Identification of tamoxifen-DNA adducts formed by 4-hydroxytamoxifen quinone methide

Tamoxifen is a liver carcinogen in rats and has been shown to increase the risk of endometrial cancer in women. Recent reports of DNA adducts in leucocyte and endometrial samples from women treated with tamoxifen indicate that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study we show that 4- hydroxytamoxifen quinone methide reacts with DNA to form covalent adducts. The major products, which result from 1,8-addition of the exocyclic nitrogen of deoxyguanosine to the conjugated system of 4- hydroxytamoxifen quinone methide, are characterized as (E)- and (Z)- alpha-(deoxyguanosin-N2-yl)-4-hydroxytamoxifen.      

DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.     

Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen

The risk of developing endometrial cancer increases significantly for women treated with tamoxifen (TAM); the present study was designed to investigate the mechanism of this carcinogenic effect. Endometrial tissue was obtained from 16 women treated for varying lengths of time with TAM and from 15 untreated control subjects. DNA was analyzed with a (32)P-post-labeling/HPLC on-line monitoring assay capable of detecting 2.5 adducts/10(10) nucleotides. Using this sensitive and specific assay, TAM-DNA adducts were detected in eight women. The major adducts found were trans and cis epimers of alpha-(N(2)-deoxyguanosinyl) tamoxifen (dG-N(2)-TAM); levels ranged between 0.2-12 and 1.6-8.3 adducts/10(8) nucleotides, respectively. There was marked inter-individual variation in the relative amounts of cis and trans adducts present. Low levels (0.74-1.1 adducts/10(8) nucleotides) of trans and cis forms of dG-N(2)-TAM N-oxide were detected in one patient. DNA adducts derived from 4-hydroxytamoxifen quinone methide were not observed. We conclude from this analysis that trans and cis dG-N(2)-TAMs accumulate in significant amounts in the endometrium of many, but not all, women treated with this drug. The level of adducts found, coupled with the previous demonstration of their mutagenicity [Cancer Res., 59, 2091, 1999], suggest that a genotoxic mechanism may be responsible for TAM-induced endometrial cancer.     

Oxidation of eugenol to form DNA adducts and 8-hydroxy-2'- deoxyguanosine: role of quinone methide derivative in DNA adduct formation

We have investigated the activation of eugenol to form DNA adducts and oxidative base damage. Treatment of myeloperoxidase containing HL-60 cells with eugenol, produced a dose-dependent formation of three DNA adducts as detected with P1-enhanced 32P-post-labeling. Incubation of HL-60 cells with the combination of 100 microM eugenol and 100 microM H2O2 potentiated the levels of DNA adduct in HL-60 cells by 14-fold, which suggests peroxidase activation in adduct formation. In vitro activation of eugenol with either horseradish peroxidase or myeloperoxidase and H2O2 produced three DNA adducts that were inhibited by the addition of either ascorbic acid or glutathione, by 66 and 90%, respectively. The DNA adducts formed in HL-60 cells treated with eugenol were the same as those formed by in vitro peroxidase activation. In addition to adduct formation, peroxidase activation of eugenol produced a 2- to 3-fold increase in the level of oxidative base damage. Eugenol quinone methide was prepared by Ag(I)oxide oxidation of eugenol. Peroxidase activation of eugenol gave a product that had the same UV spectrum as eugenol quinone methide, which suggests that it was one of the products. Reaction of eugenol quinone methide with either DNA or deoxyguanosine-3'-phosphate produced two principal adducts (2 and 4). When DNA adduct 2 formed by incubation of eugenol quinone methide with deoxyguanosine-3'-phosphate was compared with DNA 2 adduct formed in HL-60 cells treated with eugenol results demonstrated that they were the same. This suggests that eugenol quinone methide is one of the reactive intermediates leading to DNA adduct formation in cells. Activation of eugenol with 10 microM copper sulfate resulted in the production of one principal (2) and several minor adducts. DNA adduct 2 formed by activation of eugenol with copper sulfate was the same as DNA adduct 2 formed by either peroxidase activation of eugenol or by reactions with eugenol quinone methide, which indicates that the reactive intermediates generated by these activation systems were similar. Copper sulfate produced a 95-fold increase in the level of oxidative base damage, which was significantly inhibited by the addition of either bathocuproinedisulphonic acid or catalase. The formation of oxidative base damage was consistent with a Fenton reaction mechanism. Our results demonstrate that eugenol can be activated to form both DNA adducts and oxidative base damage. We propose that the formation of this DNA damage may contribute to the observed toxic properties of eugenol.      

