Cancer Research Donn Young Genetics and Biology of Brain Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schild, L. J.
Right arrow Articles by Poirier, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schild, L. J.
Right arrow Articles by Poirier, M. C.
[Cancer Research 63, 5999-6003, September 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Formation of Tamoxifen-DNA Adducts in Multiple Organs of Adult Female Cynomolgus Monkeys Dosed with Tamoxifen for 30 Days1

Laura J. Schild, Rao L. Divi, Frederick A. Beland, Mona I. Churchwell, Daniel R. Doerge, Gonçalo Gamboa da Costa, M. Matilde Marques and Miriam C. Poirier2

Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [L. J. S., R. L. D., M. C. P.]; Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 [F. A. B., M. I. C., D. R. D.]; and Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal [G. G. d. C., M. M. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of the antiestrogen tamoxifen (TAM) is associated with an increase in endometrial cancer. TAM-induced endometrial carcinogenesis may proceed through a genotoxin-mediated pathway, although the detection of endometrial TAM-DNA adducts in exposed women is still controversial. In this study, a monkey model has been used to investigate the question of TAM-DNA adduct formation in primates. Two methods have been used to determine TAM-DNA adducts: a TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), using an antiserum that has specificity for (E)-{alpha}-(deoxyguanosin-N2-yl)-tamoxifen (dG-TAM) and (E)-{alpha}-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-desmethyl-TAM) and electrospray ionization tandem mass spectrometry (ES-MS/MS) coupled with on-line sample preparation and high-performance liquid chromatography (HPLC). Mature (19 year old) cynomolgus monkeys were given either vehicle control (n = 1) or TAM (n = 3) twice daily for a total dose of 2 mg of TAM/kg body weight (bw)/day for 30 days by naso-gastric intubation. Tissues were harvested, and DNA was isolated from uterus, ovary, liver, brain cortex, and kidney. By TAM-DNA CIA, values for uterine TAM-DNA adducts in two monkeys were 0.9 and 1.7 adducts/108 nucleotides, whereas values for ovarian TAM-DNA adducts in the same animals were 0.4 and 0.5 adducts/108 nucleotides. Liver, brain cortex, and kidney DNA samples from the three exposed monkeys had TAM-DNA levels of 2.1–4.2 adducts/108 nucleotides, 0.4–5.0 adducts/108 nucleotides, and 0.7–2.1 adducts/108 nucleotides, respectively. By HPLC-ES-MS/MS, the levels of TAM-DNA adducts detected in all tissues were comparable with those observed by TAM-DNA CIA. Thus, values for uterine TAM-DNA adducts ranged from 0.5 to 1.4 adducts/108 nucleotides, whereas values for ovarian TAM-DNA adducts, measurable in two monkeys, were 0.2 and 0.3 adducts/108 nucleotides. Liver DNA contained the highest TAM-DNA adduct levels (7.0–11.1 adducts/108 nucleotides), whereas brain cortex DNA contained lower adduct levels (0.6–4.8 adducts/108 nucleotides) and the lowest levels were measured in the kidney (0.2–0.4 adducts/108 nucleotides). This study indicates that cynomolgus monkeys are capable of metabolizing TAM to genotoxic intermediates that form TAM-DNA adducts in multiple tissues.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of every eight American women will develop breast cancer by approximately age 70, and 1,000,000 new breast cancer cases are diagnosed each year worldwide. The American Society of Clinical Oncology recently estimated that 700,000 women with breast cancer in the United States are currently receiving TAM,3 a synthetic antiestrogen containing mixed estrogen agonist and antagonist activity. TAM, an effective therapy for all stages of breast cancer, received Food and Drug Administration approval for adjuvant treatment in 1977 and for chemoprevention in healthy "at-risk" women in 1998. The current recommendation for treatment of estrogen receptor positive pre- and post-menopausal women is 20 mg of TAM/day for 5 years. At this dose, TAM was found to reduce the incidence of contralateral breast cancer by 47% (1) and to reduce breast cancer incidence in "at-risk" women by 49% (2) .

It has been well established that TAM is a potent rat hepatocarcinogen. Several laboratories have demonstrated that TAM acts as a classical genotoxic chemical carcinogen in rat liver by exerting its carcinogenic activity through TAM-DNA adduct formation (3, 4, 5, 6, 7) . Rats exposed to TAM have dose-related increases in hepatic TAM-DNA adducts (8) and liver tumors (3) , and there is an association between hepatic TAM-DNA adduct levels and liver tumor incidence (9) . In contrast, hepatic TAM-DNA adduct levels are low in TAM-exposed mice (8 , 10) , and no mouse liver carcinogenicity has been associated with TAM exposure (11) . Neonatal TAM exposures cause reproductive organ tumors in both adult rats (9 , 12) and adult mice (13) .

In humans, epidemiological evidence links TAM exposure to increases in endometrial and, rarely, uterine cancer in breast cancer patients. The relative risk of endometrial cancer for TAM users is estimated to be 1.3–7.5 (2 , 14 , 15) , and the prophylactic use of TAM is associated with a 2.5-fold increase in endometrial tumors (2) . Current estimates are that of 1000 women receiving TAM, two to three will develop endometrial cancer (16) . Furthermore, recent studies have revealed an association with an increase in uterine sarcoma (17) , although this is reported to impact only 0.01% of the women receiving TAM (18) .

