Short-term dosing of {alpha}-hydroxytamoxifen results in DNA damage but does not lead to liver tumours in female Wistar/Han rats

Ian N.H. White1,4, Philip Carthew1,1, Reginald Davies1, Jerry Styles1, Karen Brown1,2, John E. Brown1,3, Lewis L. Smith1 and Elizabeth A. Martin1

MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester, LE1 9HN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is now generally accepted that activation of tamoxifen occurs as a result of metabolism to {alpha}-hydroxytamoxifen. In this study, {alpha}-hydroxytamoxifen was given to female Wistar/Han rats (0.103 or 0.0103 mmol/kg, intraperitoneally, daily for 5 days). This resulted in liver DNA damage, determined by 32P-post-labelling, of 3333 ± 795 or 343 ± 68 adducts/108 nucleotides, respectively (mean ± SD, n = 4). Following HPLC separation, the retention times of the major {alpha}-hydroxytamoxifen DNA adducts were similar to those seen following the administration of tamoxifen. However, after rats were treated with {alpha}-hydroxytamoxifen (0.103 mmol/kg) for 5 days and the animals kept for up to 13 months, no liver tumours developed (0/7 rats), even with phenobarbital promotion (0/5 rats). GST-P foci were detected in the liver, but only after 13 months was their number or area significantly increased over the corresponding controls. When {alpha}-hydroxytamoxifen was given to female {lambda}/lacI transgenic rats (0.103 mmol/kg orally for 10 days) and the animals killed 46 days later, there was an approximate 1.8-fold increase in mutation frequency but no significant increase in G:C to T:A transversions as described after tamoxifen treatment. It is concluded that DNA damage alone, resulting from the short-term administration of {alpha}-hydroxytamoxifen, is not sufficient to initiate liver tumours even with phenobarbital promotion. As with tamoxifen, long-term exposure may be required to allow promotion and progression of transformed cells.

Abbreviations: BrdU, 5-bromodeoxyuridine; GST-P, placental form of glutathione S-transferase; PB, phenobarbital; PCNA, proliferating cell nuclear antigen.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The anti-oestrogenic drug tamoxifen is widely used as adjuvant therapy in the treatment of breast cancer in women (1). This drug may also be effective in the chemoprevention of breast cancer (2). The optimum duration of treatment has not been clearly established but results suggest at least 5 years is beneficial (3). Following long-term treatment in rats, tamoxifen is a liver carcinogen. In female Wistar rats, 11 months exposure to dietary tamoxifen (420 p.p.m.) results in about a 60% incidence of hepatocellular carcinomas (4), while in Sprague–Dawley rats, 12 months daily dosing of 22.5 mg/kg tamoxifen results in 100% incidence of liver tumours (5). The time to tumour is shortened by the promoting effects of phenobarbital in the drinking water (6). Tamoxifen has to undergo activation by CYP-dependent monooxygenases to give an electrophile that binds to DNA (7). A number of active intermediates have been proposed, but it is now generally accepted that activation occurs as a result of metabolism of tamoxifen first to {alpha}-hydroxytamoxifen that is then converted to the sulfate ester (810). When tamoxifen is given to female {lambda}/lacI transgenic rats hepatic DNA damage leads to an increase in the frequency of gene mutations at lacI (11). In treated women there is evidence for an increased incidence of uterine endometrial (2,12) but not liver tumours (13). The presence of DNA damage in the target cells of tamoxifen treated women is controversial (14), and even if low levels of damage occurs (7), it is questionable if this is causally related to the increase in endometrial tumours (15,16). The role of {alpha}-hydroxytamoxifen as a carcinogen or mutagen in rats or humans in vivo has not been established. In this study, the relationship between DNA damage resulting from the administration to rats of {alpha}-hydroxytamoxifen and the subsequent development of liver lesions is investigated.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatments
trans {alpha}-Hydroxytamoxifen was prepared according to published procedures (17,18). Female 6 week-old Wistar/Han rats were obtained from Harlan Olac (Bicester, UK). 120 animals were housed in negative pressure isolators with a 12 h light/dark cycle at 19–22°C. Animals were dosed with {alpha}-hydroxytamoxifen dissolved in tricaprylin, 40 animals at 0.103 mmol/kg and 40 animals at 0.0103 mmol/kg, intraperitoneally (40 and 4 mg/ml, respectively) daily for 5 days, while controls received tricaprylin only. At the end of the dosing period animals were divided into two groups. One group of 54 rats was subsequently given water supplemented with 0.1% (w/v) phenobarbital and 54 rats received no phenobarbital water supplement. At 24 h after the final dose and at 3 and 6 months, groups of four animals were killed for all treatments, leaving 10 rats/group. The study was terminated at 13 months. In a separate experiment, four female Wistar/Han rats were dosed with tamoxifen (0.103 mmol/kg, dissolved in tricaprylin), intraperitoneally, daily for 5 days while four controls received tricaprylin only. Animals were killed 24 h after the last dose.

