Fluorescence detection of lesions in DNA.
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7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Biomedical subjects
A Weston
Publications and source records attributed to A Weston.
Serological characterization of polycyclic aromatic hydrocarbon diolepoxide-DNA adducts using monoclonal antibodies.
Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide-DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH-DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH-DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus demonstrating their specificity for PAH-DNA adducts and not the PAHs alone. Monoclonal antibodies produced against BADE-DNA also bound to chrysene diolepoxide-DNA but not to BPDE-DNA or to two other PAH-DNA adducts. These data demonstrate that significant serological cross-reactivity exists between different PAH-DNA adducts and support our contention that human antibodies to PAH-DNA can often bind to more than a single type of PAH-DNA adduct. However, our ability to produce monoclonal antibodies with restricted specificity suggest that the production of highly monoclonal antibody reagents for use in assays designed to measure PAH-DNA adducts in biological samples is feasible.
Animals↗
Synchronous fluorescence spectroscopic, immunoaffinity chromatographic and 32P-postlabeling analysis of human placental DNA known to contain benzo[a]pyrene diol epoxide adducts.
Human placenta readily catalyzes the biotransformation of polycyclic aromatic hydrocarbons (PAHs) and other carcinogens to reactive metabolites that can damage DNA through formation of covalent adducts. Placenta is widely available for epidemiologic studies and may be a useful dosimeter for carcinogen exposures in humans. However, previous studies of human placental DNA have yielded discrepant results with respect to PAH-DNA adducts. In order to resolve some of the issues surrounding these discrepancies, placental DNA samples known to contain benzo[a]pyrene diol epoxide adducts were also analyzed by 32P-postlabeling and immunoaffinity chromatography. Results indicate that previous discrepancies can be accounted for by methodologic factors affecting the specificities of adduct assays in biological samples and suggest that human placental DNA contains adducts derived from multiple PAHs.
Benzo(a)pyrene↗
Molecular mechanics and antibody binding in the structural analysis of polycyclic aromatic hydrocarbon-diol-epoxide--DNA adducts.
Analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts using monoclonal antibodies raised against DNA that had been modified with (+-)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in an enzyme-linked immunosorbent assay, as well as analysis using human serum antibodies and antibodies raised in laboratory animals, have suggested the presence on these adducts of both common and unique immunological epitopes. The molecular mechanics studies reported here establish a model for the analysis of PAH-DNA adducts through the identification of energetically favored binding conformations and they further reveal structural alterations in DNA due to the presence of carcinogen adducts. The data explain the antibody reactivity patterns by defining different molecular presenting surfaces that are available for antibody binding. The preferred orientation of the aromatic portions of the adducts, which align either 3' or 5' in the minor groove, were found to be correlated with antibody reactivity patterns. Examination of the topographical characteristics of the adducts facilitated correlation of adduct-antibody recognition and adduct presenting surface. Significant differences were found between benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts, which align 5' in the minor groove, and benz[a]anthracene-diol-epoxide (BADE)-DNA and dibenz[a,c]anthracene-diol-epoxide-DNA adducts, which align 3' within the minor groove. Chrysene-diol-epoxide-DNA adducts were found to have only a weak preference for 5' alignment and therefore share topographical characteristics with both BPDE-DNA and BADE-DNA adducts.
Animals↗
Human debrisoquine hydroxylase gene polymorphisms in cancer patients and controls.
