Immunohistochemical localization and semi-quantitation of hepatic tamoxifenDNA adducts in rats exposed orally to tamoxifen
Rao L. Divi,
Yvonne P. Dragan,1,
Henry C. Pitot,2 and
Miriam C. Poirier,3
National Cancer Institute, NIH, Bethesda, MD 20892,
1 Ohio State University, Columbus, OH 43210 and
2 University of Wisconsin, Madison, WI 53706, USA
 |
Abstract
|
---|
Administration of tamoxifen (TAM) has been shown to induce hepatocellular carcinogenesis and TAMDNA adduct formation in rat liver. Here we present TAMDNA adduct localization and semi-quantitation in hepatic tissue of rats by immunohistochemical staining followed by image analysis. We have also used a quantitative immunoassay to provide a validation for the immunohistochemical values. Rats were fed diets containing 0, 5, 50, 150 or 500 p.p.m. TAM for 45 weeks. Serial sections of paraffin-embedded liver were stained for TAMDNA adducts using a polyclonal TAMDNA antiserum. Subsequently, visualization of TAMDNA adducts was performed by peroxidase-conjugated secondary antibody-mediated signal amplification using biotinyl tyramide followed by streptavidinalkaline phosphatase and fast red. Semi-quantitation of nuclear color intensity was achieved with an Automated Cellular Imaging System (ACIS), with a detection limit of 1 TAMDNA adduct per 107 nt for these experiments. In parenchymal cells of liver sections from TAM-exposed animals a dose-dependent increase in nuclear staining was observed by ACIS and the TAMDNA adduct levels determined by ACIS were validated in liver DNA by quantitative chemiluminescence immunoassay (CIA). Comparison of semi-quantitative values determined by ACIS with quantitative values determined by CIA showed a strong correlation (r = 0.924) between the two methods. At 45 weeks of TAM exposure the liver cytoplasm contained placental glutathione S-transferase (GST-p)-positive foci, as indicated by new fuchsin staining. Staining of serial sections revealed a relative lack of TAMDNA adducts within these enzyme-altered foci. In addition, some GST-p foci contained islands of cells that did not stain for GST-p but were positive for TAMDNA adduct formation. This study validates the use of ACIS for TAMDNA adduct formation and demonstrates that steady-state TAMDNA adduct levels observed in livers of rats chronically fed TAM for several months increase in relation to dose. In addition, unlike the normal surrounding liver, preneoplastic GST-p-positive foci have virtually no TAMDNA adducts.
Abbreviations: ACIS, Automated Cellular Imaging System; CCD, charge coupled display; CIA, chemiluminescence immunoassay; GST-p, glutathione S-transferase, placental form; PBS, phosphate-buffered saline; TAM, tamoxifen.
 |
Introduction
|
---|
The non-steroidal anti-estrogen tamoxifen (TAM) is widely used as adjuvant therapy for all stages of breast cancer. In addition, it is administered as a prophylactic drug for the prevention of breast cancer in high-risk women (1,2). However, TAM therapy is associated with an increase in incidence of both premalignant and malignant lesions of the human endometrium (35). Chronic oral exposure to TAM has been shown to induce tumors in rodents (68) and these observations have raised concerns regarding use of the drug in large-scale prophylactic trials involving healthy women considered to be at high risk of developing breast cancer.
The association of TAMDNA adduct formation with TAM rodent tumorigenicity has been widely documented (912) as TAMDNA adducts have been detected in target organs of exposed experimental animal models. However, localization and visualization of TAMDNA adducts in target tissues have not been previously described. The 32P-post-labeling, accelerator mass spectrometry, HPLC and immunoassay methods used currently for quantitating TAMDNA adducts require the isolation of genomic DNA and therefore do not allow direct morphological localization of TAMDNA adducts. The immunohistochemical approach offers localization of TAMDNA adducts at the cellular level and takes into account the tissue architecture.
