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 aim of this double-blind randomized placebo-controlled trial was to examine the effect of oral ranitidine on duodenal polyposis in a group of patients with familial adenomatous polyposis. METHODS: Twenty-six patients with familial adenomatous polyposis were randomly assigned to ranitidine 300 mg daily or placebo for six months after baseline endoscopy. Polyp counts were performed and biopsy specimens taken to detect DNA adducts by 32P-postlabeling. RESULTS: No difference was seen in polyp numbers (P = 0.9) or relative adduct labeling (P = 0.7) after treatment with ranitidine or placebo. DISCUSSION: Acid suppression therapy does not seem to improve duodenal polyposis despite in vitro findings. On the other hand, ranitidine does not exacerbate actual (or markers of) neoplasia in this highly tumor-prone condition.

The use of short- and medium-term tests for carcinogens and data on genetic effects in carcinogenic hazard evaluation.

It evaluates the use of short- and medium-term assays with end-points of neoplasia or lesions that are precursors to neoplasia, and attempts to define the role of data from genetic toxicology in the prediction of carcinogenic hazard.

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. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7, 12-dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked comet formation even in the presence of HU/ara-C. Aniline and o-toluidine produced significant levels of comet formation in the absence of HU/ara-C, but in their presence comet formation was markedly increased. Benzene, lindane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride induced comet formation in the absence of HU/ara-C, but, with the exception of cisplatin, their presence enhanced comet formation. Neither sucrose nor DES elicited comet formation under the conditions used in this study. Many more agents need to be tested in order to determine how well the comet assay using MCL-5 cells (or modified versions of it) can distinguish genotoxins from non-genotoxins.

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. Concentration levels of 50 microM 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 100 microM 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (4,8-DiMeIQx), 50 microM 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 100 microM 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 100 microM 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 15 microM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 10(4) surviving cells, respectively. Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019. Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with AalphaC, 2.9 revs/ng with MeAalphaC and 5 revs/ng with PhIP were observed. Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells. Dose-dependent induction of micronuclei was observed for all HAAs tested with 1-5.4% of cells containing micronuclei at 10 ng/ml. Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeAalphaC > PhIP > AalphaC. DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay. The lowest effect doses for significant increases (P < or = 0.0007, Mann-Whitney test) in comet tail length (microm) were 45.5 microg/ml (200 microM) for PhIP, 90.9 microg/ml (410-510 microM) for 4,8-DiMeIQx, IQ, MeAalphaC and AalphaC, and 454.5 microg/ml (2130 microM) for 8-MeIQx. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens.

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. We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested alpha-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls. In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats. Alpha-hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase. All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S. typhimurium. None activated alpha-hydroxytamoxifen to a mutagen. These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.

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 0.27, 0.33, 0.07, 0.29, 0.21, 0.00, 0.07, and 0.13 transformed foci/treated dish, respectively. Although the lipid-extraction procedure used was originally designed to extract heterocyclic aromatic amines (HAAs), liquid chromatography/mass spectroscopy (LC/MS) with selective ion monitoring has failed to detect HAAs in any of the lipid extracts so far examined. Genotoxicities were also assessed in S. typhimurium TA98 and in metabolically competent human (MCL-5) cells by the micronucleus and by the alkaline single-cell gel ("comet") assays. The MLEs induced bacterial mutagenicity rates ranging from 0 to 498 revertants/plate/g-lipid equivalent and micronucleus-formation rates from 0 to 20 micronuclei/500 binucleate cells/g-lipid. Median comet tail lengths (induced with MLEs of 8.0 g-lipid equivalent) ranged from 6.0 to 74.0 micrometer. The results demonstrate the presence of as-yet-unidentified transforming agents in mammary lipid.

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. Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls. Mitoxantrone was mutagenic to S. typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant. By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined. Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.

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. Telomerase activity was assayed in desmoids from patients with and without FAP to assess the role of telomerase in the development of these lesions, and its potential as a prognostic marker and possible target for treatment. METHODS: Protein extracts from 11 desmoids from nine patients with FAP, and ten desmoids from ten patients without FAP, were analysed for telomerase activity by the telomeric repeat amplification protocol, a sensitive polymerase chain reaction-based assay. Six fibrosarcomas and a fibrosarcoma cell line were used as positive controls; all displayed telomerase activity. RESULTS: No telomerase activity was detected in any of the 21 desmoids studied. CONCLUSION: These results indicate that desmoid tumours are one of the intriguing exceptions to the emerging view that re-expression of telomerase activity accompanies the development of preneoplastic and neoplastic tissues, and suggest that alternative mechanisms may operate in these proliferative neoplasms.

