Radon, mycotoxins, p53, and uranium mining.
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Carcinoma, Squamous Cell↗
Biomedical subjects
S Venitt
Publications and source records attributed to S Venitt.
Recommendations for the performance of bacterial mutation assays.
At the International Workshop on the Standardisation of Genotoxicity Test Procedures, in Melbourne (27-28 February 1993), the current international guidelines for the correct conduct of bacterial mutation assays were considered, and the major differences between them were examined. An attempt was made to construct a scientifically based, internationally harmonized protocol. The main points of agreement were as follows. The consensus opinion was that there are currently insufficient data to justify a preference for either the preincubation or plate-incorporation methodologies as the initial test. Whichever method is used there was consensus agreement that the bacterial test battery should consist of S. typhimurium TA1537, TA1535, TA98 and TA100. There was also consensus that the 3 strains TA97a, TA97 and TA1537 could be used interchangeably. Although it was not possible to achieve a consensus, the majority of the working group members agreed that strains for the detection of mutagens acting specifically on AT base pairs should be routinely included within the test battery. These strains may be S. typhimurium TA102 or E. coli WP2 strains (WP2 pKM101 and WP2 uvrA or WP2 uvrA pkM101). With regard to study design it was universally agreed that 5 doses of test compound should be used in each experiment, and a majority agreement was obtained for 3 plates per dose. The use of 2 plates per dose is acceptable ONLY if the experiment is repeated. It is recommended that the negative controls may consist of solvent control alone provided that historical data are available to demonstrate lack of effect of the solvent in question. Positive control compounds should be included in all experiments, although the nature of these control compounds need not be specified in the guidelines. There was consensus agreement that for non-toxic freely soluble test agents, an upper limit of 5 mg/plate should be tested (5 microliters per plate for liquids). For insoluble or toxic compounds, the recommendations were the same as those for other in vitro tests (see appropriate paper). A consensus agreement was reached on the need to carry out further tests if equivocal results are obtained in the initial test, although it was generally agreed that the design of the repeat study should be left flexible. As there are little or no data to support the use of an exact repeat assay, a majority of the group recommended that negative results in the first test should be further investigated by either conducting a modified repeat (e.g. S9 titration) or by conducting the alternative methodology.(ABSTRACT TRUNCATED AT 400 WORDS)
Biotransformation↗
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). The DNA was then purified and assayed for the formation of DNA adducts using the nuclease P1 method of 32P-postlabelling. Under the conditions of the assay none of the bile acids tested with or without metabolic activation produced any evidence of DNA adduct formation. It is therefore unlikely that the adduct-forming ability of human bile can be attributed to bile acids or their metabolites.
Adenomatous Polyposis Coli↗
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. It was found that the deuterated compound [D5-ethyl]-tamoxifen is significantly less genotoxic than tamoxifen. Administration of 0.06 and 0.12 mmol/kg [D5-ethyl]-tamoxifen to female Fischer rats by gavage resulted in a 2.5- and 1.7-fold reduction respectively in the levels of hepatic DNA adducts present 24 h after treatment, compared with the non-deuterated compound. Treatment of MCL-5 cells with [D5-ethyl]-tamoxifen resulted in a 2- to 3-fold decrease, compared with tamoxifen, in the number of micronuclei induced in cells arrested in cytokinesis by cytochalasin-B. Further evidence is provided by UV irradiation of the major hepatic DNA adducts isolated from tamoxifen-treated rats, which caused a large shift in the chromatographic mobility of the adducts, consistent with the presence of a 1,2-olefinic linkage in the tamoxifen residue of the adduct leading to cyclization to a phenanthrenylic compound, and consistent with this adduct having arisen from reaction with DNA at the alpha-position of tamoxifen. Taken together, these data strongly suggest that tamoxifen is metabolized to a liver carcinogen by alpha-hydroxylation of its ethyl group.