For mutation assays, 10 female {lambda}/lacI transgenic rats (Big Blue rats, 6–8 weeks of age) homozygous for the lacI gene (Stratagene, La Jolla, CA) were dosed orally with {alpha}-hydroxytamoxifen dissolved in tricaprylin at 0.103 mmol/kg. In this assay, animals were dosed by gavage for 10 days. A control group of 10 animals received tricaprylin (1 ml/kg/day) for 10 days. For the lacI mutation assay, all the animals were killed 46 days after the last dose. The diet was withdrawn 24 h prior to killing.

Tissue preparation
Livers were removed, weighed and a sample snap frozen in liquid nitrogen for DNA isolation. Sections of all major lobes of the liver and other organs were fixed in formal buffered saline for histological examination and in Carnoy's fluid and ice-cold acetone for immunohistochemical studies.

Mutant frequency at lacI gene in the liver
The lacI mutation assay and the DNA sequence analysis of mutant lacI genes was performed as described previously (11). Sequencing of the double-stranded DNA was carried out in both directions for 25 of the control-derived mutants and 23 of the {alpha}-hydroxytamoxifen-derived mutants.

Isolation of hepatocytes
Hepatocytes were isolated from rats treated with {alpha}-hydroxytamoxifen, 0.103 mmol/kg intraperitoneally for 5 days or tricaprylin vehicle and killed on day 6. A two stage collagenase perfusion was used (19). This yields cell preparations consisting of >90% hepatocytes. Only preparations of >85% viability were used, as judged by Trypan blue exclusion and counting with a haemocytometer.

32P-Post-labelling analysis
Adducts were determined in DNA isolated from livers or hepatocytes using the 32P-post-labelling assay as described previously (20).

Immunodetection of liver GST-P and cell proliferation by PCNA expression
For the detection of GST-P foci, paraffin sections (5 µm) from acetone-fixed livers were rehydrated and GST-P proteins were located immunohistochemically using an anti-GST-P polyclonal antibody (1:100 dilution) followed by an anti-rabbit alkaline phosphatase conjugated second antibody (1:50 dilution; Sigma). Bound antibody was detected using naphthol AS/BI phosphate and Fast Red TR as described previously (6). GST-P foci were defined as groups of five or more cells. Foci were counted on at least 1 cm–2 of liver sections from animals at each time point. Areas of foci were calculated using an Analytical Measuring Systems VIDS V Imaging System (Synopics, Cambridge, UK) and were expressed as number/cm–2. For cell proliferation determination using PCNA, 5 µM paraffin sections from Carnoy's-fixed liver were rehydrated and PCNA antigen detected using a monoclonal mouse anti-PCNA antibody (1:25 dilution; Novacastra, Newcastle on Tyne, UK) followed by a rabbit anti-mouse Ig2a peroxidase-conjugated antibody (1:50 dilution; Serotec, Oxford, UK) and 3,3'-diaminobenzidine/H2O2 substrate. Sections were lightly counterstained with haematoxylin. Fields were selected for counting by grid overlay and representative areas of all the lobes were examined. Sections of duodenum, processed at the same time served as a positive control. At least 4000 nuclei were counted on each section to derive the labelling index/1000 nuclei.

