DNA and protein adducts in human tissues resulting from exposure to tobacco smoke.

Tobacco smoke contains a variety of genotoxic carcinogens that form adducts with DNA and protein in the tissues of smokers. Not only are these biochemical events relevant to the carcinogenic process, but the detection of adducts provides a means of monitoring exposure to tobacco smoke. Characterization of smoking-related adducts has shed light on the mechanisms of smoking-related diseases and many different types of smoking-derived DNA and protein adducts have been identified.

Randomized, placebo-controlled trial of gastric acid-lowering therapy on duodenal polyposis and relative adduct labeling in familial adenomatous polyposis.

Purpose: Bile has been implicated in the pathogenesis of duodenal polyps in patients with familial adenomatous polyposis. In vitro experiments have shown that familial adenomatous polyposis bile is capable of producing DNA adducts. This effect can be ameliorated by increasing the pH of the incubate.

The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells.

We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair.

Heterocyclic aromatic amines induce DNA strand breaks and cell transformation.

Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk. We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays. The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line.

Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture.

Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage. Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials. To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how alpha-hydroxy-tamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA-adduct-forming species.

Morphological transformation of C3H/M2 mouse fibroblasts by extracts of human mammary lipid.

Mammary lipid may act as a reservoir for genotoxins. Mammary lipid extracts (MLEs), obtained from eight UK women (21-41 years) undergoing reduction mammoplasty, were examined for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Resultant transformation rates were 0.

Anthracene-9,10-diones as potential anticancer agents: bacterial mutation studies of amido-substituted derivatives reveal an unexpected lack of mutagenicity.

Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254. Six of the compounds were also tested in S. typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution.

Lack of telomerase in desmoids occurring sporadically and in association with familial adenomatous polyposis.

Background: Telomerase activity may be required for unlimited growth of cells and is repressed in most somatic tissues, but is detectable in immortal cell lines, germ cells, many malignancies and some benign lesions. Desmoids are proliferative, locally invasive, non-metastasizing fibromatous tumours which rarely regress. They occur frequently in familial adenomatous polyposis (FAP), causing significant morbidity and death.

The metabolic activation of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species in rat hepatocytes proceeds via sulphation.

The biotransformation pathway of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species was investigated in rat hepatocytes in vitro. Rat hepatocytes were isolated by in situ collagenase perfusion and then maintained in sulphate-free Dulbecco's modified Eagle's medium. Magnesium sulphate was added to the medium to give concentrations of 0-10 microM, prior to treatment for 18 h with solvent vehicle (DMSO), tamoxifen (10 microM), alpha-hydroxytamoxifen (1 microM) or benzo[a]pyrene (BaP) (10 and 50 microM).

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.

The use of 32P-postlabelling in studies of the nature and origin of DNA adducts formed by bile from patients with familial adenomatous polyposis and from normal patients.

32P-postlabelling is a highly sensitive technique for the detection of DNA adducts. It is unique in that it requires no prior knowledge of the nature of adducts or adduct-forming species under investigation. In the past, we have used this technique to investigate the role of bile in the production of foregut adenomas in patients with familial adenomatous polyposis (FAP).

Detection of telomerase activity in human prostate: a diagnostic marker for prostatic cancer?

Objective: To assess the role of telomerase activity as a marker for the development of prostate cancer in men with existing benign prostatic hyperplasia (BPH), a known risk factor for prostatic carcinoma.

Materials And Methods: Telomerase activity was assayed, using a highly sensitive polymerase-chain reaction-based assay, in nine biopsies from patients with prostatic cancer, 16 from patients clinically diagnosed with BPH and 11 from patients with no evidence of prostatic disease.

Results: Telomerase activity was detectable in eight of the nine prostate cancer biopsies, in none of the normal prostates and in six of the 16 BPH biopsies.

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.

Mechanisms of spontaneous human cancers.

The causes of much of human cancer remain obscure. The fraction that is spontaneous is unknown and cannot be calculated until all known external causes have been accounted for. This is not a feasible proposition.

