Biomedical subjects

D H Phillips

Publications and source records attributed to D H Phillips.

At least 73 records · Page 4Linked to original sources

PubMed1997

Detection of DNA modifications by the 32P-postlabelling assay.

The 32P-postlabelling assay is widely used for detection of carcinogen-DNA adducts and other types of modified nucleotides in DNA. The principle of the method is the enzymatic digestion of DNA to nucleotides, 5'-labelling of these nucleotides with an isotopically labelled phosphate group, and the resolution and detection of the labelled products. Since the development of the original procedure in the early 1980s, many methods have been developed to increase the sensitivity of the method by selection of the modified nucleotides prior to labelling. In favourable circumstances, the method can achieve a level of detection as low as 1 modification in 10(10) nucleotides and requires relatively small quantities of DNA (less than 10 microg). It has been used to detect and characterise DNA adducts formed by numerous genotoxic carcinogens in bacterial and mammalian cells, in animals and, in some cases, in human tissues. Most classes of carcinogen have been subjected to 32P-postlabelling analysis, ranging from bulky and/or aromatic compounds to small and/or aliphatic compounds; it has also been used, with modifications, to detect apurinic sites in DNA, oxidative damage to DNA, UV-induced photodimers and, to a lesser extent, DNA damage caused by cytotoxic drugs. It has provided the first clear evidence for the DNA-damaging properties of several synthetic carcinogenic hormones. It has revealed the DNA-damaging potential of complex mixtures such as coal-tar and tobacco smoke. It has been used in human biomonitoring studies to detect DNA damage from occupational exposure to carcinogens, and also from environmental (i.e. non-occupational) exposures. It has also led to the discovery of the presence of numerous modifications in DNA arising from endogenous processes. The rapid expansion in the use of the assay has resulted in some divergence of procedures and there is a case to be made for the use of more standardised protocols, particularly where human exposure to carcinogens is being measured and where such results may be required for risk assessment. While the procedure is quantifiable, the efficiency of adduct labelling is, in many cases, not quantitative, and the lack of adduct standards has, in many cases, limited the interpretation of data to a demonstration of higher adduct levels in exposed groups compared with unexposed groups. Future developments are expected in automation, standardisation and, in combination with other analytical methods, elucidation of the structures of the many DNA lesions whose existence has been revealed by the 32P-postlabelling technique.

Animals↗

PubMed1997

Generation of putative intrastrand cross-links and strand breaks in DNA by transition metal ion-mediated oxygen radical attack.

Generation of putative intrastrand cross-links and strand breaks was investigated in salmon sperm DNA exposed to Fenton-type oxygen radical-generating systems. 32P-Postlabeling analysis of DNA treated with hydrogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(II), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radical-mediated oxidative DNA lesions. The copper Fenton system generated the highest total yield of these DNA lesions (75.6 per 10(8) nucleotides), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (21.7), and vanadium (17.1). Two spots, common to all these Fenton systems, were the major oxidation products in each case. Similar Fenton-type treatment of the purine dinucleotides dApdG and dApdA resulted in products that were chromatographically identical on anion-exchange TLC and on reverse-phase HPLC to the two major products generated in DNA. These results extend our earlier studies suggesting that these products were the result of a free radical-mediated intrastrand cross-linking reaction. Incubations involving cadmium(II), chromium(III), or zinc(II) ions with hydrogen peroxide did not generate DNA oxidation products at levels greater than in incubations with hydrogen peroxide alone. Generation of the putative intrastrand cross-links increased in a concentration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) ions. However, in experiments with copper, iron, or vanadium ions, maximum levels were obtained at 250, 150, and 150 microM, respectively, and the yield declined with higher concentrations of these three metal ions. Agarose gel electrophoresis demonstrated extensive DNA strand breakage with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results demonstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independent mechanisms.

Animals↗

PubMed1997

Covalent DNA adducts formed by benzo[c]chrysene in mouse epidermis and by benzo[c]chrysene fjord-region diol epoxides reacted with DNA and polynucleotides.