There are two possible mechanisms of endometrial/uterine tumor induction: (a) an estrogenic pathway and (b) a pathway involving genotoxic intermediates that damage DNA and induce mutations. As an estrogen agonist in the uterus, TAM can promote cellular proliferation. As a genotoxin, TAM is metabolized to reactive intermediates capable of binding to DNA. DNA adduct formation constitutes an initial step in the carcinogenic process, as replication of a damaged DNA template can lead to the incorporation of an incorrect base, mutagenesis in critical genes, and a heritable loss of growth control.

Several laboratories have investigated TAM-DNA adduct formation in human endometrium and arrived at conflicting conclusions (5 , 19 , 20, 21, 22, 23, 24) . To examine this issue in a primate model, three elderly (19 year old) cynomolgus monkeys were dosed by gavage for 30 days with 2 mg of TAM/kg bw/day, a daily dose ~6-fold greater than the human daily dose. We quantified dG-TAM and dG-desmethyl-TAM, the major DNA adducts detected in the livers of rats administered TAM, by TAM-DNA CIA (25) and by HPLC-ES-MS/MS (26) . We hypothesized that if TAM-DNA adducts were measurable in tissues from TAM-exposed monkeys, primates, including humans, may have the capability to metabolize TAM into genotoxic intermediates.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
TAM and calf thymus DNA were from Sigma (St. Louis, MO). Opaque 96-well high binding plates were purchased from Greiner Labortechnik (PGC Scientific, Frederick, MD). Biotinylated antirabbit IgG, streptavidin-alkaline phosphatase, I-Block (Casein) and CDP-Star with Emerald II were from Tropix (Bedford, MA). Amyloglucosidase was purchased from Boehringer Mannheim (Indianapolis, IN). Reacti-Bind DNA coating solution was obtained from Pierce (Rockford, IL). CIA wash buffer was obtained from KD Medical (Columbia, MD). PBS was from Life Technologies, Inc. (Grand Island, NY).

Animals.
Four adult (19 year old) retired breeder female cynomolgus (Macaca fascicularis) monkeys were housed and treated at Corning Hazelton Laboratories (Vienna, VA). Animal care was provided in accordance with the standards established by the Association for Assessment and Accreditation for Laboratory Animal Care. The experimental protocols were approved by the Hazleton Animal Care and Use Committee. TAM (1 mg/kg bw) was administered to three monkeys twice daily (8 h apart) on weekdays and once on Saturday and Sunday by naso-gastric intubation as a suspension in 0.5% methyl cellulose at 2 ml/kg bw/dose. The total dose administered during the 30-day treatment was 52 mg of TAM/kg bw. The animals were euthanized 16–18 h after the last dose, and organs (brain cortex, liver, kidney, ovary, and uterus) were harvested for DNA isolation. DNA was isolated by nonorganic extraction (DNA Extraction Kit; Stratagene, La Jolla, CA), followed by digestion with 1 unit/ml amyloglucosidase (30 min at 37°C) in the case of DNA isolated from liver tissue. DNA was quantified by UV spectrophotometry at A260.

TAM-DNA CIA.
Rabbit antiserum elicited against DNA modified to 2.4% with dG-TAM, as determined by 32P-postlabeling, was used in the TAM-DNA CIA as described previously (25) . In brief, DNA samples for coating plates were sonicated for 20 s and heat denatured for 5 min at 95°C. Each well of a 96-well microtiter plate was coated with 8.2 pg TAM-DNA in Reacti-Bind DNA coating solution. The coating was for 24–48 h at room temperature, and the plates were stored at -20°C until use. Nonspecific binding was reduced by incubating with 300 µl of casein solution/well (0.33% I-Block in PBS, 0.05% Tween 20, and 0.1% NaN3) for 90 min at 37°C. After each incubation, the plates were washed three times with CIA wash buffer [137 mM PBS, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 3 mM KCl, 0.02% NaN3, and 0.05% Tween 20 (pH 7.4)]. A TAM-DNA standard curve, which was included on each plate used for TAM-DNA CIA, was generated from a TAM-DNA standard, modified to 4.8 adducts/106 nucleotides, and serially diluted from 6.63 to 0.0091 fmol dG-TAM. Competition was achieved by mixing anti-TAM-DNA antiserum with either TAM-DNA standard plus carrier or biological sample DNA in PBS, so that each well contained 10 µg of total DNA. Equal volumes of anti-TAM-DNA (final dilution 1:1,000,000) in casein solution and TAM-DNA standard plus carrier or biological sample DNA in PBS were mixed and incubated at 37°C for 15–20 min before adding 100 µl to each microtiter well and incubating for 90 min at 37°C. After washing, biotinylated antirabbit IgG (100 µl, 1:3000 dilution in casein solution) was added, and the plates were incubated for 90 min at room temperature. After washing, streptavidin-alkaline phosphatase (100 µl, 1:3750 dilution in casein solution) was added and incubated for 60 min at room temperature. After a final wash, 100 µl of CDP-Star containing Emerald II enhancement solution was added. The plates were kept for 20 min at room temperature and overnight at 4°C. The next day, the plates were warmed to room temperature for 20 min, and the light emission was measured at 542 nm using a TR717 Microplate Luminometer (PE Applied Biosystems, Foster City, CA).