In the present study we report detection and visualization of TAMDNA adducts in liver tissue, a target for TAM-induced carcinogenesis in the rat. Polyclonal antibodies elicited against TAMDNA (13) were used for direct semi-quantitation of TAMDNA adducts in sections of paraffin-embedded liver from rats exposed to TAM. The localization of TAMDNA adducts was achieved by immunohistochemistry and semi-quantitation was achieved using an Automated Cellular Imaging System (ACIS). The ACIS is a robotic bright field microscope module equipped with advanced color space transformation software that allows sensitive and specific identification of the color of interest in nuclei or cytoplasm of cells or tissue sections. A doseresponse relationship for hepatic TAMDNA adducts determined by ACIS has been validated by quantitative comparison of TAMDNA adducts in extracted rat liver DNA using a chemiluminescence immunoassay (CIA) (13). We also investigated TAMDNA adduct formation in hepatic glutathione S-transferase, placental form (GST-p)-altered foci, which increased in number with TAM dose level.
 |
Materials and methods
|
---|
Rat maintenance and exposure
Timed pregnancy, late gestation SpragueDawley rats were obtained from Harlan Sprague Dawley. At 4 weeks of age neonatal female pups were retained for the study described. At the time of weaning, 28 days of age, female animals were fed a Teklad mouse/rat powdered basal diet containing 4% fat (Teklad Test Diets; Harlan Teklad, Madison, WI). Fifteen animals per group were fed this basal diet, to which TAM (Sigma-Aldrich Chemcial Co., St Louis, MO) was added at levels of 0, 5, 50, 150 or 500 mg TAM/kg diet, (P.P.M.) The animals were exposed continuously until the experiment was terminated after 45 weeks of TAM feeding.
The livers were removed, sliced and fixed in 10% neutral buffered formalin. The tissue slices were processed, embedded in low melting point paraffin and sectioned with a microtome. Serial sections of 5 µm thickness were obtained. Sequential sections were stained for hematoxylin and eosin, GST-p and TAMDNA adducts.
TAMDNA staining by immunohistochemistry
Liver sections were deparaffinized as follows: xylene, two changes of 3 min each; 100% ethanol, two changes of 1 min each; 90% ethanol, 1 min; 80% ethanol, 1 min; 70% ethanol, 1 min; 50% ethanol, 1 min; 25% ethanol, 1 min; deionized water, 1 min; phosphate-buffered saline (PBS) without Ca2+ and Mg2+, two changes of 1 min followed by one change of 30 min. For antigen retrieval by microwaving, slides were incubated with 10 µg/ml proteinase K (Boehringer Mannheim GmbH, Mannheim, Germany) at room temperature (22°C) for 10 min, rinsed in deionized water and placed in a plastic staining holder. Empty slots in the holder were filled with blank slides. The holder was placed in a microwave-resistant container containing 250 ml of 1x Antigen Retrieval Citra solution (BioGenex, San Ramon, CA). Microwaving was performed at high power for 2 min to bring the solution to a quick boil, followed by 15 min on low power (50% level) to simmer the slides. During simmering, a glass beaker containing 500 ml of deionized water was kept along with the slide container to absorb excess heat and maintain a constant temperature. At the end of microwaving the container holding the slides was allowed to reach room temperature and the slides were rinsed with several changes of deionized water followed by equilibration in PBS for 3 min at 22°C. Non-specific binding was blocked by incubation of slides with 0.25% casein (containing ultra low alkaline phosphatase; Tropix, Bedford, MA) for 20 min. Slides were then incubated with anti-TAMDNA rabbit antiserum (13) diluted 1:5000 in 0.25% casein in PBS with 0.05% Tween 20, applied by hand and incubated for 12 h at 4°C. Amplified color development was achieved with one cycle of amplification using a peroxidase-linked anti-rabbit IgG antibody (1:2000 dilution, 30 min at 22°C), biotinylated tyramide and H2O2 (Dako Corp., Carpinteria, CA), streptavidin linked to alkaline phosphatase (30 min at 22°C) (BioGenex), fast red as a substrate (610 min at 22°C) (BioGenex) and aqueous hematoxylin (BioGenex) as a counterstain (20 s).