The metabolic activation in mouse skin of benzo[c]chrysene (B[c]C), a weakly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar and crude oil, was investigated. Male Parkes mice were treated topically with 0.5 mumol of B[c]C, and DNA was isolated from the treated areas of skin at various times after treatment and analyzed by 32P-postlabeling. Seven adduct spots were detected, at a maximum level of 0.89 fmol of adducts/microgram of DNA. Four B[c]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 mumol of the optically pure putative proximate carcinogens (+)- and (-)-trans-benzo[c]chrysene-9,10-dihydrodiols [(+)- and (-)-B[c]C-diols] led to the formation of adducts which comigrated on TLC and HPLC with some of those formed in B[c]C-treated mice. The major adduct formed in mouse skin treated with B[c]C coeluted on TLC and HPLC with an adduct formed in mouse skin treated with (-)-B[c]C-diol. These results suggested that the detected adducts were formed by the fjord-region B[c]C-9,10-dihydrodiol 11,12-epoxides (B[c]CDEs). To test this, the four optically pure synthetic B[c]CDEs were reacted in vitro with DNA and with synthetic polynucleotides and these samples were 32P-postlabeled. Cochromatography, both on TLC and HPLC, of in vitro and in vivo adducts indicated that B[c]C is activated in mouse skin through formation of the (-)-anti- and (+)-syn-B[c]CDE with 9R,10S,11S,12R- and 9S,10R,11S,12R- absolute configuration, respectively, both of which formed two DNA adducts in vivo. However, the major adduct present in the B[c]C-treated skin DNA was not a fjord-region B[c]CDE adduct but was possibly derived from a bay region B[c]CDE at the 1,2,3,4-position. The extent of DNA adduct formation by B[c]C in mouse skin DNA was lower than that of moderately carcinogenic PAHs previously studied by this method, suggesting a correlation between extent of DNA adduct formation and carcinogenic potential.

Animals↗

PubMed1997

Metabolic competence and susceptibility of intestinal epithelium to genotoxic injury during regeneration.

The carcinogenic potency of many mutagens is increased in conditions of tissue regeneration. This involves fundamental changes of cellular division and differentiation, in intestinal epithelium. However, effects on epithelial capacity for carcinogen metabolism and susceptibility to genotoxic injury are unknown. Using a novel rat model, this study assessed expression of cytochrome P450 mono-oxygenases (Cyps), glutathione S-transferases (GSTs) and uridine diphosphoglucuronosyl transferase (UGT) in intestinal epithelium during sequential stages of regeneration. Enzyme induction and DNA adduct formation were also assessed after benzo[a]pyrene (BaP) exposure. Control assays were carried out in normal intestinal epithelium. Fewer phase I and II xenobiotic metabolizing enzymes were expressed in regenerating intestinal epithelium than in normal control intestinal epithelium (GSTA3, UGT in regeneration vs Cyp2B, GSTA1/2, GSTA4, GSTP1, UGT in control). Benzo[a]pyrene induced GSTA3 and UGT in regeneration vs Cyp1A, Cyp2B, GSTA1/2, GSTA3, GSTA4, GSTP1 and UGT in control normal intestinal epithelium. Benzo[a]pyrene induced low levels of GSTA3 in early regenerating intestinal epithelium but induction increased by >2-fold at late stage regeneration. Higher levels of benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE) DNA adducts were formed at early stages of regeneration, than at later stages. Intestinal epithelium displayed reduced metabolic competence and differential susceptibility to genotoxic injury from BaP, during regeneration.

Animals↗

PubMed1997

DNA damage in breast epithelial cells: detection by the single-cell gel (comet) assay and induction by human mammary lipid extracts.