Statistical analysis
Statistical analysis was carried out were carried out using Minitab version 10 (Minitab, PA). Difference between groups was tested using analysis of variance (ANOVAR) with Dunnett's test for significance at the 5% level. For gene mutation assays, Fisher's exact test was used to test the difference between the groups. Results were considered to be significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Life time effects and mortality
Dosing for 5 days with {alpha}-hydroxytamoxifen at 0.103 or 0.0103 mmol/kg did not result in subsequent reduced body weight gain, known to be associated with long-term anti-oestrogen treatment of female rats (21). Additionally, there was not the alopecia seen in rats during long-term dosing with tamoxifen (22). There were unscheduled deaths in all treatment groups. Between 9 and 12 months after dosing, 10 animals developed overgrown incisors associated with jaw misalignment. This effect was not treatment related. On an animal welfare basis, the study was terminated at 13 months.

Liver pathology and GST-P foci with {alpha}-hydroxytamoxifen treatment
At the 3, 6 and 13 month kill periods in the livers of {alpha}-hydroxytamoxifen treated rats, there was a low incidence of GST-P positive foci. These foci increased in number and area with time after treatment but became significant, relative to the corresponding controls, only in animals killed at 13 months (Table IGo). In the {alpha}-hydroxytamoxifen treated groups, phenobarbital treatment did not result in any significant difference in the number or area of foci relative to animals not exposed to phenobarbital. At the lower dose of {alpha}-hydroxytamoxifen (0.0103 mmol/kg for 5 days), occasional GST-P foci were seen at 6 and 13 months after dosing, but the number and areas were not significantly different from the corresponding control values. Rats given 0.103 mmol/kg {alpha}-hydroxytamoxifen and killed at 3, 6 and 13 months showed varying degrees of bile duct proliferation in the presence or absence of phenobarbital treatment. There was no biliary proliferation in either of the corresponding control groups. There was no difference in overall cell proliferation in the liver BrdU labelling index between the {alpha}-hydroxytamoxifen dosed rats and their controls (not shown).


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Table I. Effects of {alpha}-hydroxytamoxifen treatment on the numbers and areas of GST-P foci
 
Liver DNA damage caused by {alpha}-hydroxytamoxifen and tamoxifen in Wistar/Han rats
After dosing rats with {alpha}-hydroxytamoxifen at 0.103 or 0.0103 mmol/kg for 5 days, total liver DNA damage, as determined by 32P-post-labelling 24 h after the last dose and at 3 months after cessation of dosing, is shown in Figure 1Go. Following the separation of these adducts by HPLC, qualitatively, the pattern was very similar to that seen in DNA extracted from the livers of tamoxifen dosed animals with two major and up to 10 minor components (Figure 2Go). The pattern of adducts did not change between day 6 and day 90 time points (not shown). At 6 and 13 month time points, adduct levels in the livers of dosed rats did not differ from those of controls. Rats given tamoxifen (0.103 mmol/kg, intraperitoneally for 5 days) showed less total DNA damage by 32P-post-labelling of 372 ± 232 adducts/108 nucleotides, than the 3334 ± 795 adducts/108 nucleotides seen in rats given an equimolar dose of {alpha}-hydroxytamoxifen (Figure 1Go). Since {alpha}-hydroxytamoxifen is conjugated and excreted via the bile (23), we wanted to establish if hepatic DNA damage was present in hepatocytes and not confined to bile duct cells. Hepatocytes prepared from {alpha}-hydroxytamoxifen dosed rats (0.103 mmol/kg for 5 days) by collagenase perfusion, showed DNA damage of 4266 ± 349 adducts/108 nucleotides while control hepatocytes showed 11 ± 4 adducts/108 nucleotides. These values are similar to those seen in whole liver. At day 6, no damage could be detected in uterine DNA from treated rats above control values.