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.      

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

Daily intraperitoneal treatment of female Sprague-Dawley ratswith either 5, 10 or 20 mg/kg tamoxifen (TAM) for 1 weeks increasedthe level of peroxidase activity in the uterus 2- to 10-foldcompared to the control level. Using uterine extracts preparedfrom control and TAM treated animals, we investigated the activationof 4-hydroxytamoxifen (4-HO-TAM) and (E, Z)-1, 2-dipheny-1-(4-hydroxyphenyl)-but-1-ene(cis/trans-metabolite E) to form DNA adducts. Activation of4-HO-TAM by uterine extracts prepared from either control orTAM-treated rats produced one major (a) and Two minor DNA (band c) adducts. A similar activation of cis/trans-metaboliteE produced two adducts (d and e). There was good correlationbetween levels of uterine peroxidase activity and levels ofDNA adducts formed by 4-HO-TAM and cis/trans-metabolite E. Activationof 4-HO-TAM and cis/trans-metabolite E with horseradish peroxidase(HRP) produced the same adducts as observed by activation withuterine extract Treatment of Sprague-Dawley rats with 5 and10 mg/kg for 7 days produced eleven DNA adducts in the liverwith no adducts detected in the uterus. However, treatment ofrats with 20 mg/kg of TAM for 7 days produced the same adductpattern in the liver and also one major adduct (1) in the uteruswith a relative adduct level of 6.4 ±4.1x10^–9.Tamoxifen-DNA adduct 1 detected both in the liver and in theuterus of treated rats was similar to adducts produced by activationof 4-HO-TAM with either uterine extract or HRP. The resultsof these studies suggest a general model whereby the tamoxifenmetabolite 4-HO-TAM is further activated in the uterus by peroxidaseenzymes to form DNA adducts.     

Induction of covalent DNA adducts in rodents by tamoxifen.

The antiestrogen tamoxifen, increasingly used as adjuvant treatment for breast cancer, has been found to covalently modify DNA of rodents. For instance, the liver DNA of female Sprague-Dawley rats treated with a single injection of tamoxifen contained two DNA adducts. Four additional DNA adducts were formed and adduct concentrations increased 5- 7- and 10-15-fold after three and six tamoxifen injections, respectively, from levels observed after a single dose. The accumulation of DNA adducts with repeated administrations of tamoxifen to rodents may make this drug a poor choice for the chronic preventative treatment of breast cancer.     

Analysis of polychlorinated biphenyl-DNA adducts by 32P-postlabeling

Previous studies reported that metabolic activation of certainpolychlorinated biphenyls (PCBs) resulted in binding to protein,RNA and DNA fractions. However, the formation of DNA adductshas not been demonstrated nor have methods been optimized forthe detection of such adducts. In the present study we investigatedactivation and binding to DNA of lower chlorinated biphenylsusing ³²P-postlabeling. The incubation of 2-chloro-, 3-chloro-,3, 4-dichloro- and 3, 4, 5-trichlorobiphenyl with calf thymusDNA and liver microsomes from rats treated with pheno-barbitaland 3-methylcholanthrene, followed by oxidation with a peroxidase,produced 1–3 major adducts. Reaction of deoxyguanosine3'-monophosphate with metabolites of the congeneric chlorinatedbiphenyls produced adducts with similar chromatographic mobilityas those with DNA, suggesting that guanine was the preferentialsite of attack. Furthermore butanol and nuclease P1 enrichmentsshowed different adduct recoveries, depending upon the the chloro-biphenyl.Adducts derived from incubations with mono-chlorobiphenyls wererecovered 2- to 3-fold higher with butanol, while the recoveryof di- and tri-chlorobiphenyl adducts was 5- to 7-fold higherwith nuclease P1. DNA adducts formed during the metabolism of3, 4-dichlorobi-phenyl were reduced by the sulfur nucleophiles,glutathione and N-acetyl-L-cysteine, suggesting that reactivesemiquin-one(s) or quinone(s) are involved. In contrast, theaddition of superoxide dismutase increased adduct formation,suggesting that the quinone metabolites are responsible forthe major adducts formed. Our results are consistent with thehypothesis that lower chlorinated biphenyls are metabolicallyactivated to electrophilic quinoid species which bind to DNA.     

