Antitumour 2-(4-aminophenyl)benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo.

2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 microM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 microM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 microM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) > or = 100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 microM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 microM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (< or = 10 microM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg(-1)). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse.

Cumulative exposure to tamoxifen: DNA adducts and liver cancer in the rat.

Tamoxifen is a potent rat liver carcinogen, currently being used as a long-term chemopreventative for breast cancer in healthy women. The mechanism by which tamoxifen causes liver cancer in rats is known to be associated with the accumulation of tamoxifen DNA adducts in this organ. We have examined the dose-response relationship of tamoxifen-induced DNA adducts in the liver and the subsequent increase in the development of liver cancer, with and without phenobarbital promotion. Female Wistar (Han) rats were fed 420 ppm tamoxifen in the diet for 0, 1, 4, 8 or 12 weeks after which time rats were either examined immediately for hepatic tamoxifen-induced DNA damage using the 32P-Postlabelling assay, or left for lifetime for tumour assessment. A proportion of rats left for lifetime study were given phenobarbital in their drinking water. There was a clear dose-response relationship with respect to duration of tamoxifen exposure for both accumulation of DNA adducts and lifetime risk of liver cancer. In the absence of phenobarbital promotion there was a threshold value for tamoxifen-induced DNA adducts (180 adducts/10(8) nucleotides) and the subsequent induction of liver cancer. This study demonstrates the relationship between the accumulation of hepatic tamoxifen-induced DNA adducts and the development of liver cancer and establishes the threshold for hepatocarcinogenesis in terms of DNA adduct formation. These data could provide useful information in interpreting the relevance of low levels of DNA adducts in humans.

Tamoxifen induces endometrial and vaginal cancer in rats in the absence of endometrial hyperplasia.

Tamoxifen was administered orally to neonatal rats on days 2-5 after birth and the subsequent effects on the uterus were characterized, morphometrically, over the following 12 months. Tamoxifen inhibited development of the uterus and glands in the endometrium, indicating a classical oestrogen antagonist action. Between 24 and 35 months after tamoxifen treatment there was a significant increase in the incidence (26%) of uterine adenocarcinomas and a 9% incidence of squamous cell carcinomas of the vagina/cervix in the absence of any oestrogen agonist effect in the uterus. This demonstrates that an oestrogen agonist effect is not an absolute requirement for the carcinogenic effect of tamoxifen in the reproductive tract of the rat. The unopposed oestrogen agonist effect of tamoxifen on the endometrium may not be the only factor involved in the development of endometrial cancers. It is possible that tamoxifen causes these tumours via a genotoxic mechanism similar to that seen in rat liver. However, using (32)P-post-labelling we failed to find evidence of tamoxifen-induced DNA adducts in the uterus. Tamoxifen may affect hormonal imprinting of oestrogen receptor responses in stem cells of the uterus, causing reproductive tract cancers to arise at a later time, in the same way as has been proposed for diethylstilbestrol. If these rodent data extrapolate to humans, then women who are taking tamoxifen as a chemopreventative may have an increased risk of vaginal/cervical cancer, as well as endometrial cancer.

Further characterization of the DNA adducts formed in rat liver after the administration of tamoxifen, N-desmethyltamoxifen or N, N-didesmethyltamoxifen.

The present study compares the formation of DNA adducts, determined by (32)P-postlabelling, in the livers of rats given tamoxifen and the N-demethylated metabolites N-desmethyltamoxifen and N, N-didesmethyltamoxifen. Results show that after 4 days treatment (0.11 mmol/kg i.p.), similar levels of DNA damage were seen after treatment with either tamoxifen or N-desmethyltamoxifen [109 +/- 40 (n = 3) and 100 +/- 33 (n = 4) adducts/10(8) nucleotides, respectively], even though the concentration of tamoxifen in the livers of tamoxifen-treated rats was about half that of N-desmethyltamoxifen in the N-desmethyltamoxifen-treated animals (51 +/- 16 and 100 +/- 8 nmol/g, respectively). Administration of N, N-didesmethyltamoxifen to rats resulted in a 5-fold lower level of damage (19 adducts/10(8) nucleotides, n = 2). Following (32)P-postlabelling and HPLC, hepatic DNA from rats treated with tamoxifen and its metabolites showed distinctive patterns of adducts. Treatment of rats with N,N-didesmethyltamoxifen gave a major product that co-eluted with one of the minor adduct peaks seen in the livers of rats given tamoxifen. Following dosing with N-desmethyltamoxifen, the major product co-eluted with one of the main peaks seen following treatment of rats with tamoxifen. This suggests that tamoxifen can be metabolically converted to N-desmethyltamoxifen prior to activation. However, analysis of the (32)P-postlabelled products from the reaction between alpha-acetoxytamoxifen and calf thymus DNA showed two main peaks, the smaller one of which ( approximately 15% of the total) also co-eluted with that attributed to N-desmethyltamoxifen. This indicates that N-desmethyltamoxifen and N,N-didesmethyltamoxifen are activated in a similar manner to tamoxifen leading to a complex mixture of adducts. Since an HPLC system does not exist that can fully separate all these (32)P-postlabelled adducts, care has to be taken when interpreting results and determining the relative importance of individual adducts and the metabolites they are derived from in the carcinogenic process.