The extensive metabolizer phenotype of debrisoquine has been associated with increased risk of lung cancer, and it has been proposed that a molecular test for this phenotype is feasible. DNA restriction fragment length polymorphisms of the human debrisoquine 4-hydroxylase gene locus (CYP2D6), and the metabolic phenotype for debrisoquine have been studied in a group of healthy volunteers, a group of lung cancer patients and two control groups (chronic obstructive pulmonary disease patients and patients with cancers at sites other than the lung). Confirmation of four distinct XbaI allelic fragments (44, 29, 16/9 and 11.5 kb), previously identified among caucasians, was obtained. The 29 kb alleles were the most frequently observed in both poor and extensive metabolizers of debrisoquine. Alleles of 44 kb were found with approximately equal frequency among both poor and extensive metabolizers. The data are consistent with the hypothesis that the 11.5 and 44 kb fragments are associated with mutant alleles of the CYP2D6 gene, but the power of phenotype prediction by these alleles was less than that previously reported for a European (Swiss-German) population. Similarly, the data also show that 8% of 29 kb homozygotes are poor metabolizers (indicating that at least 28% of 29 kb fragments are also associated with mutant alleles) and are not therefore informative for predicting the debrisoquine phenotype. The 16/9 allele may represent either wild-type or mutant alleles. Restriction fragments of 44 kb were found more frequently among cancer patients and chronic obstructive pulmonary disease patients (30%) than among the healthy volunteer group (7%). Genotypes observed were not related to lung tumor histology. Furthermore, at least three EcoRI alleles were found to be in linkage disequilibrium with the 'mutant' 44 kb allele. These data suggest that the 44 kb allele can comprise three distinct haplotypes, in contrast to studies of a European population. These studies indicate that no single mutant CYP2D6 allele as determined by EcoRI appears to be associated with lung cancer, despite the findings that these patients are invariably of the extensive metabolizer phenotype.
Adenocarcinoma↗
Tumor suppression studies of human lung cancer.
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Alleles↗
The expression and purification of human rhinovirus protease 3C.
Human rhinovirus type 14 protease 3C was expressed as a soluble and active protein in Escherichia coli. The protease was purified by a cationic-exchange step followed by gel filtration on a TSK 3000 column. The final yield of purified protease was in the range 0.5-1.0 mg/l culture grown to A550 = 1.0. Sequence analysis revealed that greater than 90% of the N-terminal residues were methionine. The enzyme activity of the purified protease was measured by cleavage of a synthetic peptide representing a predicted Gln/Gly viral polyprotein cleavage site. A mutant protease (Cys146----Ser) was produced and purified in the same way. The yield of mutant protease 3C was approximately 150 micrograms/l from a culture grown to A550 = 1.0. This mutant protease 3C did not cleave the synthetic peptide substrate.
3C Viral Proteases↗
Interleukin-1 alpha gene intron containing variable repeat region coding for the SP1 transcription factor recognition sequence is polymorphic.
Interleukin-1 alpha (IL-1 alpha) is a cytokine produced by a number of cell types including macrophages, fibroblasts, keratinocytes, and mesangial cells. We were interested in identifying a DNA restriction fragment length polymorphism (RFLP) for the IL-1 alpha gene for use in studies of genetic alteration in various human cancers. Human genomic DNA from 32 unrelated individuals was digested with various restriction enzymes, alone and in combination, and subjected to Southern blot analysis. Hybridization to 32P-labeled IL-1 alpha cDNA revealed an insertion-deletion-type polymorphic pattern. After digestion with RsaI, insertion-deletion-type polymorphic bands with sizes of 3.4 kb, 3.1 kb, and 2.8 kb and one invariant band of 0.8 kb were observed. These three alleles, designated A1, A2, and A3, had relative frequencies of 0.18, 0.06, and 0.78 with heterozygosity observed in 38% of the unrelated individuals studied. Evaluation of nine related individuals for this RsaI polymorphism was consistent with a Mendelian inheritance. Comparison of restriction patterns following Southern analysis of DNA digested with several different enzymes showed that the polymorphic region resides within the sixth intron. Furthermore, this RFLP results from a variable length region containing multiple copies of a recognition sequence for SP1, an imperfect copy of viral enhancer elements, and an inverse and complementary sequence of the glucocorticoid receptor binding site. The identified polymorphism may be of value in analyses of chromosome 2 and may help to elucidate mechanisms by which IL-1 alpha transcription is regulated.
Binding Sites↗
Differential DNA sequence deletions from chromosomes 3, 11, 13, and 17 in squamous-cell carcinoma, large-cell carcinoma, and adenocarcinoma of the human lung.