The presence of DNA damage in primary cultures of human mammary epithelial cells (HMECs), and the ability of extracts of human mammary lipid to cause such damage, has been investigated. Lipid extracts, prepared by a solid-phase procedure, and HMECs were obtained from breast tissue removed from healthy women (ages 18-50 years) who were resident in the UK and undergoing elective reduction mammoplasties. DNA single strand breaks (SSBs) were detected using the single-cell gel assay (comet assay) with alkaline electrophoresis (pH 12.3) and quantified by measuring comet tail length (CTL) (microm). Untreated HMECs and HMECs incubated (30 min, 37 degrees C) with a mammary lipid extract, with or without DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C), were examined. Ionizing radiation was used as a positive control. An active lipid extract gave a linear dose-response over the range 2.0-12.2 g equivalents. When MCL-5 cells, a line of metabolically-competent human lymphoblastoid cells, were used to compare the DNA-damaging properties of lipid extracts from six different donors, significant interindividual variations (median CTLs were 15.0, 53.5, 32.5, <4.0, <4.0 and 77.5 microm respectively) were observed. In eight subjects, the donors' HMECs were examined both before and after treatment with extracts of that donor's own lipid. Pre-existing DNA damage was detected in untreated HMECs from some donors (median CTLs 22.0-37.5 microm) that was not present in others (median CTLs 4.0-11.5 microm), and increases in CTL could be induced by incubation with the matching lipid extract (8 g equivalent) in more than half (five out of eight) the subjects examined (median CTL up to 111.0 microm). There was a tendency for the most active lipid extracts to be those obtained from donors whose HMECs also contained the most pre-existing DNA SSBs. The results of this pilot study may prove to be significant in relation to the initiation of breast cancer.

Adult↗

PubMed1997

Minor products of reaction of DNA with alpha-acetoxytamoxifen.

The drug tamoxifen shows evidence of genotoxicity and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen and these arise, at least in part, from its metabolite alpha-hydroxytamoxifen. This probably undergoes conjugation in the liver tissue to give an ester, which alkylates DNA. We have prepared alpha-acetoxytamoxifen as a model for this reactive intermediate and studied its reaction with DNA in vitro. The products of this reaction were chromatographically identical to DNA adducts found in the liver of rats treated with tamoxifen. We have isolated three of these products as the nucleosides TG1, TG2 and TA1 and identified them by ultraviolet, mass and proton magnetic resonance spectroscopy. TG1 and TG2 were tamoxifen-deoxyguanosine adducts in which the alpha-position of tamoxifen was linked to the amino group of guanine; TG1, (E)-4-[4-[2-(dimethylamino)ethoxy]phenyl]-3,4-diphenyl-2-(9beta-de oxyribofuranosyl-6-oxopurin-2-ylamino)-3-butene; TG2, (Z) isomer of TG1. In TG2, the tamoxifen group had undergone trans-cis isomerization. The minor product TA1 was a tamoxifen-deoxyadenosine adduct, where linkage was through the amino group of adenine: (E)-4-[4-[2-(dimethylamino) ethoxy]phenyl]-3,4-diphenyl-2-(9beta-deoxyribofuranosylpurin -6-ylamino)-3-butene. These three adducts accounted for >90% of the reaction products (approximately 67% TG1, 18% TG2 and 7% TA1); trace products included other stereoisomers of these and dinucleotide adducts which resisted enzymatic digestion.

Animals↗

PubMed1997

Genotypes of glutathione transferase M1 and P1 and their significance for lung DNA adduct levels and cancer risk.

The A-G polymorphism at codon 104 in the glutathione S-transferase P1 (GSTP1) gene was examined in 138 male lung cancer patients and 297 healthy controls. The patients had significantly higher frequency of the GG genotype (15.9%) and a lower frequency of AA (38.4%) than the controls (9.1% and 51.5%, respectively). The level of hydrophobic DNA-adducts were determined in lung tissue from 70 current smokers. Patients with the GG genotype had a significantly higher adduct level than patients with AA (15.5 +/- 10.2 vs 7.9 +/- 5.1 per 10(8) nucleotides, P = 0.006). We also analyzed the deletion polymorphism in the GSTM1 gene in 135 male patients and 342 controls. The patients were stratified according to histology, smoking dose, age, adduct level and mutational types found in the tumors (Ki-ras and p53 genes). The results consistently indicated that the GSTM1 null genotype was associated with a slightly increased lung cancer risk. When the combined GST M1 and P1 genotypes were examined, patients with the combination null and AG or GG had significantly higher adduct levels than all other genotype combinations (P = 0.011). The distribution of combined genotypes was also significantly different in cases and controls, mainly due to increased frequency of the combination GSTM1 null and GSTP1 AG or GG among patients.