Animals↗
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. However, the contribution that genetic susceptibility to cancer makes to the total cancer burden is unknown. Genetic susceptibility may be important in occupational settings, where exposure to carcinogens is presumed to be restricted to concentrations believed to confer "acceptable" risks. This approach takes no account of individual susceptibility. The range over which metabolic polymorphisms exert their effects suggests that differences in pharmacogenetics between individuals may be important in occupational carcinogenesis. If this range were extrapolated to risk of human disease, at a given exposure one person might be 10-200 times more sensitive than another. Several metabolic polymorphisms have been linked to susceptibility to cancer. For some of these polymorphisms, genes have been cloned and genotypic tests based on use of the polymerase chain reaction have been developed. Limited data suggest that the effects of these polymorphisms on human cancer are complex. The genotypic testing of individuals occupationally exposed to carcinogens may provide firmer information on the relative contributions of nature and nurture on chemically induced cancer. The use to which such information is put demands wide debate.
Biotransformation↗
Safety of prophylactic tamoxifen.
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Animals↗
DNA adducts, detected by 32P-postlabelling, in DNA treated in vitro with bile from patients with familial adenomatous polyposis and from unaffected controls.
Patients with familial adenomatous polyposis (FAP) have a high risk of developing duodenal adenomas and carcinomas. The distribution of these neoplasms resembles mucosal exposure to bile, suggesting that bile may play a role in adenoma development. Our previous results, using DNA adducts detected by 32P-postlabelling as an index of genotoxicity, have supported this hypothesis. We found significantly higher adduct labelling in the duodenum of FAP patients than in the duodenum of control patients and significantly higher labelling in the small bowel of rats gavaged with FAP bile than in rats given control bile. We have now investigated the ability of human bile to form adducts with DNA in vitro. Bile obtained from the gallbladder of 18 FAP patients immediately before colectomy, and from 18 control patients, was incubated with salmon sperm DNA in solution at 37 degrees C for 1 h, after which the purified DNA was analysed for DNA adducts, using the nuclease P1 method of 32P-postlabelling. Relative adduct labelling values (RAL, adducts per 10(9) nucleotides) produced by FAP bile samples were significantly higher than RAL values produced by control bile samples (medians 197 versus 86, P = 0.0016, Mann-Whitney test). We found a consistent pattern of adduct labelling, varying in intensity between samples. Adduct spots were eluted from TLC plates and analyzed by reverse-phase HPLC. Each major spot gave several peaks that were consistent between bile samples from different patients and were similar in FAP and control bile. These results indicate that bile from FAP and control patients contains similar, directly acting genotoxic compounds but that levels are higher in FAP than in control patients. This suggests that bile from FAP patients is more genotoxic than bile from control patients. Incubation of bile with free-radical scavengers and deconjugating enzymes failed to influence adduct labelling in this system.
Adenomatous Polyposis Coli↗
Does a genotoxic carcinogen contribute to human breast cancer? The value of mutational spectra in unravelling the aetiology of cancer.
The p53 tumour suppressor gene is turning out to be a useful reporter for the stigmata of past genotoxic exposure. About half of all human cancers contain p53 mutations most of which occur in those regions (exons 5-8) of the gene that are highly conserved during evolution. Mutations are mainly of the missense type and their frequency and distribution vary among different kinds of cancer. The ability to detect all six possible base-substitution mutations in the p53 gene in human tumours makes it possible to construct mutational spectra for different cancers at a locus clearly implicated in carcinogenesis. Transitions at one particular hotspot--the CpG dinucleotide--occur frequently in many cancers and may reflect endogenous mutation. A reduction in the proportion of CpG mutations at the expense, for example, of an increase in GC to TA transversions may signal the effect of an exogenous mutagen. We exploited these features of the p53 gene to examine the evidence that a previously unsuspected genotoxic exposure may contribute to the high incidence of breast cancer in women living in rich industrialized countries. We compiled a mutational spectrum of p53 from 120 breast cancers and compared it with the spectrum from 145 colorectal cancers and 246 lung cancers. A germline p53 spectrum was constructed using data from 27 patients. Two hundred germline mutations in the haemophilia B gene served as a 'background' spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)
Breast Neoplasms↗
Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromes P-450 and microsomal epoxide hydrolase.