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Fig. 1. DNA damage in the livers of rats given {alpha}-hydroxytamoxifen. {alpha}-Hydroxytamoxifen was dosed (0.103 or 0.0103 mmol/kg, intraperitoneally) to rats for 5 days and the animals killed on day 6, or 3 months later. Controls received tricaprylin vehicle. DNA was extracted from the liver and DNA damage determined by 32P-post-labelling. The data shown are DNA adducts/108 nucleotides (bars, SD) in animals treated with {alpha}-hydroxytamoxifen at 0.103 mmol/kg ({square}), 0.0103 mmol/kg ({blacksquare}) or vehicle dosed controls ({blacksquare}).

 


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Fig. 2. HPLC separation of 32P-post-labelled adducts from liver DNA of rats dosed with tamoxifen or {alpha}-hydroxytamoxifen. Equimolar doses of {alpha}-hydroxytamoxifen or tamoxifen (0.103 mmol/kg, intraperitoneally) were given to rats for 5 days and the animals killed on day 6. DNA was extracted from livers, post-labelled and subjected to HPLC as described in Materials and methods. Trace A, 32P-post-labeled adducts from {alpha}-hydroxytamoxifen treated rats; B, tamoxifen treated animals. Although retention times for the major components are not identical between traces A and B, peaks 4, 5, 6, 11 and 12 co-elute.

 
Mutant frequency at the lacI gene and DNA adducts in the livers of Big Blue rats
Forty-six days following oral dosing with {alpha}-hydroxytamoxifen (0.103 mmol/kg, for 10 days), the average mutation frequency, in the treated group (9.48x10–5) was approximately 1.8-fold higher than in the corresponding controls (5.04x10–5, P < 0.05) (Table IIGo). The type and location of lacI mutations from control and {alpha}-hydroxytamoxifen treated animals are shown in Figure 3Go. The frequency of point mutations was 93 ± 4% in the treated group and 81 ± 4% in the control group. GC to AT transitions were the most common point mutations in both control and treated groups. GC to TA transversions in the tamoxifen treated group (17 ± 6%) were not significantly higher than in the control group (9 ± 5%). Immediately after dosing with {alpha}-hydroxytamoxifen (0.103 mmol/kg), hepatic DNA damage in these animals, determined by 32P-post-labelling was 411 ± 192 adducts/108 nucleotides (mean ± SD, n = 4). Rats killed at the time of the mutation assay showed DNA damage of 66 ± 14 and 3.5 ± 1.7 adducts/108 nucleotides in dosed and control rats, respectively.


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Table II. Number of plaques screened and mutations found at the lacI gene in rat liver
 


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Fig. 3. The percentage of various mutation at the lacI gene in the livers of control or {alpha}-hydroxytamoxifen-treated (0.103 mmol/kg) homozygous lacI rats. The data shown are percentages (bars, SD) in the control ({square}) and tamoxifen-treated ({blacksquare}) animals.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study shows that treatment of rats with {alpha}-hydroxytamoxifen for 5 days was sufficient to cause cumulative hepatic DNA damage, as measured by 32P-post-labelling, in the order of 3300 adducts/108 nucleotides. When Wistar rats were given dietary tamoxifen for 3 months and then returned to a normal diet, hepatic DNA damage of about 700 adducts/108 nucleotides, determined by 32P-post-labelling, resulted in five out of seven animals developing liver tumours at 12 months but only when promoted with phenobarbital (6). In contrast, no animal had liver tumours at this time without phenobarbital promotion. DNA damage of 2800 adducts/108 nucleotides seen after 6 months continuous tamoxifen treatment, resulted in three out of five animals developing liver tumours in the absence of phenobarbital promotion (4). An investigation using female Sprague–Dawley rats found that daily treatment by gavage of 22.6 mg/kg tamoxifen for 12 months, resulted in a 100% incidence (24 out of 24 rats) of hepatocellular adenomas and carcinomas (5). Liver DNA adducts were not determined in this study.

