Co-chromatography of a tamoxifen epoxide-deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats

Tamoxifen is a potent liver carcinogen in rats and has beenshown to form covalent DNA adducts in the livers of severalspecies of rodent. We have shown previously by ³²P-postlabelling(Carcinogenesis, 13, 2197–2203) that >85% of the totaladducts detected and resolved by multi-directional TLC migrateas a single spot. In the present study, this material was furtheranalysed by reverse-phase HPLC and resolved into two approximatelyequal components. Tamoxifen 1,2-epoxide, a postulated metaboliteof tamoxifen, was reacted with DNA and polydeoxyribonucleotidesand the products analysed. ³²P-Postlabelling revealed threemajor adduct spots on TLC which comigrated with the three majoradduct spots seen with DNA from livers of tamoxifen-treatedrats. Moreover, the major epoxide adduct, which contained guanineas the modified base, eluted on HPLC as a single major peakcoincident with one of the major peaks derived from the liverDNA of tamoxifen-treated rats. These results demonstrate that{small tilde}40% of the tamoxifen-DNA adducts formed in vivoare chromatographically indistinguishable with the major productof the reaction of tamoxifen epoxide with guanine residues inDNA and provide important clues to the mechanism of activationof tamoxifen to a genotoxic carcinogen.     

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.     

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.     

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest 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 compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats.     

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.     

DNA adduct formation in liver following the administration of [3H]2-nitrofluorene to rats in vivo.

The formation of RNA and DNA adducts by the environmental pollutant 2-nitrofluorene (2-NF) has been investigated in rat liver in vivo. The adduct pattern was studied after trifluoroacetic acid hydrolysis of DNA or RNA, followed by analysis of the adducts by HPLC. This was also done by enzymatic hydrolysis of DNA, followed by 32P-postlabeling. Both after oral and i.v. administration of [3H]2-NF, one major adduct was found. This adduct did not co-migrate with one of the known adducts of 2-(acetyl)-aminofluorene, N-deoxyguanosin-8-yl-2-aminofluorene (dG-C8-AF), which could have been formed after nitroreduction of 2-NF. 32P-Postlabeling revealed that two minor adducts were also formed, one of which was dG-C8-AF. The observation that the major adduct was also formed after i.v. administration of 2-NF to bile duct-catheterized rats makes a role for the intestinal microflora in the formation of this adduct very unlikely. In vitro experiments with inhibitors of the enzyme epoxide hydrolase indicated that epoxidation of 2-NF may play a role in the microsomal bioactivation of this compound.     

DNA adduct formation by the anticancer drug ellipticine in rats determined by 32P postlabeling

Ellipticine is a potent antineoplastic agent whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts in vitro and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). Here, we investigated the capacity of ellipticine to form DNA adducts in vivo. Male Wistar rats were treated with ellipticine, and DNA from various organs was analyzed by (32)P postlabeling. Ellipticine-specific DNA adduct patterns, similar to those found in vitro, were detected in most test organs. Only DNA of testes was free of the ellipticine-DNA adducts. The highest level of DNA adducts was found in liver (19.7 adducts per 10(7) nucleotides), followed by spleen, lung, kidney, heart and brain. One major and one minor ellipticine-DNA adducts were found in DNA of all these organs of rats exposed to ellipticine. Besides these, 2 or 3 additional adducts were detected in DNA of liver, kidney, lung and heart. The predominant adduct formed in rat tissues in vivo was identical to the deoxyguanosine adduct generated in DNA by ellipticine in vitro as shown by cochromatography in 2 independent systems. Correlation studies showed that the formation of this major DNA adduct in vivo is mediated by CYP3A1- and CYP1A-dependent reactions. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in vivo and confirm the formation of covalent DNA adducts as a new mode of ellipticine action.     

Molecular dosimetry of DNA adducts in C3H mice treated with glycidaldehyde.

The formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde (2 or 10 mg/animal) in acetone has been investigated by HPLC coupled with fluorescence detection and 32P-postlabelling analysis. Following a 24 h exposure period, epidermal DNA was isolated from treated dorsal skin and enzymically digested to nucleoside-3'-monophosphates. HPLC-32P-postlabelling analysis of the DNA hydrolysate indicated that a single major cyclic adduct was formed from the reaction of glycidaldehyde with deoxyadenosine residues in mouse skin DNA. This adduct was identified as 3-beta-D-deoxyribofuranosyl-7-(hydroxymethyl)-3H- imidazo[2,1-i]purine-3'-monophosphate by comparison with a synthetic standard. This adduct was stable, strongly fluorescent and readily detected by HPLC with fluorescence detection. There was no evidence for the formation of deoxyguanosine adducts in epidermal DNA of treated animals. Glycidaldehyde also reacted with calf thymus DNA in vitro at pH 7.0 to give the same deoxyadenosine adduct observed in vivo. At pH 10, however, this was a relatively minor product and the major adduct was 5,9-dihydro-7-(hydroxymethyl)-9- oxo-3-beta-D-deoxyribofuranosyl-3H-imidazo[1,2-a]purine-3'- monophosphate formed by the initial reaction of glycidaldehyde with deoxyguanosine residues.     