Site-specific tamoxifen-DNA adduct formation: lack of correlation with mutational ability in Escherichia coli.

We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.

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.

A novel HPLC system has been developed that has allowed the separation of tamoxifen DNA adducts formed in the livers of rats and mice treated with this drug. At least 13 different peaks have been separated from 32P-post-labelled DNA, with two major peaks jointly accounting for >60% of the total adducts formed by tamoxifen in the livers of treated rats and mice. This is a great improvement on the resolution obtained by thin layer chromatography, which separates the adducts into one main product consisting of a group of major adduct spots eluting together, plus several other minor spots. Identification of the nature of some of the peaks has been investigated. Comparisons of the products formed when alpha-acetoxytamoxifen is reacted with DNA in vitro with 32P-post-labelled liver DNA adducts from rats treated with tamoxifen or alpha-hydroxytamoxifen in vivo, appear to confirm that a major route of activation of tamoxifen in vivo is via alpha-hydroxylation. The resolving power of this HPLC system has further extended this result to show that six of the peaks, including the two major peaks, are formed by the reaction of an activated alpha-hydroxytamoxifen with DNA. Activation of 4-hydroxytamoxifen by the peroxidase/H2O2 system in vitro gives a more polar DNA adduct seen only at trace levels in liver DNA from tamoxifen-treated rats and mice.

Investigation of the formation and accumulation of liver DNA adducts in mice chronically exposed to tamoxifen.

Tamoxifen was administered to three strains of female mice (B6C3F1, C57BL/6 and DBA/2) in short- and long-term studies to determine their ability to activate tamoxifen and cause hepatic DNA damage. 32P-Postlabelling of liver DNA from mice treated for 4 days showed a group of major adducts that increased in a dose-dependent manner and co-chromatographed with the major adducts detected in rat liver. On cessation of dosing, the majority of adducts were cleared within 3 days. Binding of [14C]tamoxifen to DNA nucleotides was demonstrated by the use of accelerator mass spectrometry. In long-term studies of 12 months to 2 years duration, dependent on strain, tamoxifen was administered continuously in the diet to give a daily dose of approximately 40 mg/kg. DNA adducts were detected after 3 months, although the number of adducts decreased with time and by 2 years were not detectable in the tamoxifen treated mice. None of the treated groups showed a significantly increased incidence of liver tumours, with or without phenobarbital promotion and there was no sustained liver cell proliferation. Tamoxifen was detected in the mouse livers, but at levels 50 times lower than those reported in a comparable rat study. These results suggest that, in contrast to the rat, tamoxifen is non-carcinogenic in mice because it does not cause sufficient cumulative DNA damage, or act as a promoter by causing cell proliferation.

Tamoxifen induces short-term cumulative DNA damage and liver tumors in rats: promotion by phenobarbital.

Tamoxifen administered in the diet (420 ppm) to Wistar rats (TOX:P) for only 3 months caused cumulative hepatic DNA damage as assessed by 32P-postlabeling, consistent with the proposal that tamoxifen is a genotoxic carcinogen in this species. Promotion of tumor development with phenobarbital after discontinuation of dietary tamoxifen resulted in the formation of liver carcinomas after 9 months. At 12 and 20 months in this study, the majority of these rats had liver carcinomas. Rats treated with tamoxifen for 3 months but not promoted with phenobarbital also developed liver tumors over a longer period of time. These tumors were predominantly adenomas, with one carcinoma, and occurred at a lower incidence than the tumors produced by promotion with phenobarbital. Rats treated with phenobarbital alone did not develop tumors after 20 months. Tamoxifen-induced DNA adducts were relatively persistent, with only a 38% decrease 3 months after tamoxifen treatment had been discontinued. This demonstrates that, in a susceptible species (the rat), tamoxifen can cause initiation of liver cancer after only 3 months exposure. It is proposed that the persistence of such DNA adducts may account for the ability of phenobarbital to promote a high incidence of liver carcinoma, even after discontinuation of tamoxifen treatment. These data are relevant to the concern for women given prophylactic tamoxifen for long periods in that even if there is a relatively small amount of cumulative tamoxifen-induced liver DNA damage, liver tumors could be promoted by other agents, even after the cessation of tamoxifen treatment.