A preliminary evaluation of accelerator mass spectrometry in the biomedical field.
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Animals↗
Biomedical subjects
R C Garner
Publications and source records attributed to R C Garner.
Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.
To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.
Aflatoxin B1↗
A hot spot for p53 mutation in transitional cell carcinoma of the bladder: clues to the etiology of bladder cancer.
Twenty-eight transitional cell carcinomas of the bladder, grade 2 or 3, were analyzed for the presence of p53 mutations. Thirteen tumors were found to contain 14 mutations. These were all base substitution mutations, of which nine were GC-->AT transitions (three at CpG sites). The remaining five mutations were transversions (three GC-->CG, one GC-->TA, and one AT-->TA). Four of the mutations were found at codon 280. A comparison with other studies of bladder tumors reveals that a region encompassing codons 280 and 285 represents a hot spot for p53 mutation in bladder cancer. The 280/285 hot spot lies within two purine-rich sequences that may provide some clues to the identity of potential bladder carcinogens. A comparison of mutations from bladder tumors of smokers and nonsmokers reveals no significant differences.
Adult↗
Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.
In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.
Accidents, Occupational↗
The Escherichia coli DNA repair protein UvrA can re-associate with the UvrB: aflatoxin B1-DNA complex in vitro.
The UvrA and UvrB proteins form part of the UvrABc endonuclease, which is responsible for nucleotide excision repair in Escherichia coli. Using a mobility shift gel assay we have studied the binding of UvrA dimer, UvrB monomer and UvA(2)B trimer complexes with 40, 50 and 136 bp (32)P-end-labelled DNA fragments adducted with aflatoxin B(1). UvrA was shown to re-associate with adduct specific UvrB: DNA complexes, a phenomenon which could be reversed by the addition of 500 mM potassium chloride or anti-UvrA anti-sera. Re-association was shown to be UvrA concentration dependent. Re-association of UvrA(2)B to the UvrB:DNA complex was not seen. We have also shown that the UvrB:DNA complex, in the case of aflatoxin B(1), is extremely stable with a half-life excess of 400 min and that fragment termini are not a specific substrate for UvrA binding.
Adenosine Triphosphatases↗
Measurement of DNA adducts in humans after complex mixture exposure.
In contrast to acute or chronic dosing experiments with a single chemical in animals, man is exposed to thousands of chemicals during a lifetime. Each of these may act alone, additively, synergistically or antagonistically in terms of biological effects, but most current risk assessment procedures fail to recognize such interactions. In carcinogenesis, a mutational process that is thought to occur through DNA damage by endogenous and/or exogenous agents, a wide variety of host factors is involved in disease outcome. These include absorption of chemicals, their distribution, metabolism and excretion. In addition, once metabolic activation has occurred, there is an array of protective mechanisms that cells have evolved to maintain DNA integrity, such as DNA repair, genetic redundancy and programmed cell death. One approach to risk assessment is to regard all DNA-damaging events as potentially leading to cancer and to measure DNA damage as the biologically relevant endpoint. The main method, if not the only method, presently available to assay a wide range of DNA adducts is 32P-postlabelling. This method has high sensitivity (limit of detection > 1 adduct per 10(10) nucleotides) and is capable of visualizing many different DNA adducts in a single analysis. Postlabelling is best suited for detecting hydrophobic adducts--low molecular weight adducts usually need a preliminary separation procedure prior to being postlabelled. This chromatographic procedure has been used to study DNA samples from human tissues of cigarette smokers, occupationally exposed groups and individuals living in polluted environments. Correlations have been found between the severity of exposure and the level of DNA adducts detected for human samples. However, most studies are single-time point studies, whereas for risk assessment purposes it may be better to use more quantitative and representative measures of long-term exposure, for example the number of adducts formed per annum. This article reviews methods of DNA adduct measurement, with particular reference to the 32P-postlabelling technique, which has been used to determine DNA adduct levels in populations exposed to complex mixtures.