Adult↗

PubMed1996

Genotoxicity of human mammary lipid.

We tested the proposition that human mammary lipid contains mutagenic/genotoxic agents that could cause DNA damage in adjacent epithelial cells. Lipid samples from breast tissue surgically removed from 40 women undergoing elective reduction mammoplasty were extracted by a solid-phase procedure. Mutagenicity was observed in Salmonella typhimurium TA98 and TA1538 in 16 of 40 (40%) extracts assayed with rat-liver S9, but not in its absence. No mutagenicity was seen in S. typhimurium TA100 or Escherichia coli WP2uvrA(pKM101). Bacterial mutagenicity correlated with micronucleus-forming activity in a metabolically competent mammalian cell line (MCL-5). This genotoxic activity merits further investigation in relation to the etiology of breast cancer.

Adipose Tissue↗

PubMed1996

DNA adducts from chemotherapeutic agents.

The guiding principle of early work was the hypothesis that the anti-cancer alkylating drugs acted through their ability to cross-link macromolecules essential for cell division. Not long afterwards, DNA was specified as the essential target, and support for the hypothesis came from evidence that the archetypal agent, mustard gas, could link guanine bases in DNA through their N-7 atoms. Quantitative correlations between alkylation of DNA and its inactivation as a template followed, with bacteriophage as a simple test object, showing that the mean lethal dose was close to a single cross-link in the genome. This conclusion applied to either mustard gas or the more recently introduced platinum drugs. Although both inter- and intra-strand cross-links were effective, it was thought that in cells the inter-strand cross-link would, by preventing the separation of the strands necessary for cell division, and by being more difficult to repair, constitute the more effectively lethal lesion. With repair-deficient bacteria, it also emerged that a single cross-link in the genome was lethal, but proficient bacteria could remove about 20 cross-links through excision repair. Mono-7-alkylguanines were not removed and were evidently inert. Thus, only a few percent of the total alkylation products were the most effective lesions. Parallel studies with cultured mammalian cells gave a rather different picture, in that the mean lethal doses of even hypersensitive cell lines were around 20 or more cross-links per genome, about the same as for resistant strains of bacteria. Most cells could withstand several hundreds of cross-links per genome, and although adducts were removed, there was incomplete removal of cross-links. Some, but not all, sensitive cell lines were deficient in excision repair. Methods were devised for measuring the extents of alkylation of DNA in cells of patients treated with chemotherapeutic drugs; these are mainly immunoassays, and were applied generally to peripheral blood leukocytes, although some tumours were studied. Extents of alkylation of leukocyte DNA were generally of the same order as, or rather less than the mean lethal doses of cultured cells of the 'normal' type, but in some reports for cisplatin-treated patients, very wide variability between individuals was found. A positive correlation between adduct levels, and particularly a very minor adduct recognised specifically by one antibody, and favourable therapeutic outcome was discerned, and suggested to have a pharmacogenetic basis. In several instances, extents of alkylation of tumours were significantly higher than the average for leukocytes; for ovarian and a testicular tumour for cisplatin, and for a plasma cell tumour for melphalan. Nevertheless, these favourable examples would not constitute more than three or four mean lethal doses in the tumour cells, assuming that they had the same sensitivity as 'normal' cell lines: the therapeutic effect would of course be much more favourable if the tumour cells resembled 'sensitive' cell lines. This lack of a favourable difference between extents of alkylation in DNA of patients and the mean lethal dose for normal cells was particularly obvious with the methylating drugs dacarbazine and procarbazine. These considerations stress the need for higher extents of alkylation to be achieved in target tumour DNA for successful chemotherapy. One approach is to give a higher overall dose, and to 'rescue' the bone marrow (known from the earliest report on mustard gas to be the most susceptible tissue) by autologous transplantation. The second, which has yet to reach the clinic, is to convert unreactive prodrugs through enzymic activation into alkylating agents specifically in tumours (see Bagshawe, 1994).