Control experiments, carried out in order to ensure that the observed staining was due to the presence of TAMDNA adducts, included: immunohistochemical staining with TAMDNA antiserum preabsorbed with the immunogen TAMDNA; treatment of sections with DNase before immunostaining; incubation with normal rabbit serum or irrelevant secondary antibodies. To prepare preabsorbed serum, TAMDNA (2.6% modified) was dissolved in PBS containing 0.1% Tween 20 and 0.25% casein, the pH was adjusted to 7.4 with 5 M NaOH and the solution was mixed with TAMDNA antiserum (1:10 dilution in PBS), giving a final concentration of 250 µg TAMDNA/ml antiserum. Two preabsorptions, one for 4 h at 22°C and one at 4°C overnight, were performed with centrifugation (3000 r.p.m.) after each incubation. The resulting supernatant was diluted to the same extent as the specific antiserum and used routinely as a control. In another control experiment the sections were pretreated with DNase (100 U/ml for 1 h at 37°C) (Promega, Madison, WI) and rinsed with water, followed by two changes of PBS before incubation with the specific antiserum. In a third type of control experiment, normal rabbit antiserum was used at a dilution equivalent to that of the TAMDNA antiserum to determine the level of non-specific staining generated by the detection system. Finally, the TAMDNA rabbit antiserum was used in combination with biotinylated anti-mouse IgG, an irrelevant secondary antibody, as an additional control.
Quantitative imaging using ACIS
Sections were scanned and images were captured using ACIS (ChromaVision, San Juan Capistrano, CA). The instrument uses an Olympus bright field microscope equipped with an automated robotic slide moving platform, 4x60x objectives, a charge coupled display (CCD) camera and ACIS 1.81 Microdensitometry Software (ChromaVision). The CCD camera produces a voltage signal proportional to transmitted light intensity, which is then converted into a numerical density (intensity) measurement. Entire tissue sections or designated regions were scanned using the 10x objective and images were captured using the 40x objective. Typically 10 00020 000 hepatic nuclei were counted and the average nuclear intensity for all nuclei in a section or defined circle/square/rectangle of tissue was expressed in arbitrary units. The ACIS advanced color space transformation software provides sensitive and specific identification of the color of interest and the pattern recognition imaging technology allows sensitive and specific identification of morphological changes. Therefore, the instrument has the capacity to select a specific color of interest (hue) within the morphological feature of interest and to express the intensity of nuclear staining (luminosity). Thresholds for nuclear color (hue) were set on a slide containing visually negatively stained cells with hematoxylin (blue) nuclear staining (hue range 160195). The nuclear threshold in cells with no adducts was able to mask the blue nuclei in positive cells and therefore only the presence of the pink color of interest was detected. The non-specific cytosolic light pink background (hue range 120) was filtered using the appropriate color (hue) threshold. The color threshold for TAMDNA adducts was high enough to detect faint to intense pink color (hue range 200246) staining in nuclei positive for adducts. Thresholds for intensity (luminosity) for light pink cytosolic background and intense pink nuclear staining were set at the ranges 180216 and 45170, respectively. Using a 7 µm morphological filter it was possible to eliminate the contribution of cytosol intensity (luminosity) to nuclear staining. The luminosity thresholding range chosen took into consideration the slight differences in counterstaining between slides, to which ACIS is very sensitive. After the thresholds were set, the entire section or specific defined areas were scanned using the10x objective and an intensity profile was obtained.
GST-p immunohistochemistry
Staining with a GST-p antiserum was used to localize enzyme-altered and non-altered regions of the liver. After deparaffinization and antigen retrieval, as described above, sections from between three and five rats of each experimental group were incubated with anti GST-p antibody (Biogenex), followed by biotinylated anti-rabbit antibody and streptavidinalkaline phosphatase, with new fuchsin as substrate and hematoxylin as counterstain. Control experiments were carried out with normal rabbit serum. The slides were scored for number of foci positive for cytoplasmic GST-p staining and the results are expressed as number of foci per 20 000 cells (measured using ACIS) in the tissue section.