DNA Adducts↗
Molecular screening of multifocal transitional cell carcinoma of the bladder using p53 mutations as biomarkers.
Thirteen of 28 patients (46%) with grade 2-3 multifocal transitional cell carcinoma (TCC) of the bladder were found to have p53 mutations using DNA sequence analysis. These were subsequently utilized as tumor-specific biomarkers. Analysis of 17 episodes of recurrence from five of the patients revealed that all but one carried the identical mutation to the primary tumor. Thirty urine samples were collected, at initial diagnosis and during follow-up screening, from eight patients with mutations over a period of 24 months. Sequence analysis of PCR products generated from DNA extracted from the urine sediments was carried out. The p53 mutation seen in the primary tumors was detectable in 24 of 30 urine samples. The remaining six cases coincided with a negative cystoscopic examination. Interestingly, 6 of the 24 urine samples in which mutations were detectable also coincided with negative cystoscopy. The results are consistent with: (a) monoclonality of multifocal TCC; (b) the spread of TCC through a seeding mechanism; and (c) the long-term persistence of tumor cell clones (up to 97 months) within the bladder, even in the absence of obvious tumor growth.
Biomarkers↗
Molecular cancer epidemiology can predict risk.
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DNA Adducts↗
Low frequency and late occurrence of p53 and dcc aberrations in colorectal tumours.
Whilst p53 aberrations have been documented in numerous malignancies, reports of alterations to the deleted in colorectal cancer (dcc) gene are infrequent, and studies investigating the status of both genes in the same colon tumour are rare. In this study we have analysed a panel of 35 pairs of normal and neoplastic human colorectal tissues for abnormalities in these tumour-suppressor genes. In contrast to previous studies we have found only a low incidence of mutations and deletions. p53 point mutations were identified in 8/35 tumours (22%). All were G.C to A.T transitions, with 7/8 occurring at CpG dinucleotides. p53 allelic loss was detected in 4/11 informative cases (36%). Although not quite attaining statistical significance, p53 alteration correlated with the adenoma/carcinoma transition. Gross dcc alterations were identified by Southern blotting in 7/35 (20%) tumours. Microsatellite analysis using two markers, one within and one proximal to the dcc gene, detected a low frequency of deletion overall (41% informative cases). 18q/dcc aberrations were associated with the progression of early to late carcinoma, rather than with increasing adenoma size, as has been previously reported. Both p53 alterations and dcc deletions were detected at a higher frequency in distal tumours than in proximal malignancies. Two tumours exhibiting microsatellite instability in both markers were each of proximal origin.
Adult↗
Cyclophosphamide: review of its mutagenicity for an assessment of potential germ cell risks.
Cyclophosphamide (CP) is used to treat a wide range of neoplastic diseases as well as some non-malignant ones such as rheumatoid arthritis. It is also used as an immunosuppressive agent prior to organ transplantation. CP is, however, a known carcinogen in humans and produces secondary tumors. There is little absorption either orally or intravenously and 10% of the drug is excreted unchanged. CP is activated by hepatic mixed function oxidases and metabolites are delivered to neoplastic cells via the bloodstream. Phosphoramide mustard is thought to be the major anti-neoplastic metabolite of CP while acrolein, which is highly toxic and is produced in equimolar amounts, is thought to be responsible for most of the toxic side effects. DNA adducts have been formed after CP treatment in a variety of in vitro systems as well as in rats and mice using 3H-labeled CP. 32P-postlabeling techniques have also been used in mice. However, monitoring of adducts in humans has not yet been carried out. CP has also been shown to induce unscheduled DNA synthesis in a human cell line. CP has produced mutations in base-pair substituting strains of Salmonella tryphimurium in the presence of metabolic activation, but it has been shown to be negative in the E. coli chromotest. It has also been shown to be positive in Saccharomyces cerevisiae in D7 strain for many endpoints but negative in D62.M for aneuploidy/malsegregation. It has produced positive responses in Drosophila melanogaster for various endpoints and in Anopheles stephensi. In somatic cells, CP has been shown to produce gene mutations, chromosome aberrations, micronuclei and sister chromatid exchanges in a variety of cultured cells in the presence of metabolic activation as well as sister chromatid exchanges without metabolic activation. It has also produced chromosome damage and micronuclei in rats, mice and Chinese hamsters, and gene mutations in the mouse spot test and in the transgenic lacZ construct of Muta Mouse. Increases in chromosome damage and gene mutations have been found in the peripheral blood lymphocytes of nurses, pharmacists and female workers occupationally exposured to CP during its production or distribution. Chromosome aberrations, sister chromatid exchanges and gene mutations have been observed in somatic cells of patients treated therapeutically with CP. In general, there is a maximum dose and an optimum time for the detection of genetic effects because the toxicity associated with high doses of CP will affect cell division. In germ cells, CP has been shown to induce genetic damage in mice, rats and hamsters although the vast majority of such studies have used male mice.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Effect of vitamin C upon gastric mucosal O6-alkyltransferase activity and on gastric vitamin C levels.