Sample quantitation was achieved by comparison of unknown samples with a TAM-DNA standard curve, in which 50% inhibition was at 0.86 ± 0.2 fmol dG-TAM (mean ± SE, n = 5). When 20 µg DNA was analyzed, the LOD was calculated to be ~10 amol of dG-TAM adduct/µg DNA or ~0.3 adducts/108 nucleotides.

HPLC-ES-MS/MS Analyses.
The DNA adduct standards, dG-TAM and dG-desmethyl-TAM, were synthesized and characterized, and the DNA samples were hydrolyzed to nucleosides and analyzed as described (26) . For each analysis, 80 µg of DNA were hydrolyzed. All hepatic DNA samples were analyzed twice, and the rest of the DNA samples were analyzed once. In brief, each sample was loaded onto a reversed phase trap column [Luna C18 (2) , 2 x 30 mm, 3 µm; Phenomenex, Torrance, CA] and eluted with 73% of 0.1% formic acid and 27% of acetonitrile at 0.2 ml/min into the mass spectrometer. A Quatro Ultima triple quadruple mass spectrometer (Micromass, Manchester, United Kingdom), equipped with an electrospray interface, was used with a source block of 150°C and a desolvation temperature of 450°C. Nitrogen was used as the desolvation (750 liters/h) and nebulizing (90 liters/h) gas. Argon was used as the collision gas, at a collision cell pressure of 1.5 x 10-3 mBar. Positive ions were acquired in the MRM mode (dwell time of 0.3 s, span of 0.02 Da, and interchannel delay of 0.03 s) for the (M +2H)2+-> (BH +2H)2+ transitions of dG-TAM (m/z 319 -> 261), dG-desmethyl-TAM (m/z 312 -> 254), and the internal standard, dG-TAM-d6 (m/z 322 -> 264). The cone voltage was 15 V, and the collision energy was 9 eV for all three transitions.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monkey Colony and TAM Exposure.
The female cynomolgus monkeys used in this study were retired breeders that had participated previously in a terminated National Cancer Institute study. At 19 years of age, they were considered beyond midlife because the average life span is ~30 years. They were therefore assumed to provide an appropriate model for TAM use in breast cancer survivors. At the time these experiments were initiated, women were receiving 20 or 40 mg of TAM/day given in two doses (~0.33–0.66 mg TAM/kg bw/day). We chose a daily TAM dose (2 mg/kg bw) that was ~3–6-fold higher than the human daily dose. In this study, monkeys (n = 3) were dosed with a total cumulative TAM dose of 52 mg/kg bw. One unexposed monkey received vehicle by naso-gastric intubation. DNA was prepared from brain cortex, liver, uterus, ovary, and kidney.

Detection of Adducts by TAM-DNA CIA.
For each assay, microtiter plates contained a TAM-DNA standard curve as well as test samples, each assayed on three wells coated with TAM-DNA and one well coated with unmodified DNA. In addition, samples were assayed on two to three different microtiter plates on different days. The TAM-DNA antibody is specific for both dG-TAM and dG-desmethyl-TAM, presumably recognizing both adducts in biological samples, and the immunoassay LOD was 0.3 adducts in 108 nucleotides (25) .

Values for TAM-DNA adducts determined using the TAM-DNA CIA are shown in Fig. 1ACitation and listed in Table 1Citation . The highest adduct levels were detected in the liver and brain cortex; all three animals gave measurable TAM-DNA levels in liver and brain cortex. Lower, but measurable, TAM-DNA levels were observed in the kidneys of all three exposed monkeys. The ovaries and uteri of two of the three animals had low but measurable levels of TAM-DNA adducts.



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. TAM-DNA formation (dG-TAM and/or dG-desmethyl-TAM adducts/108 nucleotides) measured in organs of three cynomolgus monkeys given 2 mg of TAM/kg bw/day by gavage for 30 days. In A, TAM-DNA CIA values are mean ± SE for three CIAs for brain cortex, liver, and uterus. Ovary and kidney values are mean ± range for two CIAs. Bars represent different TAM-exposed monkeys (14, gray bars; 04, black bars; and 15, white bars). B, comparison of TAM-DNA adduct levels determined in monkey organs by TAM-DNA CIA (gray bars) and HPLC-ES-MS/MS (black bars). Data points are mean ± SE of TAM-DNA adduct measurements from all three exposed animals. Animals with nondetectable values (see Table 1Citation ) were assigned a value half the LOD for the calculation of mean ± SE. Although both dG-TAM and dG-desmethyl-TAM can be detected by TAM-DNA CIA and HPLC-ES-MS/MS, dG-desmethyl-TAM was not detected in any of the samples by HPLC-ES-MS/MS.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Comparison of monkey TAM-DNA adducts (per 108 nucleotides) by two different methods

 
Detection of TAM-DNA Adducts by HPLC-ES-MS/MS.
Monkey DNA samples were also analyzed using a newly developed assay that involves on-line HPLC separation and ES-MS/MS detection of TAM-DNA adducts, using a deuterated adduct as internal standard (26) . This method recognizes dG-TAM and dG-desmethyl-TAM; the LOD for dG-TAM is ~0.2 adducts/108 nucleotides, whereas the detection of dG-desmethyl-TAM is ~10-fold less sensitive, when using 100 µg of DNA for analysis.

