivedy et al. (25). 4-OHE1(E2)-2-glutathione (-SG), 4-OHE1(E2)-2-Cys and 4-OHE1(E2)-2-NAcCys were synthesized according to published procedures of Cao et al. (26). All enzymes and chemicals were purchased from Sigma (St Louis, MO). Certify II Sep-Pak cartridges were purchased from Varian (Palo Alto, CA). The Luna(2) high-performance liquid chromatography (HPLC) column was purchased from Phenomenex (Torrance, CA).

Methods
Injection of hamsters
Groups of four hamsters were treated with 4-OHE2. For time–course studies, the animals were intraperitoneally injected with 12 µmol of 4-OHE2 dissolved in 300 µl of DMSO/trioctanoin (1:9, v/v) per 100 g body weight for periods ranging from 1 to 24 h. For dose–response studies, the animals were treated with doses between 0.5 and 16 µmol of 4-OHE2 per 100 g body weight for 2 h. Treatment with 16 µmol was toxic and was not repeated. The animals were killed at the appropriate times, and the kidneys were excised and processed as described below.

Extraction of adducts
Kidneys were minced, frozen in liquid nitrogen and ground to a fine powder. Ground tissue was suspended in 3 ml of 50 µM ammonium acetate, pH 4.4, and divided into two portions of ~2 g each. ß-Glucuronidase from Helix pomatia (10 000 U, also containing 900 U of arylsulfatase) was added to one of the portions, which was incubated for 6 h at 37°C, whereas the other portion was not incubated. For inhibition studies, 9 µM estrone sulfamate (a sulfatase inhibitor) (27), 50 mM D-saccharic acid-1,4-lactone (a ß-glucuronidase inhibitor) (28) or 10 mM leupeptin (a protease inhibitor) (29) was added to incubation mixtures containing 1 g of tissue, together with the ß-glucuronidase/sulfatase enzymes. In some studies, a portion of the homogenized tissue was incubated with sulfatase alone (Type VIII from Abalone, 900 U) under the same conditions as with ß-glucuronidase/sulfatase. After incubation, mixtures were frozen until further processing. Sufficient methanol was added to all fractions to give a final concentration of 60%, and the mixtures were extracted with 8 ml of hexane to remove any lipids. The aqueous phase was diluted with 50 µM ammonium acetate, pH 4.4, containing 1 mg/ml ascorbic acid, to an approximate final concentration of 30% methanol, and the methanol/water mixture was applied to a Certify II Sep-Pak (200 mg) cartridge. The cartridge was first eluted with 3 ml of the buffer, followed by elutions with 2 ml each of 20, 40 and 70% methanol in buffer, and fractions were collected. To minimize oxidation of the conjugates, ascorbic acid was added to the eluting buffer at a concentration of 1 mg/ml. Collected fractions were analyzed by HPLC with electrochemical detection and then by liquid chromatography/mass spectrometry (LC/MS).

HPLC analysis
Analyses were carried out by using a Luna(2) C18 reverse phase column (250x4.6 mm, 5 µm) on an HPLC system equipped with dual ESA Model 580 solvent delivery modules, an ESA Model 540 autosampler and an 8-channel ESA CoulArray electrochemical detector (ESA, Inc., Chelmsford, MA). Two different gradient systems, each using different oxidation potentials were employed for separation of the compounds of interest, the second being used to cross-validate the first. In the first gradient system, the oxidation potentials were set at 0, 30, 60, 100, 220, 300, 380 and 460 mV, with respect to the internal standard electrode, for channels 1–8. A linear gradient starting from 100% solvent A [CH3CN/CH3OH/H2O/1 M ammonium acetate, pH 4.4 (15:5:70:10)] to 90% solvent B [CH3CN/CH3OH/H2O/1 M ammonium acetate, pH 4.4 (50:20:20:10)] over 50 min was employed to separate the compounds, at a flow rate of 1 ml/min. In the second gradient system, the oxidation potentials were set at 0, 80, 160, 240, 320, 400, 480 and 560 mV for channels 1–8. A linear gradient starting from 100% solvent A [CH3CN/CH3OH/H2O/1 M ammonium acetate, pH 4.4 (15:10:65:10)] to 90% solvent B [CH3CN/CH3OH/H2O/1 M ammonium acetate, pH 4.4 (30:10:50:10)] over 45 min at a flow rate of 1 ml/min was employed to separate the compounds (Figure 3Go). Conjugates from biological samples were identified by comparison with authentic standards, based on their retention time, as well as peak height ratios between the dominant peak and the peaks in the two adjacent channels. Data analysis was carried out by using ESA CoulArray software.