The MCL-5 cell line is a human lymphoblastoid TK+/- cell line that constitutively expresses a relatively high level of native CYP1A1, four other human cytochromes (CYP1A2, CYP2A6, CYP3A4 and CYP2E1) and microsomal epoxide hydrolase, carried as cDNAs in plasmids. The aim of this study was to evaluate this cell line for its suitability for detecting chromosomal anomalies, employing micronucleus formation in cells blocked at cytokinesis as the indicator of clastogenicity. Results from two laboratories ('ICR' and 'Swansea') using different protocols are reported. In the ICR protocol, aflatoxin B1, sterigmatocystin, benzo[a]pyrene, dibenz[a,h]anthracene, 3-methylcholanthrene, cyclophosphamide, N-nitrosodimethylamine, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline, benzidine, 2-aminofluorene, benzene, tamoxifen and omeprazole were tested and gave positive results. Anthracene, phenanthrene and pyrene were negative. In the Swansea protocol, AHH-1 cells, the parent line which constitutively expresses CYP1A1, but does not contain the genetically engineered human cytochromes or epoxide hydrolase, were tested in parallel with MCL-5 cells. Aflatoxin B1, sterigmatocystin, benzo[a]pyrene, N-nitrosodiethylamine, 2-acetylaminofluorene, benzene, omeprazole and tamoxifen were tested and gave positive results. Of these, only benzo[a]pyrene was equally potent in both cell lines. Assays of tamoxifen and omeprazole included kinetochore staining. Omeprazole, but not tamoxifen, induced a significant level of kinetochore-positive micronuclei. The detection of micronucleus formation in these genetically engineered cells appears to be a rapid, eclectic and sensitive method for screening for genotoxic activity in vitro.
Cell Division↗
32P-postlabelling studies of target tissues and bile from patients with familial adenomatous polyposis and from unaffected controls.
Patients with the inherited form of colon cancer, familial adenomatous polyposis (FAP), are at high risk of developing duodenal adenomas and carcinomas. The periampullary clustering of these neoplasms suggests that bile plays a role in their development. We investigated this theory using 32P-postlabelling to detect DNA adducts. We found significantly higher adduct levels in duodenum of FAP patients than in unaffected controls, and higher levels in duodenum than stomach. Levels of adducts were significantly higher in the small bowel of rats gavaged with FAP gallbladder bile than in the small bowel of those that received control gallbladder bile. We found that bile from FAP gallbladder produced significantly more DNA adducts than control gallbladder bile when incubated with salmon sperm DNA in vitro. These results support the hypothesis that the bile of FAP patients may be involved in the development of duodenal adenomas in these patients.
Adenomatous Polyposis Coli↗
Bile and the growth of intestinal polyps.
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Acetylation↗
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.
Animals↗
DNA adducts detected by 32P-postlabelling, in the intestine of rats given bile from patients with familial adenomatous polyposis and from unaffected controls.
Duodenal adenomas are common in patients with familial adenomatous polyposis (FAP) and cluster around the papilla, suggesting the involvement of bile in their development. Using 32P-postlabelling we determined levels of DNA adducts in the small bowel and colon of rats treated with bile from FAP and control patients. We found a significantly higher level of adducts in the small bowel of rats treated with FAP gallbladder bile compared with control gallbladder bile (P = 0.0034). This result supports the hypothesis that bile plays a role in the development of neoplasia in the foregut of FAP patients.
Adenomatous Polyposis Coli↗
Professor John J. Roberts 1926-1990.