The values obtained using the HPLC-ES-MS/MS method were remarkably similar to those observed with the TAM-DNA CIA. The data are shown in Fig. 1BCitation and Table 1Citation , and representative chromotograms are shown in Figs. 2Citation and 3Citation . Again, the values in liver and brain cortex were the highest. There was an apparent discrepancy between the two methods for the values in the kidneys, because the HPLC-ES-MS/MS method gave values that were several-fold lower than those observed by TAM-DNA CIA in two of the three monkeys (Table 1)Citation . In the three exposed monkeys, DNA isolated from the uteri contained detectable dG-TAM levels by HPLC-ES-MS/MS, with values between 0.5 and 1.4 adducts/108 nucleotides (Table 1)Citation . Lower, but measurable, dG-TAM values were found in the ovaries of two monkeys. dG-desmethyl-TAM was not detected in any sample by HPLC-ES-MS/MS.



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Representative HPLC-ES-MS/MS analyses of TAM-DNA adducts in liver DNA from control (A and C) and TAM-treated (B and D) cynomolgus monkeys. The monkeys were treated as outlined in "Materials and Methods." A and B, MRM (relative signal intensity versus time) for the transition from the doubly charged protonated nucleoside molecule [(M +2H)2+] to the doubly charged protonated purine base [(BH +2H)2+] for 100 pg of the internal standard, dG-TAM-d6 (m/z 322 -> 264). C and D, MRM for the transition from [(M +2H)2+] to [(BH +2H)2+] for dG-TAM (m/z 319 -> 261).

 


View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Representative HPLC-ES-MS/MS analyses of TAM-DNA adducts in uterus DNA from control (A and C) and TAM-treated (B and D) cynomolgus monkeys. The monkeys were treated as outlined in "Materials and Methods." A and B, MRM (relative signal intensity versus time) for the transition from the doubly charged protonated nucleoside molecule [(M +2H)2+] to the doubly charged protonated purine base [(BH +2H)2+] for 100 pg of the internal standard, dG-TAM-d6 (m/z 322 -> 264). C and D, MRM for the transition from [(M +2H)2+] to [(BH +2H)2+] for dG-TAM (m/z 319 -> 261).

 
With the first set of liver DNA samples assayed using this method, the unexposed monkey (monkey number 07) gave a positive value of 5.1 adducts/108 nucleotides in the liver DNA. To determine whether this was an artifact caused by the analytical procedure, we subsequently assayed liver DNA samples from two additional unexposed monkeys (09 and 11) and did not observe positive values with either of those samples. We did, however, also find a value of 0.5 adduct/108 nucleotides in the brain of unexposed monkey 07 by HPLC-ES-MS/MS, whereas the values for brain cortex DNA from monkeys 09 and 11 were below the detection limit.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated the formation of TAM-DNA adducts in a monkey model using two methodological approaches: TAM-DNA CIA and HPLC-ES-MS/MS. Monkeys were exposed to 2 mg of TAM/kg bw daily for 30 days. The dose used (2 mg of TAM/kg bw/day) was ~6-fold higher than the current recommended daily dose for women. However, the monkeys in this study were only exposed for 1 month, whereas breast cancer patients typically receive TAM for 5 years. TAM-DNA adducts were found in most tissues examined, with the highest levels being detected in the liver and brain cortex. Although TAM-DNA CIA detected adduct levels of 0.7–2.1/108 nucleotides in kidney DNA samples, lower levels (0.2–0.4 adducts/108 nucleotides) were measured by HPLC-ES-MS/MS. If the major kidney DNA adduct was dG-desmethyl-TAM, it would not be detected by HPLC-ES-MS/MS at the levels indicated by TAM-DNA CIA. Most importantly, TAM-DNA adducts were found in the uteri and ovaries of all three exposed animals. The data demonstrate that nonhuman primates are capable of metabolizing TAM into genotoxic intermediates that form DNA adducts in suspected target tissues. The data also suggest that humans may have the metabolic capacity to form TAM-DNA adducts in organs at risk for TAM-induced cancers.