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Fig. 3. Separation of 4-OHE2 conjugates by HPLC with electrochemical detection. A linear gradient starting from 100% solvent A [CH3CN/CH3OH/H2O/1 M ammonium acetate, pH 4.4 (15:10:65:10)] to 90% solvent B [CH3CN/CH3OH/H2O/1 M ammonium acetate, pH 4.4 (30:10:50:10)] over 45 min was employed at a flow rate of 1 ml/min. The oxidation potentials of the detector channels are shown. nA, nanoamperes; mV, millivolts.

 
LC/MS analysis
Samples were also analyzed by capillary reverse-phase HPLC coupled with electrospray ionization (ESI) MS. The HPLC was an Ultra-Plus MicroLC system (Micro Tech, Sunnyvale, CA). It supplied a total flow of 4 µl/min to a 20 µl Dyna-Mix Plus mixer. The mobile phase was a binary gradient that began with a composition of 50% solvent A (H2O with 0.5% v/v acetic acid) and 50% solvent B (CH3OH with 0.5% v/v acetic acid). This mixture was kept constant for 4 min. Between 4 and 8 min, the percent of solvent A was decreased linearly to 45%, whereas B was increased to 55%. From 8 to 32 min, A and B were changed linearly to 30 and 70%, respectively, and then held constant for a further 8 min. Each analysis was followed by a 10 min purge and then a 15 min equilibration with solvent at the starting composition. Samples were admitted with a Rheodyne 7125 injector to a Luna(2) C18 column (0.3 mmx15 cm). The total column eluant was then directed to the mass spectrometer via an electrospray interface.

ESI 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 (~1.25 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
The first issue we addressed in developing an analytical method was the clean-up of tissue extracts. Solid-phase extraction was the successful method of choice. Among various solid-phase extraction cartridges tested for the initial purification of tissue samples, Certify II was found to yield the highest recovery and reproducibility for CE conjugates. Greater than 90% recovery was consistently achieved from tissue samples spiked with synthetic CE conjugate and adduct standards. Extraction with hexane prior to solid-phase extraction facilitated the selective removal of lipids from the tissue samples. Acidic pH was necessary to maintain the integrity of the adducts and conjugates in solution, and ascorbic acid was added to the elution buffer to minimize oxidation.

HPLC coupled with multichannel electrochemical detection was used for analysis because CE and their derivatives exhibit good electrochemical properties and are easily oxidizable (Figure 3Go). Phytoestrogens and polyphenols in plasma, tissue and urine were determined previously by using electrochemical 8-channel array detection (30). By carefully selecting the potentials for the different electrochemical channels, the amount of each of the compounds oxidized in the individual channels was controlled, allowing a unique, fingerprint-type identification of the compounds of interest in very small quantities and even in the presence of larger amounts of other impurities. This method is sufficiently sensitive to detect routinely 1 pmol of the conjugates and adducts injected on the column. However, we did not investigate rigorously the detection limit in this first study.

All of the assignments of the CE conjugates from hamster kidney were verified by LC/tandem MS. The product-ion mass spectra of (M+H)+ matched well with those of synthetic reference conjugates. A typical example of a match is the determination of the 4-OHE2-2-NAcCys conjugate (Figure 4Go). The product-ion spectrum of the kidney sample afforded fragment ions of m/z 432, 408, 391, 362, 345, 319, 287 and 269 (Figure 4AGo); all of these fragment ions were found in the spectrum of the 4-OHE2-2-NAcCys standard (Figure 4BGo). With this protocol, 4-OHE1-2-NAcCys, 4-OHE1-2-Cys, 4-OHE2-2-Cys, 4-OHE1-2-SG and 4-OHE2-2-SG were also positively identified.