CIA of TAMDNA adducts
DNA was extracted by a non-organic extraction method (Stratagene, La Jolla, CA) followed by digestion with 1 U/ml amyloglucosidase (Boehringer Mannheim). Subsequently, DNA (310 µg) was sonicated (15 s) and heat denatured (5 min at 95°C) before being subjected to adduct quantitation by TAMDNA CIA (13). Sample quantitation was achieved by comparison with a TAMDNA standard curve in which 0.8 ± 0.12 fmol TAM in TAMDNA gave 50% inhibition. Since up to 20 µg DNA could be analyzed, the lower limit of detection was calculated as ~10 amol adduct/µg DNA, or ~ 3 adducts/109 nt.
 |
Results
|
---|
Immunohistochemical detection and semi-quantitation of TAMDNA adducts in livers of TAM-exposed rats
A dose-dependent increase in TAMDNA adduct nuclear staining was determined by ACIS in hepatic nuclei from rats (n = 3) administered 5, 50, 150 and 500 p.p.m. TAM/kg diet for 45 weeks (Table I
). Representative specific staining for TAMDNA adducts in livers of rats exposed to 0, 150 and 500 p.p.m. TAM for 45 weeks is shown in Figure 1
. The presence of TAMDNA adducts (pink-red staining) is evident in livers from TAM-fed rats (Figure 1D and F
), but not in the unexposed control (Figure 1B
). The TAMDNA adduct staining was homogeneously distributed throughout the hepatic lobes, although a slight intralobular difference in color intensity was occasionally observed. The staining appeared primarily in parenchymal cells and no staining was observed in bile duct cells. Nuclear staining was very intense in liver sections from rats fed 150 and 500 p.p.m. TAM and at these doses a weaker cytosolic pink staining was observed.
View this table:
[in this window]
[in a new window]
|
Table I. TAMDNA adduct levels determined in livers of rats exposed to dietary TAM, at the indicated doses, for 45 weeks
|
|

View larger version (123K):
[in this window]
[in a new window]
|
Fig. 1. Representative immunohistochemical staining for TAMDNA adducts (pink-red color) in liver sections from rats fed 0 (A and B), 150 (C and D) and 500 p.p.m. TAM (E and F) in the diet for 45 weeks. Staining with rabbit TAMDNA antiserum is shown in (B), (D) and (F), while staining with normal rabbit antiserum is shown in (A), (C) and (E). Counterstaining with aqueous hematoxylin (blue color) reveals localization of the nuclei. 600x magnification.
|
|
Substitution of the TAMDNA antiserum by normal rabbit serum (Figure 1A
, C and E) or substitution of the secondary antibody by non-specific secondary antibody (data not shown) abrogated the pink nuclear staining, while the blue hematoxylin staining of the nuclei remained. The specificity of the immunostaining was further confirmed by preabsorption of the TAMDNA antiserum with an excess of the immunogen TAMDNA. Incubation of slides with preabsorbed anti-TAMDNA produced a very weak staining of hepatic cytosol but no nuclear staining (data not shown). In addition, pretreatment of slides with DNase virtually abolished nuclear TAMDNA staining (data not shown).
Validation of the ACIS for TAMDNA adduct determination
In order to validate the numbers obtained with ACIS, a quantitative TAMDNA CIA (13) was used to determine TAMDNA adducts in additional rats (n = 3 per dose) fed the same TAM doses (0, 5, 50, 150 and 500 p.p.m.) in the diet for 45 weeks. Table I
lists the values obtained using both ACIS and CIA and Figure 2
demonstrates the correlation of the data obtained by the two methods. There was a strong correlation (r = 0.9236) between the arbitrary intensity units obtained by immunohistochemistry/ACIS and values generated by quantitative CIA. Since the rats were subjected to chronic TAM feeding for 45 weeks, the TAMDNA adduct values at each dose reflect both DNA adduct formation and DNA adduct removal.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2. Correlation of TAMDNA adduct levels in liver sections measured by semi-quantitative immunohistochemistry/ACIS and in liver DNA measured by TAMDNA CIA. Rats were exposed for 45 weeks to 0, 5, 50, 150 and 500 p.p.m. TAM in the diet. The values represent means ± SD of three animals at each exposure level.