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.     

Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F1 mice [Cancer Lett. 193 (2003) 119-125

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F1 mice. We previously reported that eight dehydroretronecine (DHR)-derived DNA adducts were formed in liver DNA of rats treated with riddelliine. In order to examine the relationship between DNA adduct levels and the incidence of hemangiosarcomas, we have measured DHR-derived DNA adduct levels in purified rat and mouse liver endothelial cells, the cells of origin for the hemangiosarcomas. F344 rats and B6C3F1 mice were treated by gavage 5 days per week for 2 weeks with riddelliine at 1.0 mg/kg for rats and 3.0 mg/kg for mice. One, 3, 7, and 28 days after the last dose, liver parenchymal and endothelial cell fractions were isolated, and the quantities of DHR-derived DNA adducts were determined by 32P-postlabeling/HPLC. The DHR-derived DNA adduct levels in the endothelial cells were significantly greater than in the parenchymal cells. The DNA adduct levels in rat endothelial cells were greater than in the mouse endothelial cells. These results indicate that the levels of riddelliine-induced DNA adducts in specific populations of liver cells correlate with the preferential induction of liver hemangiosarcomas by riddelliine.     

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.      

Characterization of 4,4'-methylenebis(2-chloroaniline)--DNA adducts formed in vivo and in vitro.

4,4'-Methylenebis(2-chloroaniline) (MOCA) is a genotoxic and carcinogenic industrial chemical to which there is considerable potential human exposure. Since metabolic activation and formation of DNA adducts are believed to be important for the induction of these effects, DNA was treated in vitro with radiolabeled N-hydroxy-MOCA, the presumed proximate carcinogenic metabolite formed in vivo. Two major radioactive peaks were observed after HPLC separation of enzymatic hydrolysates. The two products were analyzed by MS and characterized as N-(deoxyadenosine-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosin-8-yl)-4-amino-3-chlorotoluene. The same adducts were also the major adducts formed in DNA of tissues from rats treated with radiolabeled MOCA. They were eliminated from rat liver with non-linear kinetics, in agreement with observations made for other carcinogens. The selective reaction of N-hydroxy-MOCA with DNA-adenine and the formation of single arylamine ring adducts suggest a substitution mechanism involving an intermediate with strong SN1 character, aided by the negative inductive effect of the ortho-chlorine. Due to tautomer formation, the initial adduct may be inherently unstable and undergo cleavage at the 1'-carbon-methylene bond to yield the observed adducts.     

Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F(1) mice

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F(1) mice. We previously reported that eight dehydroretronecine (DHR)-derived DNA adducts were formed in liver DNA of rats treated with riddelliine. In order to examine the relationship between DNA adduct levels and the incidence of hemangiosarcomas, we have measured DHR-derived DNA adduct levels in purified rat and mouse liver endothelial cells, the cells of origin for the hemangiosarcomas. F344 rats and B6C3F(1) mice were treated by gavage 5 days per week for 2 weeks with riddelliine at 1.0 mg/kg for rats and 3.0 mg/kg for mice. One, 3, 7, and 28 days after the last dose, liver parenchymal and endothelial cell fractions were isolated, and the quantities of DHR-derived DNA adducts were determined by (32)Ppostlabeling/HPLC. The DHR-derived DNA adduct levels in the endothelial cells were significantly greater than in the parenchymal cells. The DNA adduct levels in rat endothelial cells were greater than in the mouse endothelial cells. These results indicate that the levels of riddelliine-induced DNA adducts in specific populations of liver cells correlate with the preferential induction of liver hemangiosarcomas by riddelliine.     

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 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.     

