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 n M , adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 μ M ) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 μ M 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 μ M 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) ≥100 n M generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 μ M , one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 μ M 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 (⩽10 μ M , 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.

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.

Human cancer is controlled by a complex interaction between genetic and environmental factors. Such environmental factors are well defined for smoking-induced lung cancer; however, the roles of specific genes have still to be elucidated. Glutathione transferase P (GSTP) catalyzes the detoxification of electrophilic diol epoxides produced by the metabolism of polycyclic aromatic hydrocarbons such as benzo [a]pyrene (BaP), a common constituent of tobacco smoke. Activityaltering polymorphisms in Gstp have therefore been speculated to be potential risk modifiers in lung cancer development. To clearly establish a role for GSTP in lung tumorigenesis, we investigated whether deletion of the murine Gstp genes (Gstp1 and Gstp2) alters susceptibility to chemically induced lung tumors following exposure to BaP, 3-methylcholanthrene (3-MC), and urethane. Gstp-null mice were found to have substantially increased numbers of adenomas relative to wildtype mice following exposure to all three compounds (8.3-, 4.3-, and 8.7-fold increase for BaP, 3-MC, and urethane, respectively). In Gstp-null mice, the capacity of pulmonary cytosol to catalyze conjugation of the BaP diol epoxide was significantly reduced. Concomitant with this, a significant increase in the level of BaP DNA adducts was measured in the lungs of null animals; however, no increase in DNA adducts was measured in the case of 3-MC exposure, suggesting that an alternative protective pathway exists. Indeed, significant differences in pulmonary gene expression profiles were also noted between wild-type and null mice. This is the first report to establish a clear correlation between Gstp status and lung cancer in vivo.

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The drug tamoxifen, used to treat breast cancer, causes liver cancer in rats and endometrial cancer in women. Tamoxifen forms liver DNA adducts in both short- and long-term dosing of rodents, and DNA adducts have also been reported in tissues of women undergoing tamoxifen therapy. It is not known if the induction of endometrial cancer in women is through these DNA adducts or through the estrogenic nature of the drug. In this study, we have investigated the mutagenicity of two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM). These form the same DNA adducts as those found in tamoxifen-treated rats. The two compounds were used to treat the pSP189 plasmid containing the supF gene, which was replicated in Ad293 cells before being screened in indicator bacteria. Plasmid reacted with 4-OHtamQM was more likely to be mutated (2-7-fold increase) than that reacted with alpha-acetoxytamoxifen, despite having a lower level of DNA damage (12-20-fold less), as assayed by (32)P-postlabeling. The two compounds induced statistically different mutation spectra in the supF gene. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC -->TA transversions while GC-->AT transitions were formed in 4-OHtamQM-treated plasmid. 4-OHTamQM-treated DNA induced a larger proportion of multiple mutations and large deletions compared to alpha-acetoxytamoxifen. Sites of mutational hotspots were observed for both compounds. In conclusion, the quantitatively minor DNA adduct of tamoxifen (dG-N(2)-4-hydroxytamoxifen) is more mutagenic than the major tamoxifen DNA adduct (dG-N(2)-tamoxifen).

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Reaction of 2'-deoxyguanosine 3'-monophosphate with mixtures of the benzene metabolites p-benzoquinone (p-BQ) and hydroquinone (HQ) in an aqueous solution at pH 6.0 gave two main products which were isolated from the reaction mixture using reversed-phase HPLC and characterized using UV spectroscopy, negative ion electrospray mass spectrometry, and (1)H NMR. Variation of the ratio of p-BQ to HQ in the reaction mixture caused an increase in yield of one of the products. The two products were identified as (3"-hydroxy)-1,N(2)-benzetheno-2'-deoxyguanosine 3'-monophosphate and a new product, not previously characterized, (3",4"-dihydroxy)-1,N(2)-benzetheno-2'-deoxyguanosine 3'-monophosphate. Similar products were isolated from identical reactions with 2'-deoxyguanosine. Reaction of calf thymus DNA with HQ and p-BQ (1:1, w/w) resulted in four main products as identified by (32)P-postlabeling coupled with HPLC. The relative abundances of these adducts were 9%, 60%, 27%, and 4%, respectively. Co-chromatography of (32)P-postlabeled (3"-hydroxy)-1,N(2)-benzetheno-2'-deoxyguanosine 3'-monophosphate and (3",4"-dihydroxy)-1,N(2)-benzetheno-2'-deoxyguanosine 3'-monophosphate with the (32)P-postlabeled adducted calf thymus DNA identified these as the two minor products of the calf thymus DNA reaction.

No abstract