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 was isolated and analysed by 32P-post-labelling. For tamoxifen and alpha-hydroxytamoxifen, the level of DNA adduct formation was directly proportional to the concentration of sulphate in the medium. Between 0 and 10 microM MgSO4, the DNA adduct level increased 10-fold with both compounds. Rat hepatocytes were also maintained in normal Dulbecco's modified Eagle's medium and pretreated with dehydroisoandrosterone-3-sulphate (DHEAS, a sulphotransferase inhibitor) at concentrations ranging from 0-1 mM, prior to treatment with solvent vehicle (DMSO), tamoxifen (10 microM), alpha-hydroxytamoxifen (1 microM) or BaP (50 microM). For tamoxifen and alpha-hydroxytamoxifen the level of DNA adducts was reduced to approximately one-fifth by the addition of DHEAS (0.1 mM). BaP-DNA adduct formation, which proceeds by a pathway that does not require sulphation, was not significantly affected by sulphate concentration or by addition of DHEAS, which demonstrates that the general metabolic capacity and viability of the hepatocytes were not compromised. It is concluded that the activation of tamoxifen in rat liver cells to DNA binding products proceeds predominantly through hydroxylation followed by sulphate ester formation at the alpha-position of the ethyl side chain.

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. CONCLUSION: The finding of telomerase activity in six of 16 biopsies from patients with BPH could indicate early prostate cancer and suggests that telomerase activity may be of use as a biomarker in patients diagnosed with BPH and who may subsequently develop prostate cancer.

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). We have found that bile contains constituents that form DNA adducts directly, and after metabolic activation, and that the bile of FAP patients has an increased capacity for adduct formation with DNA in vitro, in human cell lines in culture, and in the gastrointestinal tract of rats given bile by gavage. The sensitivity of 32P-postlabelling is such that it is difficult to obtain sufficient quantities of DNA adducts for chemical analysis. The nature of the adducts produced by bile, or of the bile constituents that produce them is as yet undetermined. In the present studies, we have combined 32P-postlabelling with indirect methods to gain some insight into the nature of DNA adducts produced by bile and the properties of the reactive species that form them. Firstly, bile was incubated with synthetic monodeoxynucleotides or polydeoxynucleotides. Bile did not produce adducts when incubated with monodeoxynucleotides or single-stranded polydeoxynucleotides. However, it did produce adducts when incubated with double-stranded polydeoxynucleotides. The pattern of adduct formation suggested that human bile forms a mixture of adenine and guanine adducts. Secondly, bile was fractionated by extraction with blue cotton or with neutral, acid or alkaline organic solvent. Blue cotton, which efficiently and selectively absorbs mutagens having 3 or more fused aromatic rings, did not absorb biliary constituents that could form adducts with DNA in vitro or with DNA of MCL-5 cells, a metabolically competent human cell line. This suggests that biliary DNA adduct precursors are polar compounds that contain fewer than 3 aromatic rings or are non-aromatic. Acidic organic extracts of human bile produced much higher levels of DNA adducts in vitro or with DNA of MCL-5 cells than did neutral or alkaline organic extracts, suggesting that constituents of bile that form DNA adducts are acidic in nature.

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.

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.

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. However, there is substantial evidence that about 80% of human cancer could be avoided by eliminating tobacco consumption; by dietary changes; by reducing infection with certain viruses, bacteria, and parasitic worms; and, in white populations, by avoiding sunburn. Alcohol, occupational and medical carcinogens, and certain patterns of reproductive behavior also contribute to the cancer burden. Cancers that cannot be attributed to these causes, and for which no other causes can be found, could be considered spontaneous and to arise from endogenous processes. Epidemiological evidence suggests that spontaneous and induced cancers share the same mechanism. Cancer is a genetic disorder of somatic cells. An accumulation of mutant genes that control the cell cycle, maintain genomic stability, and mediate apoptosis is central to carcinogenesis. Spontaneous mutation may cause spontaneous cancer. Endogenous causes of mutation include depurination and depyrimidation of DNA; proofreading and mismatch errors during DNA replication; deamination of 5-methylcytosine to produce C to T base pair substitutions; and damage to DNA and its replication imposed by products of metabolism (notably oxidative damage caused by oxygen free radicals). Deficiencies in cellular defense mechanisms may also provoke spontaneous mutation. These include defective DNA excision-repair; low levels of antioxidants, antioxidant enzymes, and nucleophiles that trap DNA-reactive electrophiles; and enzymes that conjugate nucleophiles with DNA-damaging electrophiles. Mechanisms underlying many of those cellular defenses are under genetic control. Thus, germ line mutations or polymorphisms of genes that govern them may also contribute to spontaneous cancer.