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DNA Damage↗
N-nitrosoproline excretion in the presence and absence of gastric disease.
N-nitrosoproline (NPRO) excretion, an indicator of endogenous nitrosation, was measured in a group of hospital inpatients who were identified by endoscopy and gastric biopsy as either having gastric lesions or having healthy stomachs. NPRO was assayed in background 24-hour urine samples and samples collected after loading doses of nitrate and L-proline. The presence of gastric lesions was associated with altered gastric pH and concomitant changes in gastric juice nitrate and nitrite concentration. Gastric juice pH increased with increasing severity of gastric disease (P = 0.031) and patients with normal stomachs had a lower gastric pH than those with chronic atrophic gastritis (CAG) (3.0 vs. 6.5, P = 0.017). The changes in gastric juice nitrate concentration were in the reverse direction (P = 0.002 for trend) with normal patients having higher mean levels than CAG patients (12.7 vs. 5.5 micrograms/ml, P less than 0.0001). Nitrite concentration increased with severity of gastric disease but the results were not significant (normal, 82.9 vs. CAG, 223.4 ng/ml, P = 0.069). No association was found between the presence of gastric lesions and increased urinary NPRO excretion. Mutagenic activity was not detected in any of the gastric juice samples.
Adult↗
DNA adducts, detected by 32P-postlabelling, in the foregut of patients with familial adenomatous polyposis and in unaffected controls.
Duodenal neoplasms in patients with familial adenomatous polyposis (FAP) cluster around the ampulla; duodenal neoplasia is common but gastric neoplasia is rare. These observations suggest that bile enhances neoplasia by carrying carcinogens to target cells. A critical step in carcinogenesis is the reaction of carcinogens with DNA to form chemical adducts. Using 32P-postlabelling to measure DNA adducts we asked: (i) are there more adducts in the duodenum of patients with FAP than in the duodenum of normal patients? (ii) Are there more adducts in the duodenum than in the stomach? We measured adducts in duodenal biopsies from 51 patients with FAP and 30 age-matched controls; and paired gastric and duodenal biopsies from 31 FAP patients and six controls. The foregut of 90% of all patients studied contained DNA modifications with characteristics of aromatic non-polar DNA adducts. In duodenal biopsies, adduct labelling per 10(9) DNA nucleotides was significantly higher in FAP patients (median 15.0, range 0-162) than in normal patients (median, 7.5, range 0-40; P = 0.0002). In paired duodenal and gastric biopsies from 31 FAP patients, adduct labelling was significantly higher in duodenal DNA (median 15, range 0-109) than in stomach DNA (median 8.0, range 0-40; P = 0.0004). Age, gender or smoking did not account for these differences. Gastric adducts (median 3.5, range 0-10) were lower than duodenal adducts (median 8.5, range 0-29) in paired biopsies from six control patients, P = 0.031). These results support the hypothesis that pancreaticobiliary secretions may be involved in the pathogenesis of foregut neoplasia in FAP.
Adenomatous Polyposis Coli↗
Mutagenicity of bile and duodenal adenomas in familial adenomatous polyposis.
Duodenal adenomas occur almost inevitably in patients with familial adenomatous polyposis (FAP) whereas gastric adenomas are rare. FAP patients are also at high risk of duodenal cancer. Within the duodenum, adenomas cluster around the ampulla of Vater, as do the majority of duodenal cancers, suggesting that bile plays a role in tumour development. We therefore tested duodenal bile from 29 postcolectomy FAP patients (27 of whom had duodenal adenomas) and 24 non-FAP patients for mutagenicity, using techniques that detect point mutations in bacteria. Results which appeared to show that FAP bile was more mutagenic than control bile could be accounted for by a feeding effect, elimination of which also eliminated 'mutagenicity'. Under the conditions of our assays we conclude that if bile is an important factor in genesis of duodenal tumours, it does not act by inducing point mutation.
Adenoma↗