Animals↗

PubMed1996

Lack of genotoxicity of tamoxifen in human endometrium.

The potential for the anti-breast cancer drug tamoxifen [(Z)-1-[4-[2-( dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene] to induce genotoxic damage (DNA adducts) in the human endometrium was investigated in vivo and in vitro. Endometria from hysterectomy patients who were not on tamoxifen were sectioned and maintained in short-term organ culture. The cultures were treated with either solvent vehicle (DMSO), tamoxifen, alpha-hydroxytamoxifen [(E)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-buten-3- ol; the major DNA-reactive metabolite in the rat], or benzo(a)pyrene. DNA was isolated and analyzed by 32P postlabeling. Chromatography on polyethyleneimine-cellulose TLC plates revealed DNA adducts in endometria treated with alpha-hydroxytamoxifen identical to those seen previously in the rat liver. However, no adducts were seen from treatment with tamoxifen itself. The viability of the enzyme-metabolizing systems of the endometrial samples was demonstrated by the detection of expected DNA adducts induced by benzo(a)pyrene. Examination by liquid chromatography-mass spectrometry of the explant culture media from endometria treated with tamoxifen revealed the presence of the alpha-hydroxy metabolite in a dose-dependent manner, although apparently at levels insufficient to produce detectable DNA adducts. Endometrial DNA obtained from 18 patients undergoing daily treatment with 10-40 mg tamoxifen for 3 months-9 years was also analyzed. No evidence for any DNA adducts induced by tamoxifen was found in any of the patients examined. These data suggest that the genotoxic events observed with tamoxifen in the rat may not apply to the human endometrium.

Adult↗

PubMed1996

Identification of the major tamoxifen-deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen.

The antiestrogenic drug tamoxifen induces liver tumors in rats by a genotoxic mechanism. The key step has been proposed to be the formation of a reactive carbocation from the metabolite alpha-hydroxytamoxifen. This compound reacts with DNA in vitro to a small extent (1 in 10(5) DNA bases), giving products identical to those found in rat liver cells treated with tamoxifen. Now we have prepared the more reactive alpha-acetoxytamoxifen, which reacts with DNA in vitro to a much greater extent (1 in 50 bases). The products of this reaction were subjected to 32P postlabeling and shown by both TLC and reverse-phase liquid chromatography to be identical to those isolated from DNA treated with alpha-hydroxytamoxifen and to those found in the liver DNA of rat hepatocytes treated with tamoxifen or of the livers of rats treated with tamoxifen. The major product was also isolated as the nucleoside and characterized by UV, mass, and proton magnetic resonance spectroscopy. It is an adduct of tamoxifen and deoxyguanosine in which the alpha position of tamoxifen is linked covalently to the exocyclic amino group of deoxyguanosine.

Animals↗

PubMed1996

Visual perception elicited by electrical stimulation of retina in blind humans.

OBJECTIVE: To evaluate the feasibility of bypassing damaged photoreceptors and electrically stimulating the remaining viable retinal layers to provide limited visual input to patients who are blind because of severe photoreceptor degeneration. METHODS: In the operating room with the patient under local anesthesia, focal electrical stimulation of the retinal surface with brief biphasic pulses was performed using small probes inserted through the sclera. The procedure was performed in five subjects who had little or no light perception. Three subjects had retinitis pigmentosa, one had age-related macular degeneration, and one had unspecified retinal degeneration from birth. RESULTS: Stimulation elicited visual perception of a spot of light (phosphene). Subjects who previously had useful vision accurately localized the phosphenes according to the retinal area stimulated. Two subjects could track the movement of the stimulating electrode by reporting movement of the elicited phosphene, and could perceive two simultaneous phosphenes on independent stimulation with two electrodes. In a resolution test, one of the subjects with no light perception in his left eye resolved phosphenes at 1.75 degrees center-to-center distance (ie, 4/200 OS visual acuity). CONCLUSIONS: Local electrical stimulation of the retinal surface in patients blind from outer retinal disease results in focal light perception that seems to arise from the stimulated area. Such findings in an acute experiment warrant further research into the possibility of prolonged retinal stimulation, improved resolution, and ultimately, an intraocular visual prosthesis.