Activation of protooncogenes and inactivation of putative tumor suppressor genes are genetic lesions considered to be important in lung carcinogenesis. Fifty-four cases of non-small-cell lung cancer (23 adenocarcinomas, 23 squamous-cell carcinomas, and 8 large-cell carcinomas) were examined for loss of DNA sequences at 13 polymorphic genetic loci. Loss of heterozygosity was seen more frequently in squamous-cell carcinoma than in adenocarcinoma. The loss of DNA sequences from the short arm of chromosome 17 (D17S1 locus) was detected in 8 of 9 heterozygous cases of squamous-cell carcinoma and in only 2 of 11 heterozygous cases of adenocarcinomas. Furthermore, in 7 of these 8 squamous-cell carcinomas, loss of heterozygosity from chromosome 17 was accompanied by loss of DNA sequences from chromosome 11. The spectrum of allelic sequences lost from chromosome 11 was, however, similar in every type of carcinoma studied, and the data show two regions commonly deleted from chromosome 11 (11pter-p15.5 and 11p13-q13) that may have a role in the pathogenesis of all these types of non-small-cell bronchogenic carcinoma. Loss of DNA sequences from chromosome 3 was seen in 16 of 31 cases where the constitutive DNA was heterozygous-i.e., informative. These data included only 6 of 16 cases where loss of heterozygosity involved a chromosomal locus previously shown to be lost consistently in small-cell lung cancer (DNF15S2). Loss of heterozygosity at the chromosome 13q locus, D13S3, was seen in 9 of 21 informative cases, and in 2 cases, both adenocarcinomas, duplication of the intact DNA sequences suggested the possibility that mitotic recombination had occurred. Frequent DNA sequence deletions, including those from chromosome 17, in squamous-cell carcinomas may reflect the extensive mutagenic and clastogenic effects of tobacco smoke that may lead to inactivation of putative tumor-suppressor genes.
Adenocarcinoma↗
Alkyl and aryl carcinogen adducts detected in human peripheral lung.
Human peripheral lung tissue samples were obtained at autopsy from 17 individuals of known occupational and smoking histories. A spectrum of different carcinogen-DNA adducts was detected using a variety of sensitive techniques. High-pressure liquid chromatography-linked synchronous fluorescent spectrophotometry and an ultrasensitive enzyme radioimmunoassay detected adducts derived from benzo[a]pyrene diol epoxide and other apparent polycyclic aromatic hydrocarbons. An amplified enzyme-linked immunosorbent assay demonstrated the presence of 4-aminobiphenyl-DNA adducts in many of these samples. A number of these specimens also contained O6-alkyldeoxyguanosine as measured by 32P-postlabeling techniques. Thus this pilot study indicates not only that human lung contains a spectrum of carcinogen-DNA adducts, but also that a full scale molecular dosimetry study of human exposure to both aryl and alkyl chemical carcinogens is warranted.
Adolescent↗
Fluorescence and mass spectral evidence for the formation of benzo[a]pyrene anti-diol-epoxide-DNA and -hemoglobin adducts in humans.
Highly specific methods are required to detect and quantitate carcinogen-macromolecular adducts in humans who are exposed to complex mixtures of chemical carcinogens. High performance liquid chromatography and fluorescence spectroscopy have been used successfully to detect and identify residues of benzo[a]pyrene-7,10/8,9-tetrahydrotetrol (BP-7,10/8,9-tetrol) that were released upon mild acid hydrolysis of human DNA or hemoglobin. Synchronous fluorescence spectroscopy data indicate that levels of benzo[a]pyrene-diol-epoxide-DNA (BPDE-DNA) adducts as high as 1.54 fmol BPDE/micrograms DNA are formed (1 adduct in 5 million nucleotides) in peripheral blood lymphocytes of coke-oven workers; these data were subsequently corroborated by gas chromatography/mass spectroscopy single ion monitoring analysis (m/z 404+). Additionally, among lung cancer patients, 5 samples of tumor DNA were found to be negative and 1 of 4 samples of corresponding lung tissue was found to be positive. Extraction and purification of BP-7,10/8,9-tetrol from the hemoglobin of smokers suggested levels of bound carcinogen in excess of 1 ng BPDE/gm of hemoglobin. High performance liquid chromatography combined with synchronous fluorescence spectroscopy provides a highly specific method for the detection of covalently bound BP residues in both human hemoglobin and DNA.
Benzo(a)pyrene↗
The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli.