In addition to the TAM-DNA CIA and HPLC-ES-MS/MS analyses reported here, DNA samples from this study have been assayed by 32P-postlabeling-HPLC by Shibutani et al. (27) . The method differentiates between dG-TAM and dG-desmethyl-TAM and determines both adducts with similar sensitivity. The combined values for the two adducts were very similar to those reported here using TAM-DNA CIA and HPLC-ES-MS/MS. In the liver, all three exposed monkeys were positive, with a value of 4.43 ± 0.37 adducts/108 nucleotides (mean ± SD), whereas the brain cortex had 1.45 ± 0.91 adducts/108 nucleotides. One monkey had measurable adducts in the uterus (0.52 adducts/108 nucleotides), and two monkeys had measurable adducts in the ovaries (0.20 and 0.42 adducts/108 nucleotides). There were no adducts detected in the kidneys. For 32P-postlabeling-HPLC, the LOD varied with the tissue examined and was between 0.1 and 0.4 adducts/108 nucleotides. Because all three methods for determination of TAM-DNA adducts gave consistent positive values for monkey liver, brain cortex, uterus, and ovary, it is clear that the cynomolgus monkey is capable of metabolizing TAM into intermediates that form DNA adducts in many different organs, including organs at risk for cancer induction in humans.

Rats exposed to TAM develop liver (3 , 28) and uterine tumors (12) and have high levels of hepatic TAM-DNA adducts (4 , 8 , 29 , 30) . However, some laboratories have found no evidence of rat uterine TAM-DNA adducts (31 , 32) , whereas other groups have reported TAM-DNA adduct formation in this tissue (33 , 34) . Unlike rats, TAM-exposed mice are not susceptible to liver tumor induction (11) and have low levels of TAM-induced hepatic DNA adducts (8 , 10) . Similar to rats, the exposed mice develop uterine tumors, but there has been no evidence of uterine TAM-DNA adducts (35) . One study examined TAM-DNA adduct formation in rhesus monkeys (36) , but because this was a short-term exposure, it was not possible to ascertain tumor formation in either the liver or uterus. Rhesus monkeys were given two daily doses of 25 mg/kg bw for 1 week, and exposed male and female monkeys had hepatic TAM-DNA adduct levels of 10 and 14/108 nucleotides, respectively, when assessed by 32P-postlabeling. In this study, the unexposed controls had background levels of ~3.5 adducts/108 nucleotides (36) . In addition, the daily TAM dose chosen was 10-fold higher than the dose used for the experiments reported here.

There are strong associations between DNA damage, mutagenesis, and carcinogenesis. These associations are clearly evident in livers of rats exposed to TAM; however, in humans, there is controversy surrounding the detection of endometrial TAM-DNA adducts. Several groups have reported the formation of TAM-DNA adducts in leukocytes (37 , 38) and human endometrial tissue (19, 20, 21) . However, other groups have not detected TAM-specific human DNA adducts in either endometrium or leukocytes (5 , 22 , 23 , 24 , 39 , 40) . Humans exposed to TAM do not demonstrate an increased incidence of liver cancer (1) , and no TAM-specific DNA adducts have been detected in liver tissue from biopsies of several patients on TAM therapy (41) . In one study, {alpha}-hydroxy-TAM was present in human endometrial explant cultures, although no DNA adducts were detected in explants exposed to 20–500 µM TAM (22) . Therefore, although the formation of TAM-DNA adducts in human endometrial tissue may be possible, sufficiently sensitive methods for their reliable detection may not be available.

Methods used for human TAM-DNA adduct quantitation have primarily involved 32P-postlabeling. In 1996, Hemminki et al. (19) found positive TAM-DNA adducts in five of seven human endometrium samples at an adduct level of 2.7 in 109 nucleotides. However, the TAM-specific radioactivity was only twice the background, and its significance has been questioned (5 , 42) . Carmichael et al. (23 , 43) did not find TAM-DNA adducts in human endometrium samples, even when using the protocol of Hemminki et al. (19) . Shibutani et al. (20) first reported that 6 of 13 TAM-exposed patients had endometrial TAM-DNA levels of 1.5–13.1 adducts/108 nucleotides, determined by 32P-postlabeling-TLC. In a second study (21) , these same 13 samples were reanalyzed with three additional samples by an improved 32P-postlabeling-HPLC method, and 0.2–18 TAM-DNA adducts/108 nucleotides were detected in 8 of 16 samples. However, the cis/trans ratios of the TAM-DNA adducts reported in these studies were different from those reported to form chemically in tissue culture and animal models (5) . Martin (44) et al. used the highly sensitive accelerator mass spectrometry, with a single 14C-labeled TAM exposure in hysterectomy patients, and reported very low levels of uncharacterized adducts in the endometrium and myometrium. These controversial findings may be influenced by multiple factors, including variable 32P-postlabeling phosphorylation efficiencies, and the detection procedures used in different laboratories.

Overall, the data presented here suggest that a genotoxic mechanism could be partially involved in the etiology of endometrial cancer in breast cancer patients receiving TAM therapy. Although TAM is very effective in inhibiting breast cancer induction, the use of this drug in healthy "at-risk" women may require monitoring for endometrial abnormalities. Additional studies are needed to understand fully the link between TAM exposure, TAM-DNA adduct formation in endometrium, and endometrial cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Mariana Gerschenson for assisting in the collection of cynomolgus monkey tissues. We also thank Bettie Sugar for her editorial assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by a research grant from program POCTI, FCT, Portugal (to M. M. M.). Back

2 To whom requests for reprints should be addressed, at Carcinogen-DNA Interactions Section, National Cancer Institute, Building 37, Room 4032, NIH, 37 Convent Drive, Bethesda, MD 20892-4255. Phone: (301) 402-1835; Fax: (301) 402-8230; E-mail: poirierm{at}exchange.nih.gov Back