Intrinsic reactivity of tamoxifen and toremifene metabolites with DNA

The antiestrogen tamoxifen is known to cause liver cancer in rats. This may be due to the formation of abundant DNA adducts in rat liver. A likely precursor to some of the tamoxifen adducts in rats is -hydroxytamoxifen. It is not clear whether the rat data are relevant to human exposure. In the present study, we show that one of the major metabolites in humans reacts with double-stranded DNA in vitro in the absence of any metabolizing enzymes or activating chemicals. At least two distinct adduct spots resulting from 4-hydroxy-N-desmethyltamoxifen (metabolite Bx) were detected by ³²P postlabeling and thin layer chromatography. The adduct level increases dramatically when metabolite Bx is irradiated with UV light to fuse into a phenanthrene ring system. 4-hydroxy-N-desmethyltoremifene, which differs from Bx by a single chlorine atom,forms fewer DNA adducts without irradiation but similar amounts after irradiation. These results suggest that the chlorine atom may interfere with drug-DNA interactions which facilitate adduct formation.     

Genotoxic effects of the novel mixed antiestrogen FC-1271a in comparison to tamoxifen and toremifene

Tamoxifen has been used for the treatment of breast cancer since the 1970s, but is considered a carcinogen because it has been linked to liver cancer in rats and an increased risk of endometrial cancer in patients. In rats, DNA adducts appear to be responsible for carcinogenesis, but their contribution to carcinogenesis in humans is not clear. FC-1271a and toremifene are mixed antiestrogens similar to tamoxifen. In order to compare the genotoxicity of these different triphenylethylenes, we treated mice for 28 days with 50 mg/kg of either tamoxifen, toremifene, FC-1271a or vehicle control. DNA from liver and uterus was assayed by standard ³²P-postlabeling and thin layer chromatography for the presence of DNA adducts. Two methods of drug administration (oral and subcutaneous) and two strains of mice were compared and the plasma and tissue concentrations of the drugs and three metabolites of tamoxifen and toremifene were determined. Regardless of the conditions, only tamoxifen-treated mice showed DNA adducts in the liver. Adduct levels did not correlate with drug or metabolite levels and adducts were present even when drug was not detectable. Mice were also treated orally with either 50, 100, or 200 mg/kg of drug for 7 days. Again, adducts were found only in liver tissue of mice treated with tamoxifen, and adduct levels were dose-dependent. In conclusion, the chlorinated triphenylethylene FC-1271a did not cause DNA adducts under various conditions in mice, suggesting a low carcinogenic potential.     

32P-postlabeling analysis of DNA adducts in rats during estrogen-induced hepatocarcinogenesis and effect of tamoxifen on DNA adduct level.

DNA adduct formation in the liver, pancreas, kidneys and uterus in ethynylestradiol (EE)-induced carcinogenesis and the effect of tamoxifen (TAM) on DNA adduct formation were evaluated in female Wistar JCL rats using the 32P-postlabeling method. Hyperplastic nodules were noted in the liver of all rats 4 months after the first oral administration of 0.075 mg of EE, and hepatocellular carcinoma was detected in 8.1% of rats treated with EE for 12 months. DNA adducts increased in the liver for 4 months, reaching a level of 7.3 adducts/10(7) nucleotides and decreasing thereafter. Formation of DNA adducts was also noted in the pancreas and kidney, but the adduct levels were lower than those in the liver. TAM inhibited estrogen receptors (ER) in liver tissues and completely suppressed the development of hyperplastic nodules or hepatocellular carcinoma but did not affect DNA adduct formation in the liver. In this model, therefore, EE is considered to cause mutations of hepatocytes due to DNA adduct formation without mediation by ER and to induce initiated cells to develop into hepatocellular carcinoma in the presence of ER-mediated hormonal activities.     

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells.

Chronic administration of tamoxifen to female rats causes hepatocellular carcinomas. We have investigated damage to liver DNA caused by the administration of tamoxifen to female Fischer F344/N rats or C57B1/6 or DBA/2 mice using 32P-postlabelling. Following the administration of tamoxifen for 7 days (45 mg/kg/day) and extraction of hepatic DNA, up to 7 radiolabelled adduct spots could be detected after PEI-cellulose chromatography of the 32P-labelled DNA digests. Tamoxifen caused a time-dependent increase in the level of adduct detected up to a value of at least 1 adduct/10(6) nucleotides after 7 days dosing. A dose response relationship was demonstrated over the range of 5-45 mg/kg/day (0.013-0.12 mmol/kg/day). On cessation of dosing there was a loss of adducts from the liver DNA. These adducts were not detected in DNA from vehicle-dosed controls or in DNA from kidney, lung, spleen, uterus or peripheral lymphocytes. Pyrrolidinotamoxifen caused a similar level of adduct formation as tamoxifen. In contrast, no significant adduct formation could be detected in liver DNA from rats given droloxifene or toremifene. Mice given tamoxifen (45 mg/kg/day for 4 days) showed levels of adducts in the liver which were 30-40% of those present in rats. Exposure of rat hepatocytes to tamoxifen in vitro, resulted in induction of unscheduled DNA synthesis, when preparations from rats which had been pretreated with tamoxifen in vivo were used. No such increase could be detected in hepatocytes from control rats, suggesting tamoxifen may induce enzymes responsible for its own activation. Tamoxifen induced a significant increase in micronucleus formation in a dose dependent manner in cultures of MCL-5 cells, a human cell line that expresses 5 different human cytochrome P450 isoenzymes, as well as epoxide hydrolase.     