|
|
GST-p-altered foci
The foci positive for expression of GST-p in liver sections from TAM-exposed rats (n = 5 per dose) were examined for TAMDNA adduct formation. Representative GST-p staining of unexposed liver and TAM-exposed liver are shown in Figure 3A and B
, respectively. No foci were observed in livers from five unexposed rats. Expression of GST-p is observed as red staining localized in the cytoplasm with intense nuclear membrane staining and faint nuclear staining. The number and size of the GST-p foci (Figure 3B
) increased with increasing TAM dose with an average of 253 foci/liver, including all three liver lobes, observed in rats fed 500 p.p.m. TAM.

View larger version (124K):
[in this window]
[in a new window]
|
Fig. 3. Sections of livers from rats fed TAM (500 p.p.m.) for 45 weeks were stained for GST-p (AC and E) and TAMDNA (D and F), respectively. No GST-p-positive enzyme-altered foci were seen in unexposed animals (A), while (B) shows a representative GST-p-positive focus from a TAM-exposed animal. (C and D) Serial sections of a focus stained for GST-p and TAMDNA, respectively, demonstrating a comparative lack of TAMDNA adducts (blue arrow in D) in the GST-p-positive focus (green arrow in C). (E and F) Close-ups of the region outlined in (C) and (D) demonstrating islets of cells within the focus that are positive for TAMDNA adducts (black arrows in F) and negative for GST-p (yellow arrows in E). Magnifications: (A) and (B), 600x; (C) and (D), 300x; (E) and (F), 1200x.
|
|
The levels of TAMDNA adducts and GST-p foci increased with increasing TAM dose (Figure 4
). In addition, examination of many foci revealed that there was virtually no TAMDNA staining (Figure 3D
) within the GST-p focal areas (Figure 3C and D
). However, within some of the GST-p foci there were clusters of cells that were negative for GST-p (Figure 3C and E
) and that showed intense staining for TAMDNA adducts (Figure 3D and F
). Similar to studies with other hepatocarcinogens, including aflatoxins and N-2-acetlyaminofluorene (14), these data suggest that the GST-p foci induced by chronic TAM administration can either detoxify TAM more efficiently than normal liver or fail to activate the drug. In addition, the small adduct-positive focal areas within the GST-p foci have presumably undergone a second mutagenic event that alters the original change and results in the formation of TAMDNA adducts.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4. Comparison of TAMDNA adduct levels (by ACIS) (·· ··) with numbers of GST-p-positive foci () in sections of livers from rats fed 0, 5, 50, 150 and 500 p.p.m. TAM in the diet for 45 weeks. Liver sections from between three and five rats at each exposure level were scored for GST-p-positive foci and values are expressed as numbers of foci per 20 000 cells.
|
|
 |
Discussion
|
---|
In this report we have described and validated a unique semi-quantitative immunohistochemical staining method for the determination of TAMDNA adducts. This immunohistochemical semi-quantitation is made possible through the use of the ChromaVision ACIS, a novel bright field microscope and image analyzer that provides arbitrary numbers for the intensity of nuclear color staining for tissue sections and is able to count 10 00020 000 cells within minutes. An entire tissue section is scanned, using an automated bright field microscope equipped with a robotic slide platform, and the program calculates an average color intensity score per nucleus. Photographic images are obtained using a CCD camera and stored for permanent record. The values determined by ACIS in liver sections from rats fed TAM at different levels in the diet were compared with quantitative determination (CIA) of TAMDNA adducts in liver DNA from similarly exposed rats and results from the two assays were highly correlated. The data therefore suggest that semi-quantitative immunohistochemical staining for TAMDNA adducts will be a powerful tool for many different types of studies where only small amounts of tissue are available.