3 The abbreviations used are: TAM, tamoxifen; bw, body weight; dG-TAM, (E)-{alpha}-(deoxyguanosin-N2-yl)-tamoxifen; dG-desmethyl-TAM, (E)-{alpha}-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen; ES-MS/MS, electrospray ionization tandem mass spectrometry; LOD, limit of detection; MRM, multiple reaction monitoring; HPLC, high-performance liquid chromatography; CIA, chemiluminescence immunoassay; TAM-DNA, DNA modified with tamoxifen. Back

Received 2/27/03. Revised 5/23/03. Accepted 7/ 1/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet, 351: 1451-1467, 1998.[Medline]
  2. Fisher B., Costantino J. P., Wickerham D. L., Redmond C. K., Kavanah M., Cronin W. M., Vogel V., Robidoux A., Dimitrov N., Atkins J., Daly M., Wieand S., Tan-Chiu E., Ford L., Wolmark N., et al Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl. Cancer Inst. (Bethesda), 90: 1371-1388, 1998.[Abstract/Free Full Text]
  3. Greaves P., Goonetilleke R., Nunn G., Topham J., Orton T. Two-year carcinogenicity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res., 53: 3919-3924, 1993.[Abstract/Free Full Text]
  4. Carthew P., Rich K. J., Martin E. A., De Matteis F., Lim C-K., Manson M. M., Festing M. F. W., White I. N. H., Smith L. L. DNA damage as assessed by 32P-postlabelling in three rat strains exposed to dietary tamoxifen: the relationship between cell proliferation and liver tumour formation. Carcinogenesis (Lond.), 16: 1299-1304, 1995.[Abstract/Free Full Text]
  5. Phillips D. H. Understanding the genotoxicity of tamoxifen?. Carcinogenesis (Lond.), 22: 839-849, 2001.[Abstract/Free Full Text]
  6. White I. N. H. Anti-oestrogenic drugs and endometrial cancers. Toxicol. Lett., 120: 21-29, 2001.[Medline]
  7. Brown K. Breast cancer chemoprevention: risk-benefit effects of the antioestrogen tamoxifen. Expert Opin. Drug Saf., 1: 253-267, 2002.[Medline]
  8. White I. N. H., De Matteis F., Davies A., Smith L. L., Crofton-Sleigh C., Venitt S., Hewer A., Phillips D. H. Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells. Carcinogenesis (Lond.), 13: 2197-2203, 1992.[Abstract/Free Full Text]
  9. Carthew P., Lee P. N., Edwards R. E., Heydon R. T., Nolan B. M., Martin E. A. Cumulative exposure to tamoxifen: DNA adducts and liver cancer in the rat. Arch. Toxicol., 75: 375-380, 2001.[Medline]
  10. Martin E. A., Carthew P., White I. N. H., Heydon R. T., Gaskell M., Mauthe R. J., Turteltaub K. W., Smith L. L. Investigation of the formation and accumulation of liver DNA adducts in mice chronically exposed to tamoxifen. Carcinogenesis (Lond.), 18: 2209-2215, 1997.[Abstract/Free Full Text]
  11. Tucker M. J., Adam H. K., Patterson J. S. Tamoxifen Laurence D. R. McLean A. E. M. Weatherall M. eds. . Safety Testing of New Drugs, 125-161, Academic Press New York 1984.
  12. Carthew P., Edwards R. E., Nolan B. M., Martin E. A., Heydon R. T., White I. N. H., Tucker M. J. Tamoxifen induces endometrial and vaginal cancer in rats in the absence of endometrial hyperplasia. Carcinogenesis (Lond.), 21: 793-797, 2000.[Abstract/Free Full Text]
  13. Newbold R. R., Jefferson W. N., Padilla-Burgos E., Bullock B. C. Uterine carcinoma in mice treated neonatally with tamoxifen. Carcinogenesis (Lond.), 18: 2293-2298, 1997.[Abstract/Free Full Text]
  14. Rutqvist L. E., Johansson H., Signomklao T., Johansson U., Fornander T., Wilking N. Adjuvant tamoxifen therapy for early stage breast cancer and second primary malignancies. J. Natl. Cancer Inst. (Bethesda), 87: 645-651, 1995.[Abstract/Free Full Text]
  15. Curtis R. E., Boice J. D., Jr., Shriner D. A., Hankey B. F., Fraumeni J. F., Jr. Second cancers after adjuvant tamoxifen therapy for breast cancer. J. Natl. Cancer Inst. (Bethesda), 88: 832-834, 1996.
  16. Jordan V. C., Morrow M. Should clinicians be concerned about the carcinogenic potential of tamoxifen?. Eur. J. Cancer, 30A: 1714-1721, 1994.
  17. Bergman L., Beelen M. L. R., Gallee M. P. W., Hollema H., Benraadt J., van Leeuwen F. E. Risk and prognosis of endometrial cancer after tamoxifen for breast cancer. Assessment of Liver and Endometrial Cancer Risk Following Tamoxifen. Lancet, 356: 881-887, 2000.[Medline]
  18. Twombly R. FDA issues warning about ‘new’ tamoxifen risk. J. Natl. Cancer Inst. (Bethesda), 94: 1122 2002.[Free Full Text]
  19. Hemminki K., Rajaniemi H., Lindahl B., Moberger B. Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 56: 4374-4377, 1996.[Abstract/Free Full Text]
  20. Shibutani S., Suzuki N., Terashima I., Sugarman S. M., Grollman A. P., Pearl M. L. Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol., 12: 646-653, 1999.[Medline]
  21. Shibutani S., Ravindernath A., Suzuki N., Terashima I., Sugarman S. M., Grollman A. P., Pearl M. L. Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen. Carcinogenesis (Lond.), 21: 1461-1467, 2000.[Abstract/Free Full Text]
  22. Carmichael P. L., Ugwumadu A. H. N., Neven P., Hewer A. J., Poon G. K., Phillips D. H. Lack of genotoxicity of tamoxifen in human endometrium. Cancer Res., 56: 1475-1479, 1996.[Abstract/Free Full Text]