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). 32P-Postlabeling assays showed that MCL-5 cells (genetically engineered to express five human cytochromes P450 and microsomal epoxide hydrolase) formed characteristic adduct spots with benz[a]pyrene, benzo[g]chrysene, 7,12-dimethylbenz[a]anthracene, benzidine, sterigmatocystin and 3-methylcholanthrene, whereas CCRF cells did not. Accordingly, we assayed the ability of bile from FAP patients and controls to form DNA adducts in MCl-5 and in CCRF cells. Relative adduct labelling (RAL) in MCL-5 cells treated with FAP bile (12 patients, median 10, range 1-74) was significantly higher than in cells treated with control bile (12 patients, median 4, range 0-9; P = 0.0007) as was RAL for the two major adduct spots. These two major adduct spots were not observed when bile was incubated with CCRF cells. The adduct spots in CCRF DNA appeared in positions similar to some of the minor adduct spots produced by bile in MCL-5 DNA and to some of the adduct spots seen previously when bile was incubated with salmon sperm DNA in vitro. RAL for CCRF cells incubated with FAP bile (seven patients, median 23.0, range 0-49) was significantly higher than in cells treated with control bile (seven patients, median 2.0, range 0-26; P = 0.0034). These results indicate that the bile obtained from FAP and control patients contains adduct-forming substances, some of which are direct acting and some of which require metabolic activation. In both cell lines, FAP bile produced significantly higher adduct labelling than control bile, adding to the evidence that bile can induce DNA damage in vitro and plays a role in neoplastic development in the FAP foregut.

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. (Carcinogenesis 1994, 15, 1911-1915), also using postlabelling, claimed that chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid bound covalently to DNA in vitro. To investigate this discordance we incubated solutions of salmon sperm DNA for 1 h at 37 degrees C with 1 mg/ml of cholic acid, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid or taurolithocholic acid. Each incubate was extracted extensively with diethyl ether after which a sample of DNA was taken and 32P-postlabelled using the nuclease P1 method. The DNA in the remaining incubate was precipitated from high salt solution with ethanol. Aliquots of this DNA were postlabelled. The remainder of the DNA was purified with proteinase-K, ribonuclease, phenol-chloroform, precipitated and postlabelled. Parallel incubates were made with the same bile acids, under the same conditions but in the absence of DNA and were then extracted, precipitated and postlabelled as described above. When DNA was present in the incubate but was not precipitated, chenodeoxycholic acid, lithocholic acid, glycolithocholic acid and taurolithocholic acid, but not cholic acid, produced spots similar to those reported by Hamada et al. No such spots were seen when DNA was postlabelled after precipitation, or after precipitation and purification. These same bile acids produced spots when postlabelled in the absence of DNA, but spots were absent when these incubates were precipitated and purified before postlabelling. We conclude that the spots obtained when bile acids are incubated with DNA which is not precipitated from high salt before it is postlabelled are technical artefacts, and cannot be regarded as evidence that bile acids bind covalently to DNA to form adducts. We also confirm reports (Vulimiri et al. Carcinogenesis 1994, 15, 2061-2064) that bile acids alone can produce spots when incubated with T4 polynucleotide kinase and [gamma-32P]ATP.

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. In this study it is shown that the excess of adduct labelling produced by FAP bile in vitro depends on the pH of the incubate. While adduct labelling at pH 6-8 did not differ significantly between bile from six FAP patients and six controls, at pH 4-5 FAP bile, but not control bile, produced a near threefold excess of adduct labelling over that at pH 6-8. Therapy that increases duodenal pH may therefore alleviate duodenal polyposis.

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). To shed light on the mechanisms underlying these different patterns of bladder cancer we looked for mutations in the p53 gene in SBC from 92 patients in Egypt, where schistosomiasis is hyperendemic. Patients' mean age at presentation of bladder cancer was 49.4 +/- 9.9 years and 90% had a clinical history of schistosomiasis and/or histological evidence of schistosomal eggs adjacent to the carcinoma. There were 53 SCC, 23 TCC, 13 adenocarcinomas and three other carcinomas. Thirty patients had tumours with mutations in exons 5-8 of the p53 gene: 17/53 SCC, 8/23 TCC, 4/13 adenocarcinomas and 1/3 other tumours. Of 19 mutations in SCC, 16 were base pair substitutions (BPS), two were deletions and one an insertion. Two tumours each contained two mutations. Of the BPS, nine were transitions at CpG dinucleotides and two were G-->T transversions. All the mutations in TCC were BPS: four were transitions at CpG dinucleotides and three were G-->C transversions. One TCC had two mutations. Of four adenocarcinomas with mutations, two had transitions at CpG dinucleotides. Of the 30 BPS mutations, 16 were transitions at CpG dinucleotides, of which 12 were C-->T. We combined these 33 mutations with six obtained from Egyptian SCC reported by Habuchi et al. (Cancer Res., 53, 3795-3799, 1993) to compile a mutational spectrum. This was compared with a NSBC spectrum assembled from 118 mutations reported in the literature. The proportion of BPS at CpG dinucleotides was significantly higher in SBC than in NSBC (18/34 versus 25/103, P = 0.003). There was also a bias away from mutations in exons 7 and 8 towards mutations in exons 5 and 6. We suggest that the excess of transitions at CpG dinucleotides in SBC results from nitric oxide (NO) produced by the inflammatory response provoked by schistosomal eggs. NO could produce such mutations directly, by deamination of 5-methylcytosine, and indirectly, following conversion to nitrate, bacterial reduction to nitrite and endogenous formation of urinary N-nitroso compounds. These produce O6-alkylguanines in DNA, leading to very high rates of G:C-->A:T transitions, a process possibly augmented by inefficient repair of alkylated bases at CpG dinucleotides.