Adult↗

PubMed1996

Activation of tamoxifen and its metabolite alpha-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes.

The metabolic activation of tamoxifen and its metabolite alpha-hydroxytamoxifen in primary cultures of rat, mouse and human hepatocytes has been compared. The extent of formation of DNA adducts in these cells was measured by 32P-postlabelling, using either nuclease P1 digestion or sorbent extraction of DNA digests to enhance the sensitivity of the assay. DNA adducts were readily detected in rat hepatocytes treated with 1 or 10 microM tamoxifen (mean levels 18.2 and 89.8 adducts/10(8) nucleotides respectively). Similar levels of adducts were formed by mouse hepatocytes (15.0 +/- 1.8 adducts/10(8) nucleotides, 10 microM tamoxifen). However DNA adducts were not detected in tamoxifen-treated human hepatocytes with a detection limit for the assay of 4 adducts/10(10) nucleotides. Treatment of rat cells with alpha-hydroxytamoxifen resulted in 15- to 63-fold higher levels of adducts than with comparable concentrations of tamoxifen. A similar level of adducts was also seen in mouse hepatocytes treated with alpha-hydroxytamoxifen at the 1 microM concentration (173.9 +/- 4.1 adducts/10(8) nucleotides). Treatment of human cells with alpha-hydroxytamoxifen resulted in DNA adduct formation at levels (1.94 +/- 0.89 and 18.9 +/- 17.9 adducts/10(8) nucleotides at 1 and 10 microM respectively) approximately 300-fold lower than those in rat hepatocytes. The presence of alpha-hydroxytamoxifen in the culture medium from experiments where cells were incubated with tamoxifen was monitored by mass spectrometry. Concentrations were found to be approximately 50-fold lower in the medium from human hepatocytes than from rat and mouse hepatocytes.

Adolescent↗

PubMed1996

p53 mutations in lung tumours: relationship to gender and lung DNA adduct levels.

Human lung cancer exhibits a high frequency of transversion mutations at G:C base pairs of the p53 gene, possibly the result of DNA damage by cigarette smoke constituents, most notably benzo[a]pyrene. We have investigated gender differences in the p53 mutational spectrum and levels of hydrophobic DNA adducts. Tumour tissue was obtained from 115 non-small cell lung cancer tumours and examined for mutational alterations in the p53 gene (exons 4-9) using PCR and single-strand conformational polymorphism analysis. We have previously examined exons 5-8 in lung cancer. Sequence analysis of exons 4 and 9 revealed that almost 20% of the mutations were located in exons 4 and 9. The levels of hydrophobic DNA adducts in non-tumorous lung tissue of 55 of the patients were analyzed by the 32P-postlabelling assay. There were both a higher frequency of G:C-->T:A mutations and a higher average hydrophobic DNA adduct level in females than in male patients, even though the level of exposure to carcinogens from cigarette smoking was lower among the females than among the males. Frameshift mutations were more common in women than in men (30 versus 15%). These preliminary findings lend support to epidemiological evidence that women may be at greater risk than men of contracting tobacco-induced lung cancer.

Adenocarcinoma↗

PubMed1996

Tamoxifen does not form detectable DNA adducts in white blood cells of breast cancer patients.

DNA from white blood cells of seven women receiving tamoxifen as adjuvant therapy for breast cancer and of three women who served as healthy controls was analysed for the presence of tamoxifen-DNA adducts using 32P-postlabelling with a limit of detection of 8 adducts/10(10) nucleotides. No postlabelled adducts with the chromatographic properties of known tamoxifen-DNA adducts were detected in any of the samples. It is concluded that at therapeutic levels of exposure there is no significant formation of DNA adducts by tamoxifen or its metabolites in circulating white blood cells.

Adult↗

PubMed1996

Covalent DNA adducts formed in mouse epidermis by benzo[g]chrysene.