The repair enzyme O6-alkyltransferase will repair O6-methylguanine adducts in human DNA. In gastric mucosal DNA these adducts may be formed as a result of exposure to nitrosamines within the gastric lumen. The formation of these nitrosamines may be inhibited by vitamin C. We have examined the effect of oral vitamin C supplementation upon intragastric vitamin C levels and gastric mucosal O6-alkyltransferase levels in 48 patients. Intragastric vitamin C levels were significantly elevated in those patients with normal gastric mucosal histology after treatment, although a variable response in intragastric vitamin C to supplementation was seen in the presence of chronic atrophic gastritis. Gastric mucosal O6-alkyltransferase activities ranged from 100 to 950 fmol/mg protein before vitamin C administration. The range of enzyme activity was similar after the course of vitamin C (62-1137 fmol/mg) but O6-alkyltransferase activities were found to be higher in 33 of the 48 patients following treatment (P < 0.01). Once again this effect was more pronounced in patients with normal gastric mucosa than those displaying evidence of chronic atrophic gastritis. We speculate that inhibition of intragastric nitrosation by vitamin C results in decreased formation of O6-methylguanine-DNA. In consequence, less O6-alkyltransferase is consumed in repairing these adducts resulting in higher tissue levels of this enzyme.
Ascorbic Acid↗
DNA damage as measured by 32P-postlabelling in normal and pre-malignant gastric mucosa.
DNA was extracted from the gastric mucosa of 69 patients and analysed for the presence of DNA adducts by 32P-postlabelling. Adduct levels found in patients with histologically normal gastric mucosa were compared with levels found in patients displaying evidence of chronic atrophic gastritis or intestinal metaplasia, both of which may be considered pre-malignant conditions. Adduct patterns were the same for all patients, but the highest adduct levels were found in the latter two groups. Mean adduct levels were also higher in patients with abnormal gastric mucosa, but there was no statistically significant difference in adduct levels between the normal and pre-malignant groups (P > 0.05, Mann-Whitney U test). Thus DNA adduct levels do not correlate with the presence of histological abnormalities in the stomach and are not useful as a marker of malignant potential.
Adult↗
Activation of promutagens in a human bronchial epithelial cell line stably expressing human cytochrome P450 1A2.
Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T-antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T-antigen-positive cells (BEAS-2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S-transferase pi, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS-2B cells either by infection with a high-titer recombinant retrovirus (pXT-1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B-1A2 and B-CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50- to 400-fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B1 (AFB1) than the corresponding control cell lines. The cytotoxic effects of AFB1 were paralleled by increased metabolism of AFB1 and enhanced formation of the AFB1-N7 guanine adduct in B-CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter-driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase I enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens.
Aflatoxin B1↗
Effect of vitamin C supplementation on gastric mucosal DNA damage.