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Fig. 4. LC/MS/MS analysis of hamster kidney tissue. (A) Product-ion spectrum of the m/z 448 ion of 4-OHE2-2-NAcCys found in the tissue sample. (B) Product-ion spectrum of the standard 4-OHE2-2-NAcCys. Samples were introduced to an ion-trap mass spectrometer by HPLC.

 
Dose–response
Hamsters were treated with different amounts of 4-OHE2 for 2 h to determine the relationship between the level of treatment and formation of CE conjugates (Figure 5Go) and CE–DNA adducts in the kidneys. GSH, Cys and NAcCys conjugates of 4-OHE2 and 4-OHE1 were detected in the kidney tissue at all doses of 4-OHE2. The conjugates were formed even at the lowest dose of 0.5 µmol 4-OHE2/100 g body weight. In untreated animals and control animals treated with solvent, none of the conjugates or DNA adducts were detected.



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Fig. 5. Dose–response for treatment of hamsters with 4-OHE2 for 2 h. Tissue homogenates were not incubated at 37°C.

 
The highest levels of GSH conjugates, 350 and 85 pmol/g tissue for 4-OHE2-2-SG and 4-OHE1-2-SG, respectively, were observed after treatment of hamsters with 1 µmol of 4-OHE2 (Figure 5Go). At higher doses (i.e. 2–16 µmol), their levels stayed fairly constant over all the doses employed. The levels of Cys and NAcCys conjugates increased in an approximately linear fashion up to a dose of 12 µmol of 4-OHE2. With 12-µmol treatment, 130, 30, 260 and 165 pmol of 4-OHE2-2-Cys, 4-OHE1-2-Cys, 4-OHE2-2-NAcCys and 4-OHE1-2-NAcCys were formed, respectively. At all doses, more NAcCys than Cys conjugates were detected.

To determine whether the CE conjugates were further conjugated by either glucuronidation or sulfation, half of the kidney tissue from each sample was treated for 6 h at 37°C with an enzyme preparation containing both ß-glucuronidase and sulfatase (Figure 6Go). This incubation of the tissue resulted in a dramatic increase in the levels of the Cys conjugates (Figure 6Go). At each dose level studied, the amount of both 4-OHE2-2-Cys and 4-OHE1-2-Cys increased between 12- and 16-fold compared to the amounts found in unincubated samples (Figure 5Go). The amounts of NAcCys conjugates did not change, but the levels of GSH conjugates decreased dramatically. Similar results were obtained when the tissues were simply incubated at 37°C for 6 h without added ß-glucuronidase/sulfatase (see Table IGo and `Inhibition studies' following). The increase in Cys conjugates and concomitant decrease in GSH conjugates may be due to enzymic hydrolysis of the GSH tripeptide to give both the dipeptide and ultimately the Cys conjugate (Figure 2Go) and not to removal of glucuronic acid or sulfate groups from hypothesized doubly-conjugated products (this is explained in the section on `Inhibition studies').



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Fig. 6. Dose–response for treatment of hamsters with 4-OHE2 for 2 h. Tissue homogenates were incubated at 37°C for 6 h with ß-glucuronidase/sulfatase.