Differences in the levels and pattern of DNA-adduct labelling in human cell lines MCL-5 and CCRF, proficient or deficient in carcinogen-metabolism, treated in vitro with bile from familial adenomatous polyposis patients and from unaffected controls.

In patients with familial adenomatous polyposis (FAP), duodenal adenomas cluster around the ampulla and their distribution closely resembles mucosal exposure to bile, suggesting a role for bile in their development. Previous studies using 32P-postlabeling to detect DNA adducts, have provided evidence to support this hypothesis. We have now investigated the role of metabolic activation in influencing the levels and patterns of adduct formation by incubating precolectomy gallbladder bile from FAP patients and bile from unaffected controls with human lymphoblastoid cell lines that are metabolically proficient (MCL-5), or deficient (CCRF).

Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding.

We reported (Scates et al. Carcinogenesis 1994, 15, 2945-2948) that incubating a range of bile acids with DNA in vitro, with or without exogenous metabolic activation, gave no evidence of DNA adduct formation as judged by the nuclease P1 method of 32P-postlabelling. In contrast Hamada et al.

High pH reduces DNA damage caused by bile from patients with familial adenomatous polyposis: antacids may attenuate duodenal polyposis.

Patients with familial adenomatous polyposis (FAP) develop periampullary duodenal tumours, suggesting that bile contributes to their formation. The hypothesis that bile contains carcinogens has been tested by looking for DNA adducts (markers of carcinogen exposure) in the duodenum of patients with or without FAP and by determining whether bile can produce DNA adducts in vitro. Using 32P-postlabelling to detect adducts, there was an excess (compared with unaffected patients) of DNA adducts in the duodenum of FAP patients and an excess of DNA adducts in the small bowel of rats treated with FAP bile, while bile from FAP patients formed significantly more DNA adducts in vitro than did bile from controls.

Mutations in the p53 gene in schistosomal bladder cancer: a study of 92 tumours from Egyptian patients and a comparison between mutational spectra from schistosomal and non-schistosomal urothelial tumours.

Much of bladder cancer in East Africa and the Middle East is attributed to chronic urinary infection with Schistosoma haematobium ('schistosomiasis'). Most schistosomal bladder cancer (SBC) is squamous cell carcinoma (SCC) and occurs in the fifth decade of life. In contrast, nonschistosomal bladder cancer (NSBC) in Western countries usually occurs in the seventh decade of life and is largely transitional cell carcinoma (TCC).

Bile acids do not form adducts when incubated with DNA in vitro.

Bile acids have been implicated in the aetiology of colon cancer. We have previously found, using 32P-postlabelling, that bile from control patients and from patients with familial adenomatous polyposis (FAP) produces DNA adducts when incubated with salmon sperm DNA in vitro. In the present study we have incubated the common primary and secondary, conjugated and unconjugated bile acids with salmon sperm DNA in vitro, in both the presence and absence of metabolic activation (Aroclor-induced rat liver S9).

Reduced genotoxicity of [D5-ethyl]-tamoxifen implicates alpha-hydroxylation of the ethyl group as a major pathway of tamoxifen activation to a liver carcinogen.

A proposed mechanism for the metabolic activation of tamoxifen to electrophilic species that form DNA adducts leading to liver cancer involves alpha-hydroxylation of the ethyl group in the critical first step. This mechanism predicts that tamoxifen deuterated at the alpha-position would be less genotoxic than the non-deuterated compound owing to an isotope effect that would reduce the rate of oxidative metabolism at this position. This hypothesis has now been tested with experiments conducted in rats in vivo and in human cells in vitro.

Mechanisms of carcinogenesis and individual susceptibility to cancer.

The population can be divided into four groups, or "oncodemes," depending on the relative contributions of environment and genetics to their risk of cancer. These oncodemes are: 1) background (random mutations in normal people); 2) environmental (environmental carcinogens acting on normal people); 3) environmental/genetic (environmental carcinogens acting on genetic susceptibility); and 4) genetic, with genetic susceptibility being more important than environmental exposure. Most cancer probably occurs in oncodemes 2 and 3.