N-demethylation accompanies alpha-hydroxylation in the metabolic activation of tamoxifen in rat liver cells.

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via alpha-hydroxylation leading to modification of the N(2)-position of guanine in DNA and to a lesser extent the N(6)-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N(2)-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or alpha-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, alpha-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the alpha position was confirmed in experiments in which [alpha-D(2)-ethyl]tamoxifen, but not [beta-D(3)-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and alpha-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both alpha-hydroxylation and N-demethylation, which is followed by Phase II activation at the alpha-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by (32)P-postlabelling is achieved with >90% labelling efficiency.     

32P-Postlabeling Analysis of DNA Adducts in Rats during Estrogen-induced Hepatocarcinogenesis and Effect of Tamoxifen on DNA Adduct Level

DNA adduct formation in the liver, pancreas, kidneys and uterus in ethynylestradiol (EE)-induced carcinogenesis and the effect of tamoxifen (TAM) on DNA adduct formation were evaluated in female Wistar JCL rats using the ³²P-postlabeling method. Hyperplastic nodules were noted in the liver of all rats 4 months after the first oral administration of 0.075 mg of EE, and hepatocellular carcinoma was detected in 8.1% of rats treated with EE for 12 months. DNA adducts increased in the liver for 4 months, reaching a level of 7.3 adducts/10⁷ nucleotides and decreasing thereafter. Formation of DNA adducts was also noted in the pancreas and kidney, but the adduct levels were lower than those in the liver. TAM inhibited estrogen receptors (ER) in liver tissues and completely suppressed the development of hyperplastic nodules or hepatocellular carcinoma but did not affect DNA adduct formation in the liver. In this model, therefore, EE is considered to cause mutations of hepatocytes due to DNA adduct formation without mediation by ER and to induce initiated cells to develop into hepatocellular carcinoma in the presence of ER-mediated hormonal activities.     

Cutaneously applied 4-hydroxytamoxifen is not carcinogenic in female rats.

Tamoxifen is widely used to treat oestrogen-dependent carcinoma of the breast. Previous long-term studies have shown that oral administration of tamoxifen induces hepatoproliferative lesions and hepatocellular tumours in rats. 4-hydroxytamoxifen is an active metabolite of tamoxifen undergoing clinical evaluation for the treatment of various non-malignant breast diseases by topical application. In the present study, 4-hydroxytamoxifen was administered daily by cutaneous application for 101 weeks to groups of 50 female Sprague-Dawley rats at 20, 140 or 1000 microg/kg/day. The product was applied with no occlusive bandage and oral ingestion was avoided by application of an Elizabethan collar for 6 h after administration. Treatment with 4-hydroxytamoxifen was clinically well tolerated and induced changes such as decreased food consumption and body weight gain, uterine and ovarian atrophy, mucification of vaginal epithelium and reduced mammary development, all of which were attributed to its pharmacological action. Mortality was significantly lower in the treated animals. The number of animals with palpable masses was similarly reduced. The incidence of mammary tumours and hypophyseal tumours was markedly lower in 4-hydroxytamoxifen-treated animals. The incidence of chronic tubulo-interstitial nephropathies, a common cause of mortality, was also lowered. There was no evidence of a carcinogenic action of 4-hydroxytamoxifen on the liver, genital organs or skin. Plasma levels of 4-hydroxytamoxifen were stable over the duration of the study and were proportional to the administered dose, exceeding clinical plasma levels by 60-fold at the high dose-level. In conclusion, 4-hydroxytamoxifen is not carcinogenic in the rat and reduces the incidence of spontaneous mammary and hypophyseal tumours.     