 

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Table I. Hamster kidney treated with 12 µmol 4-OHE2/100 g body weight for 2 h: Effect of inhibitors on conjugate levels
 
Time–response
The persistence of conjugates and adducts in the kidneys was studied over a period of 24 h by treating animals with 12 µmol of 4-OHE2 (Figures 7 and 8GoGo). The highest amounts of all the conjugates were observed 1 h after treatment. As expected, the levels of all the conjugates decreased over time due to excretion, and only trace amounts were detectable after 24 h (data not shown). In tissues not incubated for 6 h at 37°C with ß-glucuronidase/sulfatase (Figure 7Go), GSH conjugates were the dominant CE conjugates detected at all exposure times. The highest amounts of 700 and 115 pmol 4-OHE2-2-SG and 4-OHE1-2-SG, respectively, were found after 1 h. The Cys conjugates were detected in the smallest amounts. However, upon incubation for 6 h at 37°C with ß-glucuronidase/sulfatase (Figure 8Go), the amount of Cys conjugates increased and that of the GSH conjugates decreased, as explained above. The amount of NAcCys conjugates did not change significantly.



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Fig. 7. Time–response for treatment of hamsters with 12 µmol of 4-OHE2. Tissue homogenates were not incubated at 37°C.

 


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Fig. 8. Time–response for treatment of hamsters with 12 µmol of 4-OHE2. Tissue homogenates were incubated at 37°C for 6 h with ß-glucuronidase/sulfatase.

 
Inhibition studies
Since the amount of Cys conjugates increased when tissue samples were incubated at 37°C, with or without added ß-glucuronidase/sulfatase, we investigated the source of the increased level of Cys conjugates (Table IGo). The similar results with or without added ß-glucuronidase/sulfatase suggest that the incubation temperature and activation of endogenous enzymes are most likely responsible for the change in the amount of conjugates. To eliminate definitively the possibility of a second conjugation event, tissue samples were incubated with either D-saccharic acid-1,4-lactone, a ß-glucuronidase inhibitor (28), or with estrone sulfamate, an arylsulfatase inhibitor (27), both in the presence and absence of added ß-glucuronidase/sulfatase enzyme preparations (Table IGo). Sulfatase alone was also used for the hydrolysis of tissue, either in the presence or absence of its inhibitor, estrone sulfamate (data not shown). Leupeptin, a potent protease inhibitor (29), was also used to determine the presence or absence of proteolytic activity that could result in Cys conjugates arising from hydrolysis of tissue proteins (data not shown). The inhibitors had only a small (~10%) effect on the amount of conjugates detected.

These results show that 4-OHE1(E2)-2-SG, 4-OHE1(E2)-2-Cys and 4-OHE1(E2)-2-NAcCys are not further conjugated by glucuronidation or sulfation and that the Cys conjugates do not arise from hydrolysis of modified proteins in the tissue preparation. Thus, the large increase in Cys conjugates most likely arises from the enzymic hydrolysis of the GSH tripeptide to its Cys moiety (17). Since the increase (roughly two orders of magnitude) in Cys conjugates with incubation at 37°C was greater than the initial amounts of GSH conjugates, the increase is likely to be due to hydrolysis not only of GSH conjugates, but in fact mainly of the intermediate cysteinylglycine conjugates (Figure 2Go). We are unable to prove the existence of the dipeptide intermediate because we have no standard for HPLC analysis.

4-OHE–DNA adducts
DNA nucleobases modified by 4-OHE could not be identified satisfactorily by electrochemical detection. Minor HPLC peaks indicating the probable presence of N7Gua adducts appeared at the proper retention times, but the diagnostic electrochemical peak ratios could not be determined with sufficient accuracy to validate the detection. However, analysis of the Sep-Pak fractions by LC/MS revealed that both 4-OHE2-1-N7Gua (Figure 9Go) and 4-OHE1-1-N7Gua were present in small quantities, along with the CE conjugates. The 4-OHE2-1-N7Gua standard gave a product-ion spectrum showing ions of m/z 436, 420, 403, 392, 377, 351, 324, 298, 286 and 272 (Figure 9BGo). The peak ratios were different for standard and unknown because ion statistics were low owing to the small amounts of materials. Nevertheless, all these product ions were found in the tandem MS analysis of the kidney sample (Figure 9AGo). The 4-OHE1(E2)-1-N3Ade adducts were not identified in kidney samples.