The metabolic activation in mouse skin of benzo[g]chrysene (B[g]C), a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar, was investigated. Male Parkes mice were treated topically with 0.5 micromol B[g]C and DNA was isolated from the treated areas of skin at various times after treatment and analysed by 32P-post-labelling. Seven major adduct spots were detected, at a maximum level of 6.55 fmol adducts/microg DNA. Mouse skin treated with the PAH benzo[c]phenanthrene (B[c]Ph) gave a total of 0.24 fmol adducts/microg DNA. B[g]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 micromol of the optically pure putative proximate carcinogens, the (+)- and (-)-trans benzo[g]chrysene-11,12-dihydrodiols, led to the formation of adducts which comigrated on TLC and HPLC with those formed in B[g]C-treated mice, which suggested that the detected adducts were formed by the fjord region B[g]C-11,12-dihydrodiol-13,14-epoxides (B[g]CDEs). To test this, the four optically pure synthetic B[g]CDEs were reacted in vitro with DNA and the heteroco-polymers poly(dA x dT) and poly(dG x dC) and these samples 32P-postlabelled. Co-chromatography, on both TLC and HPLC, of in vitro and in vivo adducts indicated that B[g]C is activated in mouse skin through formation of the (-)-anti-(11R,12S,l3S,14R) and (+)-syn-(11S,12R,13S,14R) B[g]CDEs. (-)-anti-B[g]CDE formed five adducts with DNA, two of them with adenine and three with guanine bases. (+)-syn-B[g]CDE formed one adduct with each of these bases in DNA. The adenine adducts accounted for 64% of the total major adducts formed in B[g]C-treated mouse skin. The route of metabolic activation or B[g]C is similar to that reported for B[c]Ph, but the extent of activation to the fjord region diol-epoxides is significantly greater in the case of B[g]C, as demonstrated by the higher levels of adduct formation in vivo.

Animals↗

PubMed1996

Metabolic activation and DNA binding of food mutagens and other environmental carcinogens in human mammary epithelial cells.

Cultures of human mammary epithelial cells were treated with one of seven heterocyclic amine food mutagens [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-di-methylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-3,4,7,8-tetramethylimidazo[4,5-f]quinoxaline (4,7,8-TriMeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP)], four nitropyrenes (1-nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP) or 1,8-dinitropyrene (1,8-DNP)] or the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). DNA isolated from the cultures was analysed by 32P-post-labelling and in each case the presence of carcinogen-DNA adducts was detected. The patterns and numbers of adducts obtained when human mammary cell DNA digests were separated on polyethyleneimine-cellulose TLC were found to closely resemble those previously demonstrated to be present in the DNA of tissues from rodents and other primates treated with the same agents. Up to six DNA adducts were detected in human breast cells treated with IQ and MeIQ. Fewer adducts (1-3) were detected following treatment with MeIQx or its methylated derivatives, whilst PhIP gave rise to at least four distinct adduct spots. Five adduct spots were detected in breast cells treated with DB[a,l]P or with 1-NP, but fewer adduct spots were formed by 1,3-, 1,6- and 1,8-DNP. These data demonstrate the ability of human breast epithelial cells to activate to DNA binding species a range of carcinogenic compounds known to be present in the human diet or to which humans are known to be exposed environmentally.

Biotransformation↗

PubMed1996

DNA adducts in human tissues: biomarkers of exposure to carcinogens in tobacco smoke.

Tobacco smoking causes millions of cancer deaths annually. Tobacco smoke is a complex mixture of thousands of chemicals including many known animal carcinogens. Because many carcinogens from DNA adducts in target animal or human tissues, the detection of the formation of adducts using such methods as postlabeling, immunoassay, fluorescence spectroscopy, and mass spectrometry is a means of monitoring human exposure to tobacco carcinogens. Smokers are at increased risk of cancer in many organs, and studies have revealed either specific adducts related to smoking or increased levels of adducts in the lung, bronchus, larynx, bladder, cervix, and oral mucosa of smokers. In a limited number of studies, the adducts and the carcinogens responsible for them have been identified. Some studies have demonstrated higher levels of adducts in the white blood cells of smokers, while other studies indicate other sources of genotoxic agents, including diet, can contribute to the DNA damage observed in these cells.

Biomarkers, Tumor↗