  23. Carmichael P. L., Sardar S., Crooks N., Neven P., Van Hoof I., Ugwumadu A., Bourne T., Tomas E., Hellberg P., Hewer A. J., Phillips D. H. Lack of evidence from HPLC 32P-post-labelling for tamoxifen- DNA adducts in the human endometrium. Carcinogenesis (Lond.), 20: 339-342, 1999.[Abstract/Free Full Text]
  24. Beland F. A., Churchwell M. I., Doerge D. R., Malejka-Giganti D., Zhang X., Divi R. L., Poirier M. C., Gamboa da Costa G., Marques M. M. High performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of tamoxifen DNA adducts in rats, monkeys, and humans. Proc. Am. Assoc. Cancer Res., 43: 343 2002.
  25. Divi R. L., Osborne M. R., Hewer A., Phillips D. H., Poirier M. C. Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling. Cancer Res., 59: 4829-4833, 1999.[Abstract/Free Full Text]
  26. Gamboa da Costa G., Marques M. M., Beland F. A., Freeman J. P., Churchwell M. I., Doerge D. R. Quantification of tamoxifen DNA adducts using on-line sample preparation and HPLC-electrospray ionization tandem mass spectrometry. Chem. Res. Toxicol., 16: 357-366, 2003.[Medline]
  27. Shibutani S., Suzuki N., Laxmi Y. R. S., Schild L. J., Divi R. L., Grollman A. P., Poirier M. C. Identification of tamoxifen-DNA adducts in monkeys treated with tamoxifen. Cancer Res., 63: 4402-4406, 2003.[Abstract/Free Full Text]
  28. Carthew P., Martin E. A., White I. N. H., De Matteis F., Edwards R. E., Dorman B. M., Heydon R. T., Smith L. L. Tamoxifen induces short-term cumulative DNA damage and liver tumors in rats: promotion by phenobarbital. Cancer Res., 55: 544-547, 1995.[Abstract/Free Full Text]
  29. Han X., Liehr J. G. Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res., 52: 1360-1363, 1992.[Abstract/Free Full Text]
  30. Martin E. A., Heydon R. T., Brown K., Brown J. E., Lim C. K., White I. N. H., Smith L. L. Evaluation of tamoxifen and {alpha}-hydroxytamoxifen 32P-post-labelled DNA adducts by the development of a novel automated on-line solid-phase extraction HPLC method. Carcinogenesis (Lond.), 19: 1061-1069, 1998.[Abstract/Free Full Text]
  31. Beland F. A., McDaniel L. P., Marques M. M. Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo. Carcinogenesis (Lond.), 20: 471-477, 1999.[Abstract/Free Full Text]
  32. Gamboa da Costa G., McDaniel-Hamilton L. P., Heflich R. H., Marques M. M., Beland F. A. DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives. Carcinogenesis (Lond.), 22: 1307-1315, 2001.[Abstract/Free Full Text]
  33. Pathak D. N., Pongracz K., Bodell W. J. Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: comparison with DNA adducts formed in the uterus of Sprague-Dawley rats treated with tamoxifen. Carcinogenesis (Lond.), 17: 1785-1790, 1996.[Abstract/Free Full Text]
  34. White I. N. H., Martin E. A., Mauthe R. J., Vogel J. S., Turteltaub K. W., Smith L. L. Comparisons of the binding of [14C]radiolabelled tamoxifen or toremifene to rat DNA using accelerator mass spectrometry. Chem. Biol. Interact., 106: 149-160, 1997.[Medline]
  35. Hellmann-Blumberg U., Taras T. L., Wurz G. T., DeGregorio M. W. Genotoxic effects of the novel mixed antiestrogen FC-1271a in comparison to tamoxifen and toremifene. Breast Cancer Res. Treat., 60: 63-70, 2000.[Medline]
  36. Comoglio A., Gibbs A. H., White I. N. H., Gant T., Martin E. A., Smith L. L., Gamalero S. R., DeMatteis F. Effect of tamoxifen feeding on metabolic activation of tamoxifen by the liver of the rhesus monkey: does liver accumulation of inhibitory metabolites protect from tamoxifen-dependent genotoxicity and cancer?. Carcinogenesis (Lond.), 17: 1687-1693, 1996.[Abstract/Free Full Text]
  37. Hemminki K., Rajaniemi H., Koskinen M., Hansson J. Tamoxifen-induced DNA adducts in leucocytes of breast cancer patients. Carcinogenesis (Lond.), 18: 9-13, 1997.[Abstract/Free Full Text]
  38. Umemoto A., Komaki K., Monden Y., Lin C-X., Momen H. D. A., Ueyama Y., Ravindernath A., Shibutani S. Formation of tamoxifen-DNA adducts in human leucocytes. Proc. Am. Assoc. Cancer Res., 43: 346 2002.
  39. Phillips D. H., Hewer A., Grover P. L., Poon G. K., Carmichael P. L. Tamoxifen does not form detectable DNA adducts in white blood cells of breast cancer patients. Carcinogenesis (Lond.), 17: 1149-1152, 1996.[Abstract/Free Full Text]
  40. Bartsch H., Phillips D. H., Nair J., Hewer A., Meyberg-Solomeyer G., Grischke E-M. Lack of evidence for tamoxifen- and toremifene-DNA adducts in lymphocytes of treated patients. Carcinogenesis (Lond.), 21: 845-847, 2000.[Abstract/Free Full Text]
  41. Martin E. A., Rich K. J., White I. N. H., Woods K. L., Powles T. J., Smith L. L. 32P-postlabelled DNA adducts in liver obtained from women treated with tamoxifen. Carcinogenesis (Lond.), 16: 1651-1654, 1995.[Abstract/Free Full Text]
  42. Orton T. C., Topham J. C. Correspondence re: Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 57: 4148 1997.[Free Full Text]
  43. Carmichael P. L. Mechanisms of action of antiestrogens: relevance to clinical benefits and risks. Cancer Investig., 16: 604-611, 1998.[Medline]
  44. Martin E. A. Tamoxifen–risks and opportunities. Toxicology, 168: 20-27, 2001.[Medline]