In addition, these experiments demonstrated that, similar to a number of other chronically administered hepatocarcinogens (1416), TAM induces the formation of hepatic enzyme-altered foci that have lost the capacity to metabolize the drug to DNA-binding species. In this study foci expressing GST-p were induced in a dose-related fashion and were lacking TAMDNA adducts. Within some of the large GST-p-positive foci occasional islets of cells were negative for GST-p and positive for TAMDNA adducts, suggesting that a further mutation may have altered expression and/or activity of drug metabolizing enzymes (17,18), resulting in a changed focus phenotype. Previous studies, both in the liver and in the skin, have demonstrated that malignant conversion of a benign tumor (skin) (19,20) or an enzyme-altered focus (liver) (21) may arise subsequent to a mutation that produces a malignant clonal expansion of cells within a larger benign focus of altered phenotype. Both in the liver (21) and in the skin (19,20) it has been possible to observe very small foci of more malignant phenotype located within the original benign clonal expansion.
The antibody used here to stain liver sections from rats given dietary exposure to TAM was elicited against TAMDNA (13). In the CIA (13) the antibody showed cross-reactivity with TAM alone at concentrations 5000-fold higher than the TAMDNA adduct concentration. The CIA 50% inhibition occurred at 5440 ± 100.2 fmol with TAM compared with 0.8 ± 0.13 fmol with the TAMDNA adduct (13). In the present study there was some cytoplasmic staining that might have been the result of reactivity of antibody either with TAM alone, with TAM-modified mitochondrial DNA or with TAM-modified protein. A decrease in cytoplasmic background was observed when the sections were incubated with proteinase K for extended periods of time, but this treatment resulted in loss of cell structural integrity and was therefore not feasible to apply routinely. Fortunately, the sophistication of the ACIS software routines made it possible to subtract the contribution of cytoplamic staining, thereby removing its influence from the nuclear intensity values.
Glutathione S-transferases are Phase II detoxification enzymes that catalytically enhance the conjugation of glutathione to electrophilic cytotoxic compounds. A high level of GST-p in placenta prevents absorption of potential toxicants by the fetus. However, the enzyme is not normally expressed in adult liver and it is one of several enzymes that are typically aberrantly expressed in hepatic preneoplastic foci in response to xenobiotic administration (2225). Events that could contribute to the lack of TAMDNA adduct formation in GST-p-altered preneoplastic foci include increased cell replication, increased DNA repair, down-regulation of the enzymes required to metabolize TAM to DNA-binding species and up-regulation of the enzymes (including GSTs) involved in TAM detoxification. Elevated levels of detoxification enzymes (GSTs) have been shown to correlate with resistance to chemotherapy in endometrial cancer (26), lymphocytic leukemia (27), kidney and lung carcinomas (28) and acute leukemias (29). Thus, the response of the liver in inducing GST-p subsequent to TAM exposure confers a growth advantage to cells resistant to the persistent chemical insult. Resistance mechanisms that have been elucidated in multiple human tumor studies (26,28,30,31) include induction of both P-glycoprotein and GST-p and topoisomerase II downregulation. The simultaneous occurrence of these events suggests a common regulatory mechanism at the genetic level. Preliminary studies from our laboratory indicate that P-glycoprotein may be expressed in tandem with GST-p in TAM-exposed rat liver.
This study demonstrates the feasibility of analyzing tissue sections for the presence of TAMDNA adducts at the cellular level. This approach facilitates the tracking of DNA adduct formation and disappearance in cells and provides for differential localization within tissues. It should be feasible for use with small amounts of human tissue, such as in molecular epidemiology studies. However, it remains to be seen whether the present method will be sufficiently sensitive to demonstrate the presence of TAMDNA adducts in tissue from TAM-exposed women. Currently we are exploring the possibility of obtaining human endometrium for immunohistochemical studies.
 |
Notes
|
---|
3 To whom correspondence should be addressed at: CarcinogenDNA Interactions Section, National Cancer Institute, Building 37, Room 2A05, NIH, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255, USA. Email: poirierm{at}exchange.nih.gov 
 |
Acknowledgments
|
---|
We thank Dr Stuart Yuspa for helpful discussions and critical reading of the manuscript. We also thank Mrs Bettie Sugar for editorial assistance and Ms Johanna Eltz for technical assistance. In addition, we would like to thank Mr Hai (Harry) Zhang and Mr Syd Mandelbaum of ChromaVision Inc. for exceptional assistance in developing and providing the microdensitometry software applications used in ACIS and Drs David H.Phillips and Martin R.Osborne for their generous gift of immunogen TAMDNA.