7H-benzo[c]fluorene: a major DNA adduct-forming component of coal tar

Coal tar is a complex mixture that exhibits high carcinogenic potency in lungs of animals when administered in the diet. Studies have noted that lung tumor induction does not correlate with the benzo[a]pyrene content of coal tar, suggesting that other hydrocarbons may be involved in the observed tumorigenicity. Our previous studies have demonstrated that a major 'unknown' chemical-DNA adduct is formed in the lung of mice exposed to coal tar. We have used an in vitro rat microsomal activation system to generate the 'unknown' adduct with neat coal tar and fractions of coal tar obtained by chemical fractionation and HPLC. Chemical-DNA adduct formation was evaluated by (32)P-postlabeling using both multi-dimensional TLC and HPLC. GC-MS analysis of the coal tar fractions obtained from HPLC, which produced the 'unknown' adduct in vitro, demonstrated that the adducting hydrocarbon had a mass of 216. A careful evaluation of candidate hydrocarbons led to the conclusion that a benzofluorene derivative may be responsible for forming the 'unknown' chemical-DNA adduct. Comparative in vitro and in vivo studies on the adducting properties of all three isomers of benzofluorene indicated that 7H-benzo[c]fluorene is responsible for producing the 'unknown' adduct observed in the lung of mice ingesting coal tar. Animal feeding studies also demonstrated that 7H-benzo[c]fluorene formed considerably more lung DNA adducts than 11H-benzo[a]fluorene and 11H-benzo[b]fluorene. These data indicate that the four-ring polycyclic aromatic hydrocarbon 7H-benzo[c]fluorene, a hydrocarbon not previously shown to form DNA adducts in lung, is in fact a potent lung DNA adductor and is a candidate PAH for causing lung tumors in animals treated with coal tar.     

Lack of evidence for tamoxifen- and toremifene-DNA adducts in lymphocytes of treated patients

Tamoxifen (TAM) is used for the adjuvant treatment of women with breast cancer and has also been recommended as a chemopreventive agent. Among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clearly understood. We studied DNA adducts in lymphocytes of female breast cancer patients treated with TAM or toremifene (TOR), a TAM analogue and compared them with adducts formed by TAM in rat liver, where the drug induces tumours. DNA adducts were measured by TLC-(32)P-post-labelling assays. After TLC, all DNA samples including DNA from untreated healthy women showed a faint radioactive zone, where the positive control DNA adducts isolated from the liver of rats treated with TAM migrated. The relative adduct levels were calculated from the radioactivity present in this zone. Means +/- SD of adduct levels per 10(8) nucleotides (associated with this area) were for untreated volunteers (control) 1.83 +/- 1.41 (n = 13), for TAM treatment 2.17 +/- 3.04 (n = 25) and for TOR treatment 1.18 +/- 1.05 (n = 8). Most of the human samples were further analysed by HPLC after labelling with (32)P in order to compare adducts in human DNA with those in liver DNA isolated from TAM-treated rats. None of the human samples showed any peaks at retention times where putative TAM-DNA adducts were eluted. In conclusion, lymphocyte DNA from female patients treated at therapeutic levels did not show evidence of the formation of TAM- or TOR-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.     

Carcinogen-DNA adduct formation in the lungs and livers of preweanling CD-1 male mice following administration of [3H]-6-nitrochrysene, [3H]-6-aminochrysene, and [3H]-1,6-dinitropyrene

6-Nitrochrysene (NC) and 6-aminochrysene (AC) have been shown to be potent lung and liver carcinogens when administered in multiple i.p. doses to preweanling mice. 1,6-Dinitropyrene has been shown to be a strong hepatocarcinogen but a weak lung carcinogen in this same bioassay. We have examined carcinogen-DNA adduct profiles in the target tissues of preweanling male CD-1 mice following administration of single or multiple doses of these compounds. Depending on the tissue and the dosing schedule, the total level of DNA modification in animals dosed with [3H]NC was 2- to 9-fold higher than in animals dosed with [3H]AC. Regardless of the dosing schedule, DNA isolated from the lungs and livers of both [3H]NC- and [3H]AC-treated preweanling male mice contained a single major and chromatographically identical adduct. This major adduct, which accounted for as much as 90% of the total carcinogen-DNA adducts in enzymatic hydrolysates from treated animals, was chromatographically distinct from the major C8-purine-substituted adducts formed from the reaction of N-hydroxy-AC with calf thymus DNA. In contrast to the results obtained with NC and AC, the major carcinogen-DNA adduct formed in the livers of mice treated with [3H]-1,6-dinitropyrene was found to cochromatograph with 1-N-(deoxyguanosin-8-yl)amino-6-nitropyrene, a product derived from N-hydroxy-1-amino-6-nitropyrene. Since NC and its nitro-reduced derivative, AC, yielded an identical carcinogen-DNA adduct in vivo and this adduct was not derived from N-hydroxy-AC, we conclude that the metabolic activation of NC in the neonatal mouse must involve some previously undescribed combination of ring-oxidation and nitro-reduction pathways. This activation pathway could be an important factor in determining the potency of NC and AC as carcinogens in this bioassay system.     

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.