This article has been cited by other articles:


Home page
 MutagenesisHome page
K. Brown
Is tamoxifen a genotoxic carcinogen in women?
Mutagenesis, September 1, 2009; 24(5): 391 - 404.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Brown, E. M. Tompkins, D. J. Boocock, E. A. Martin, P. B. Farmer, K. W. Turteltaub, E. Ubick, D. Hemingway, E. Horner-Glister, and I. N.H. White
Tamoxifen Forms DNA Adducts in Human Colon after Administration of a Single [14C]-Labeled Therapeutic Dose
Cancer Res., July 15, 2007; 67(14): 6995 - 7002.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
S. Y. Kim, N. Suzuki, Y. R. S. Laxmi, and S. Shibutani
INEFFICIENT REPAIR OF TAMOXIFEN-DNA ADDUCTS IN RATS AND MICE
Drug Metab. Dispos., February 1, 2006; 34(2): 311 - 317.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
R. Singh and P. B. Farmer
Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection
Carcinogenesis, February 1, 2006; 27(2): 178 - 196.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
S. Y. Kim, Y. R. S. Laxmi, N. Suzuki, K. Ogura, T. Watabe, M. W. Duffel, and S. Shibutani
FORMATION OF TAMOXIFEN-DNA ADDUCTS VIA O-SULFONATION, NOT O-ACETYLATION, OF {alpha}-HYDROXYTAMOXIFEN IN RAT AND HUMAN LIVERS
Drug Metab. Dispos., November 1, 2005; 33(11): 1673 - 1678.
[Abstract] [Full Text] [PDF]

Home page
 MutagenesisHome page
D. H. Phillips, A. Hewer, M. R. Osborne, K. J. Cole, C. Churchill, and V. M. Arlt
Organ specificity of DNA adduct formation by tamoxifen and {alpha}-hydroxytamoxifen in the rat: implications for understanding the mechanism(s) of tamoxifen carcinogenicity and for human risk assessment
Mutagenesis, July 1, 2005; 20(4): 297 - 303.
[Abstract] [Full Text] [PDF]

Home page
 MutagenesisHome page
L. J. Schild, D. H. Phillips, M. R. Osborne, A. Hewer, F. A. Beland, M. I. Churchwell, K. Brown, M. Gaskell, E. Wright, and M. C. Poirier
Hepatic DNA adduct dosimetry in rats fed tamoxifen: a comparison of methods
Mutagenesis, March 1, 2005; 20(2): 115 - 124.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
F. A. Beland, M. I. Churchwell, D. R. Doerge, D. R. Parkin, D. Malejka-Giganti, A. Hewer, D. H. Phillips, P. L. Carmichael, G. Gamboa da Costa, and M. M. Marques
Electrospray Ionization-Tandem Mass Spectrometry and 32P-Postlabeling Analyses of Tamoxifen-DNA Adducts in Humans
J Natl Cancer Inst, July 21, 2004; 96(14): 1099 - 1104.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schild, L. J.
Right arrow Articles by Poirier, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schild, L. J.
Right arrow Articles by Poirier, M. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online