 |
References
|
---|
-
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. and Wolmark,N. (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl Cancer Inst., 90, 13711388.[Abstract/Free Full Text]
-
Fisher,B. (1999) Highlights from recent National Surgical Adjuvant Breast and Bowel Project studies in the treatment and prevention of breast cancer. CA Cancer J. Clin., 49, 159177.[Abstract/Free Full Text]
-
Rutqvist,L.E., Johansson,H., Signomklao,T., Johansson,U., Fornander,T. and Wilking,N. (1995) Adjuvant tamoxifen therapy for early stage breast cancer and second primary malignancies. Stockholm Breast Cancer Study Group. J. Natl Cancer Inst., 87, 645651.[Abstract]
-
Dallenbach-Hellweg,G., Schmidt,D., Hellberg,P., Bourne,T., Kreuzwieser, E., Doren,M., Rydh,W., Rudenstam,G. and Granberg,S. (2000) The endometrium in breast cancer patients on tamoxifen. Arch. Gynecol. Obstet., 263, 170177.[ISI][Medline]
-
Assikis,V.J., Neven,P., Jordan,V.C. and Vergote,I. (1996) A realistic clinical perspective of tamoxifen and endometrial carcinogenesis. Eur. J. Cancer, 32A, 14641476.
-
Hirsimaki,P., Hirsimaki,Y., Nieminen,L. and Payne,B.J. (1993) Tamoxifen induces hepatocellular carcinoma in rat liver: a 1-year study with two antiestrogens. Arch. Toxicol., 67, 4954.[ISI][Medline]
-
Diwan,B.A., Anderson,L.M. and Ward,J.M. (1997) Proliferative lesions of oviduct and uterus in CD-1 mice exposed prenatally to tamoxifen. Carcinogenesis, 18, 20092014.[Abstract]
-
Newbold,R.R., Jefferson,W.N., Padilla-Burgos,E. and Bullock,B.C. (1997) Uterine carcinoma in mice treated neonatally with tamoxifen. Carcinogenesis, 18, 22932298.[Abstract]
-
White,I.N., de Matteis,F., Davies,A., Smith,L.L., Crofton-Sleigh,C., Venitt,S., Hewer,A. and Phillips,D.H. (1992) 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, 13, 21972203.[Abstract]
-
Han,X.L. and Liehr,J.G. (1992) Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res., 52, 13601363.[Abstract]
-
Li,D., Dragan,Y., Jordan,V.C., Wang,M. and Pitot,H.C. (1997) Effects of chronic administration of tamoxifen and toremifene on DNA adducts in rat liver, kidney and uterus. Cancer Res., 57, 14381441.[Abstract]
-
Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and Phillips,D.H. (1996) Identification of the major tamoxifen-deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res., 56, 6671.[Abstract]
-
Divi,R.L., Osborne,M.R., Hewer,A., Phillips,D.H. and Poirier,M.C. (1999) Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling. Cancer Res., 59, 48294833.[Abstract/Free Full Text]
-
Huitfeldt,H.S., Hunt,J.M., Pitot,H.C. and Poirier,M.C. (1988) Lack of acetylaminofluoreneDNA adduct formation in enzyme-altered foci of rat liver. Carcinogenesis, 9, 647652.[Abstract]
-
Gopalan,P., Tsuji,K., Lehmann,K., Kimura,M., Shinozuka,H., Sato,K. and Lotlikar,P.D. (1993) Modulation of aflatoxin B1-induced glutathione S-transferase placental form positive hepatic foci by pretreatment of rats with phenobarbital and buthionine sulfoximine. Carcinogenesis, 14, 14691470.[Abstract]
-
Hiruma,S., Qin,G.Z., Gopalan-Kriczky,P., Shinozuka,H., Sato,K. and Lotlikar,P.D. (1996) Effect of cell proliferation on initiation of aflatoxin B1-induced enzyme altered hepatic foci in rats and hamsters. Carcinogenesis, 17, 24952499.[Abstract]