In rats, an apparent causal relationship between DNA damage and liver tumours has been established. Dosing rats with tamoxifen analogues such as toremifene and droloxifene, compounds that cause little or no hepatic DNA damage, does not result in liver tumours (5,24,25). In tamoxifen treated rats, tumours only develop at the major site of DNA damage in the liver. A single dose of tamoxifen that leads to ~30 adducts/108 nucleotides in the liver, as determined by 32P-post-labelling (26), does not act as a tumour initiator even in the presence of phenobarbital promotion (27). The absence of liver tumours can be correlated with a low incidence of hepatic GST-P foci. In the present study, the area or number of such foci are only ~10% those seen in Wistar rats exposed to dietary tamoxifen that develop liver tumours (4).

Following the administration of tamoxifen to rats, about 0.1% of the dose is excreted via the bile as {alpha}-hydroxytamoxifen glucuronide (23). On this basis, a dose of 0.103 mmol/kg tamoxifen would be metabolized to the equivalent of 0.001 mmol/kg {alpha}-hydroxytamoxifen. The comparatively high doses of {alpha}-hydroxytamoxifen needed to get hepatic DNA damage in the present study suggests rapid inactivation occurs in the rat. Oral administration results in much lower levels of hepatic DNA damage suggesting inactivation probably associated with acidic pH values of the stomach. Although {alpha}-hydroxytamoxifen has been detected in the blood of patients given tamoxifen (28) in the present investigation, this compound was below the limit of detection in plasma 24 h after the last dose using HPLC with UV detection (29) (<100 ng/ml; I.N.H.White, unpublished).

{alpha}-Hydroxytamoxifen causes a significant increase in mutation frequency in the livers of female {lambda}/lacI transgenic rats. There was no significant increase in G:C to T:A transversions, previously characterized as typical for tamoxifen induced mutations at lacI in these animals (11). Both the in vitro reaction of {alpha}-acetoxytamoxifen, a model ester of {alpha}-hydroxytamoxifen with DNA and the administration of tamoxifen to rats lead to the same major adduct, the trans form of {alpha}-(N-2-deoxyguanosinyl)tamoxifen (30,31). A number of other products are formed in the liver including (N-2-deoxyguanosinyl) N-desmethyltamoxifen (31), but as judged by HPLC, the overall pattern of DNA adducts formed following the administration of {alpha}-hydroxytamoxifen is very similar to that seen after tamoxifen treatment of rats (18). Other metabolites, such as those formed by the peroxidase activation of 4-hydroxytamoxifen, form adducts which co-elute with minor adducts in tamoxifen treated rats (20), but these adducts are more mutagenic than those derived from the in vitro reaction of {alpha}-acetoxytamoxifen, as shown using the lacI gene polymerase STOP assay (32).

Results show that the initiation of DNA damage by {alpha}-hydroxytamoxifen is not sufficient to lead to the development of hepatocellular carcinomas even with phenobarbital promotion. While qualitatively, the pattern of adducts formed in the liver following dosing with tamoxifen or {alpha}-hydroxytamoxifen are the same, the relative proportions of the different adducts differ. Not all of the DNA damage detected may be directly associated with the subsequent development of liver tumours. This has been established, for example, with a number of N-nitrosodialkylamine alkylating agents where most of the ring nitrogen atoms and the exocyclic oxygen atoms of guanine residues in DNA are targets (33), but all are not equivalent in terms of carcinogenic potential (34). Defining the relationship between the level of DNA damage and the length of time needed to allow promotion and progression of transformed cells will be of importance in understanding the mechanism of carcinogenicity.


    Notes
 
1 Present addresses: SEAC Toxicology Unit, Unilever Research, Colworth House, Sharnbrook, Bedfordshire, MK44 1LQ, UK, Back

2 Lawrence Livermore National Laboratory, Livermore, CA 94551-9900, USA and Back

3 Pharmaceutical Chemistry, University of Bradford, Bradford, BD7 1DP, UK Back

4 To whom correspondence should be addressed Email: iw6{at}le.ac.uk Back


    Acknowledgments
 
We thank Richard and Jennifer Edwards, James Fisher, Robert Heydon, Margaret Gaskell, Siddhartha Kakar, Barbara Nolan and Nihal Razvi for their expert technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 13, 2000; revised January 18, 2001; accepted January 19, 2001.