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Fig. 9. LC/MS/MS analysis of hamster kidney tissue. (A) Product-ion spectrum of the m/z 438 ion of 4-OHE2-1-N7Gua found in the tissue sample. (B) Product-ion spectrum of the standard 4-OHE2-1-N7Gua. Samples were introduced to an ion-trap mass spectrometer by HPLC.

 
The N7Gua DNA adducts were formed in much smaller quantities than those of the conjugates, as expected, and were below the limit for identification by electrochemical detection. A further complication is that the modified nucleobases are oxidized at higher oxidation potentials than the conjugates. Nevertheless, positive identification by LC/tandem MS of these N7Gua adducts in kidney preparations from hamsters treated with 4-OHE2 establishes that DNA damage occurs upon exposure to this estrogen metabolite. DNA damage and formation of the N7Gua adduct was previously shown to occur in in vitro and in vivo studies (4) and in cultured mammary glands of rats treated with 4-OHE2 (31).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several lines of evidence suggest that oxidation of 4-OHE is the critical pathway leading to estrogen-induced cancer. The 4-OHE are formed more abundantly in hamster kidney (32,33) and other organs prone to estrogen-induced cancer (34,35). Predominant 4-hydroxylase activity was also observed in microsomes from human tissues susceptible to estrogen-induced cancer (3638).

Administration of 4-OHE2 to hamsters produces estrogenic imbalance in the kidney, as determined by the formation of GSH conjugates and depurinating N7Gua adducts of CE-3,4-Q. The largest amounts of the conjugates were observed in the hamster kidney 1 h after treatment with 4-OHE2 (Figures 7 and 8GoGo), indicating that the response to estrogenic imbalance was very fast. After 24 h, almost all of the CE conjugates were excreted, and only trace amounts were detected. Conjugate formation also occurred after treatment with only 0.5 µmol of 4-OHE2/100 g body weight (Figures 5 and 6GoGo).

The amounts of Cys conjugates increased dramatically from picomole to nanomole levels upon incubation of the tissue at 37°C for 6 h. Addition of ß-glucuronidase/sulfatase raised the amounts ~10% more (Figures 5–8GoGoGoGo and Table IGo), suggesting that further conjugation by glucuronidation or sulfation was at best borderline. It is reasonable to conclude that further hydrolysis of the GSH tripeptide and the dipeptide (Figure 2Go) during incubation of the tissue at 37°C afforded the Cys conjugates.

Measurements can also be extended to examine not only conjugates and DNA adducts, but also metabolites and methoxy derivatives of estrogens at the same time. Analyses of hamster urine to detect these estrogen metabolites, conjugates and DNA adducts are reported elsewhere (R.Todorovic, P.Devanesan, S.Higginbotham, J.Zhao, M.Gross, E.Rogan and E.Cavalieri, manuscript in preparation). Analysis of human breast samples and rat prostate tissue for estrogen conjugates and metabolites by using this method is already in progress (unpublished results).

Conclusions
The results obtained in this study clearly indicate that treatment of hamsters with an excess of 4-OHE can lead to oxidation of CE to semiquinones and CE-Q. GSH conjugation represents a mechanism to protect against the possibility that CE-Q can react with DNA. The detection of both GSH conjugates and N7Gua adducts in the kidney tissue demonstrates that the carcinogenic 4-OHE2 is oxidized to E2-3,4-Q, followed by reaction with both GSH and DNA in this target organ.

The CE conjugates and DNA adducts are potentially useful as biomarkers to indicate both estrogenic imbalance, due to chronic exposure to elevated estrogen levels in tissues and potential susceptibility to estrogen-related cancers. The ability to detect these estrogen conjugates and adducts is expected to prove valuable in studies of estrogen-induced cancer.


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


    Acknowledgments
 
We thank W.Liang for synthesis of 4-OHE2 and the CE conjugates and S.Higginbotham for treating animals and isolating tissue. This research was supported by US Public Health Service grant P01 CA49210 from the National Cancer Institute. Core support in 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 20, 2000; revised October 20, 2000; accepted October 20, 2000.