-
Nuwaysir,E.F., Daggett,D.A., Jordan,V.C. and Pitot,H.C. (1996) Phase II enzyme expression in rat liver in response to the antiestrogen tamoxifen. Cancer Res., 56, 37043710.[Abstract]
-
Nuwaysir,E.F., Dragan,Y.P., Jefcoate,C.R., Jordan,V.C. and Pitot,H.C. (1995) Effects of tamoxifen administration on the expression of xenobiotic metabolizing enzymes in rat liver. Cancer Res., 55, 17801786.[Abstract]
-
Hennings,H., Shores,R., Mitchell,P., Spangler,E.F. and Yuspa,S.H. (1985) Induction of papillomas with a high probability of conversion to malignancy. Carcinogenesis, 6, 16071610.[Abstract]
-
Hennings,H., Shores,R., Wenk,M.L., Spangler,E.F., Tarone,R. and Yuspa,S.H. (1983) Malignant conversion of mouse skin tumours is increased by tumour initiators and unaffected by tumour promoters. Nature, 304, 6769.[ISI][Medline]
-
Scherer,E., Feringa,A.W. and Emmelot,P. (2001) Initiation-promotion-initiation. Induction of neoplastic foci within islands of precancerous liver cells in the rat. IARC Sci. Publ., 19, 5766.
-
Ahotupa,M., Hirsimaki,P., Parssinen,R. and Mantyla,E. (1994) Alterations of drug metabolizing and antioxidant enzyme activities during tamoxifen-induced hepatocarcinogenesis in the rat. Carcinogenesis, 15, 863868.[Abstract]
-
Ishii,I. and Kitada,M. (1997) Multidrug-resistance by induction of inactivation for anti-cancer drugs (in Japanese). Nippon Rinsho, 55, 10441049.[Medline]
-
Carthew,P., Edwards,R.E. and Nolan,B.M. (1997) Depletion of hepatocyte nuclear estrogen receptor expression is associated with promotion of tamoxifen induced GST-P foci to tumours in rat liver. Carcinogenesis, 18, 11091112.[Abstract]
-
Dragan,V.P., Vaughan,J., Jordan,V.C. and Pitot,H.C. (1995) Comparison of the effects of tamoxifen and toremifene on liver and kidney tumor promotion in female rats. Carcinogenesis, 16, 27332741.[Abstract]
-
Schneider,J., Efferth,T., Rodriguez-Escudero,F.J. and Volm,M. (1994) Intrinsic overexpression of two different mechanisms of resistance to chemotherapy (P-glycoprotein and GST-pi) in human endometrial carcinoma. Chemotherapy, 40, 265271.[ISI][Medline]
-
Holmes,J., Wareing,C., Jacobs,A., Hayes,J.D., Padua,R.A. and Wolf,C.R. (1990) Glutathione-s-transferase pi expression in leukaemia: a comparative analysis with mdr-1 data. Br. J. Cancer, 62, 209212.[ISI][Medline]
-
Volm,M., Mattern,J., Efferth,T. and Pommerenke,E.W. (1992) Expression of several resistance mechanisms in untreated human kidney and lung carcinomas. Anticancer Res., 12, 10631067.[ISI][Medline]
-
Koberda,J. and Hellmann,A. (1991) Glutathione S-transferase activity of leukemic cells as a prognostic factor for response to chemotherapy in acute leukemias. Med. Oncol. Tumor Pharmacother., 8, 3538.[ISI][Medline]
-
Morrow,C.S., Smitherman,P.K., Diah,S.K., Schneider,E. and Townsend,A.J. (1998) Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells. J. Biol. Chem., 273, 2011420120.[Abstract/Free Full Text]
-
Thorgeirsson,S.S., Huber,B.E., Sorrell,S., Fojo,A., Pastan,I. and Gottesman,M.M. (1987) Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat liver. Science, 236, 11201122.[ISI][Medline]
Received April 4, 2001;
revised June 5, 2001;
accepted June 12, 2001.