32P-postlabeling analysis of 1-nitropyrene-DNA adducts in female Sprague-Dawley rats

The ³²P-postlabeling technique was used to qualitatively establishthe pattern of DNA adduct formation in mammary tissue and liverfollowing administration of 1-nitropyrene to female Sprague-Dawleyrats. 1-Nitropyrene (100 mg/kg b.w.) was administered by gavagein trioctanoin and the rats were sacrificed 24 h later. DNAwas isolated from mammary fat pads and liver, enzymaticallyhydrolyzed to deoxy ribonucleoside-3'-monophosphates and thenconverted to [5'-³²P]3',5'-bisphosphates. The polyethyleneimine-cellulose(PEI-cellulose) TLC ³²P-fingerprints revealed the presence ofmultiple putative adducts in the mammary fat pads and in thelivers. To investigate the role of nitroreduction in the formationof these adducts, calf thymus DNA was incubated with [³H]1-nitropyrenein vitro in the presence of xanthine oxidase. The DNA was isolatedand analyzed by the ³²P-postlabeling technique. A major adductspot was detected and confirmed as N-(deoxyguanosin-8-yl)-1-amino-pyrene.This adduct cochromatographed with a minor in vivo adduct ofDNA obtained from mammary fat pads and livers. However, themajor adducts detected in vivo did not appear to originate fromsimple nitroreduction of 1-nitropyrene. The results of thisstudy suggest that other metabolic pathways, such as ring oxidation,or ring oxidation followed by nitroreduction, may be responsiblefor the putative 1-nitropyrene—DNA adducts observed inmammary fat pads and livers of female Sprague-Dawley rats.     

Induction of lacI mutations in Big Blue rats treated with tamoxifen and alpha-hydroxytamoxifen.

The antiestrogen tamoxifen is carcinogenic in the liver and uterus of rats. Liver tumors appear to result from sequential hydroxylation and esterification of the alpha-carbon of tamoxifen followed by DNA adduct formation. The mechanism for the induction of uterine tumors is not known. Big Blue rats were treated by intraperitoneal injection with 21 daily doses of 54 micromol/kg tamoxifen or its proximate carcinogenic metabolite alpha-hydroxytamoxifen. One month after the last treatment, the mutant frequency in the lacI transgene was determined in the liver and uterus. For comparison, the mutant frequency in the hypoxanthine phosphoribosyl transferase (Hprt) gene of spleen lymphocytes was also measured. In the liver, tamoxifen (32+/-18 mutants/10(6) plaques; mean+/-SD) and alpha-hydroxytamoxifen (770+/-270 mutants/10(6) plaques) caused a significant increase in the mutant frequency of the lacI gene compared to solvent treated controls (10+/-10 mutants/10(6) plaques). 32P-Postlabeling analyses of liver DNA indicated three DNA adducts, one each from tamoxifen, N-desmethyltamoxifen, and N,N-didesmethyltamoxifen. Neither tamoxifen nor alpha-hydroxytamoxifen caused an increase in the mutant frequency in the lacI gene of the uterus or in the Hprt gene of spleen lymphocytes. These results suggest that induction of endometrial tumors in rats is not due to the genotoxicity of tamoxifen.     

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.     

Tamoxifen DNA damage detected in human endometrium using accelerator mass spectrometry

This study was aimed to establish whether tamoxifen binds irreversibly to uterine DNA when given to women. Patients were given a single therapeutic dose of [(14)C]tamoxifen citrate orally (20 mg, 0.37 or 1.85 MBq) approximately 18 h prior to hysterectomy or breast surgery. Nonmalignant uterine tissue was separated into myometrium and endometrium. DNA and protein were isolated and bound radiolabel determined by the sensitive technique of accelerator mass spectrometry. Levels of irreversible DNA binding of tamoxifen in the endometrium of treated patients were 237 +/- 77 adducts/10(12) nucleotides (mean +/- SE, n = 10). In myometrial tissues, a similar extent of DNA binding was detected (492 +/- 112 adducts/10(12) nucleotides). Binding of tamoxifen to endometrial and myometrial proteins was 10 +/- 3 and 20 +/- 4 fmol/mg, respectively. In breast tissue, sufficient DNA could not be extracted but protein binding was an order of magnitude higher than that seen with endometrial proteins (358 +/- 81 fmol/mg). These results demonstrate that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers relative to those previously reported in women after long-term tamoxifen treatment where levels, when detected, ranged from 15000 to 130000 adducts/10(12) nucleotides. Our findings support the hypothesis that the low level of DNA adducts in human uterus is unlikely to be involved with endometrial cancer development.