The effect of oral vitamin C supplementation on intragastric ascorbate levels and gastric mucosal DNA damage as measured by the 32P-postlabelling assay was assessed in 43 patients. In patients with normal gastric mucosa, treatment with vitamin C resulted in elevation of intragastric ascorbate levels in all cases. In the presence of chronic atrophic gastritis, however, the effect was variable. Gastric mucosal DNA damage was decreased in 28 of the 43 patients after vitamin C supplementation (P = 0.01; Wilcoxon sign rank test). This supports epidemiological evidence that suggests vitamin C may exert a protective effect against the development of gastric cancer.
Ascorbic Acid↗
The detection of adducts in human cervix tissue DNA using 32P-postlabelling: a study of the relationship with smoking history and oral contraceptive use.
32P-Postlabelling was used to measure DNA adducts in the human cervix. Adduct levels were compared with patient smoking histories and contraceptive use. DNA adducts were found in 43 out of 58 samples. The number of adducts ranged from 0.2 to 59.5 adducts/10(8) nucleotides, though no significant difference was found to exist between the number of DNA adducts detected and the smoking history of each patient. In contrast, a significant difference at the 1% probability level was found between the adduct levels obtained from the cervical DNA of smokers who had used oral contraceptives and smokers who did not. Autoradiograms revealed a variety of adduct patterns. Some were found to have a diagonal zone of radioactivity which migrated from the origin of the TLC plate. Other autoradiograms revealed the presence of additional adduct spots located in the upper regions of the TLC plate, whereas others revealed the presence of these adduct spots alone. The origin of the adduct spots located in the upper regions of the TLC plate is unknown.
Cervix Uteri↗
DNA adducts of aflatoxins, sterigmatocystin and other mycotoxins.
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Aflatoxins↗
Immunoaffinity concentration of human lung DNA adducts using an anti-benzo[a]pyrene-diol-epoxide-DNA antibody. Analysis by 32P-postlabelling or ELISA.
DNA, isolated from 15 human lung autopsy samples, was examined for the presence of polycyclic aromatic hydrocarbon (PAH) DNA adducts. Using the nuclease P1 modification of the 32P-postlabelling technique, between 1 and 12 adducts/10(8) nucleotides were detected prior to immunoconcentration. Autoradiograms from most of the samples revealed a diagonal smear of radioactivity consistent with complex mixture (cigarette smoking) DNA damage. The DNA samples were digested to oligonucleotides, made single-stranded and subsequently applied to immunoaffinity columns containing immobilised anti-benzo[a]pyrene (B(a)P)-7,8-diol-9,10-epoxide (BPDE) DNA polyclonal rabbit antibody. The material remaining bound to the column, in addition to that passing through, was analysed using both ELISA and 32P-postlabelling techniques. Column-bound adducts comprised between 0% and 78% of any particular sample. Immunoconcentration, followed by 32P-postlabelling of the material which had been bound to the column, revealed the presence of a number of discrete adduct spots in autoradiograms of the more heavily adducted samples. Sample DNA not retained by the columns was also analysed; the chromatographic pattern obtained was a dense zone of radioactive material migrating from the origin. This evidence suggests that the composition of PAH-DNA adducts found in human lung samples exhibits wide inter-individual variation.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
O6-alkyltransferase activity in normal and abnormal gastric mucosa.
The activity of the DNA repair enzyme O6-alkyltransferase has been studied in a series of stomachs with abnormal gastric mucosa and the activities found compared with those in normal stomachs. Enzyme activities found in stomachs with the macroscopic abnormalities of gastric ulcer, duodenal ulcer or gastric cancer were not significantly different from normal. In those stomachs where there was histological evidence of chronic atrophic gastritis or intestinal metaplasia however enzyme activities (mean 398 fmole/mg) were significantly higher than normal (mean activity 228 fmole/mg activity P < 0.001). We speculate that the conditions which stimulate these histological changes also give rise to induction of O6-alkyltransferase.
Adult↗