Human atrial natriuretic factor [ANF(1-28)] has been isolated from a fusion protein produced in Escherichia coli. ANF(1-28) was linked to a naturally occurring E. coli protein, chloramphenicol acetyltransferase, via unique cleavage sequences susceptible to either human thrombin digestion, or the chemical action of 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole). The linker sequences were Gly-Val-Arg-Gly-Pro-Arg and Trp respectively. The liberated ANF was purified by reversed-phase HPLC. Optimised cleavage conditions released 5-10% (by mass) of the maximal yield of ANF(1-28) from the fusion protein with the thrombin-susceptible linker, whilst a 2-5% (by mass) yield was observed from the fusion protein with the tryptophan linker after BNPS-skatole treatment. The purified cleavage products were biologically active and shown to comprise intact ANF(1-28). Fast-atom-bombardment mass spectrometry confirmed [MH]+ of 3079 m/z, consistent with ANF(1-28).
Acetyltransferases↗
The application of immunoassays and fluorometry to the detection of polycyclic hydrocarbon-macromolecular adducts and anti-adduct antibodies in humans.
The metabolic activation of polycyclic aromatic hydrocarbons (PAH) to chemical species that form covalent adducts with cellular macromolecules (DNA and protein) is central to theories of carcinogenesis. Assays are currently being developed that will accurately reflect human macromolecular exposure to these carcinogens. Immunoassays are capable of detecting low levels of PAH-DNA adducts and antibodies directed against these adducts in humans and HPLC/spectrophotofluorimetry allows the detection of carcinogen-DNA or carcinogen-protein adducts in human peripheral blood. Both types of method have inherent advantages and disadvantages, and the use of more than one type of corroborative assay is a feature in our work. Simplified but highly specific synchronous fluorescence spectra have been obtained for BP-tetrols after mild acid hydrolysis and HPLC of sample materials. When using a wavelength difference of 34 nm to drive the excitation and emission monochromators simultaneously, the pyrene fluorophore, when present, has a signature peak at 345 nm (excitation). The results of immunoassays demonstrate human exposure to PAH as a class of carcinogen, since serological cross-reactivity of antibodies does not limit detection in this system to a single chemical compound. In addition the formation in humans of anti-PAH-DNA antibodies has been shown, presumably in response to both past and present exposure to the parent compounds. In summary the results of each assay can indicate human exposure to PAH and have the potential for molecular dosimetry of this exposure.
Antibodies↗
Detection of benzo[a]pyrene diol epoxide-DNA adducts in human placenta.
Human placenta is a readily available organ that responds to maternal environmental insult and has been previously used to investigate metabolism and bioactivation of procarcinogens, for example, benzo[a]pyrene. HPLC in combination with synchronous fluorescence spectroscopy was used to examine 28 placentas for the presence of benzo[a]pyrene diol epoxide-DNA adducts, and 10 of these were found to be positive. DNA samples from these placentas were subsequently pooled and subjected to partial enzymatic digestion to oligonucleotide fragments. Concentration of those DNA fragments containing benzo[a]pyrene diol epoxide-DNA adducts was achieved by immunoaffinity chromatography with polyclonal antibodies raised against these adducts. Column eluates were hydrolyzed under mild acid conditions and extracted with an organic solvent. The presence of benzo[a]pyrene-7,10/8,9-tetrahydrotetrol residues in the extracts was determined by HPLC and synchronous fluorescence spectroscopy and was confirmed by GC/MS. The results unequivocally confirm bioactivation and formation of DNA adducts from benzo[a]pyrene in human placenta in vivo and establish a methodological approach to direct measurement of carcinogen-DNA adducts that are formed as a result of human environmental exposure.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Interlaboratory comparison of antisera and immunoassays for benzo[a]pyrene-diol-epoxide-I-modified DNA.
An interlaboratory comparison of immunoassays using antisera elicited against benzo[a]pyrene-diol-epoxide-modified DNA (BPDE-I-DNA) was carried out resulting in standardization of antisera, competitors and assay conditions. The assays used included competitive enzyme-linked immunosorbent assays (ELISA) with color and fluorescence endpoint detection and an ultrasensitive enzyme radioimmunoassay (USERIA) with a radioactive endpoint. Three different antisera were compared, two of which were obtained from different rabbits immunized with the same BPDE-I-DNA and a third from an animal immunized with another BPDE-I-DNA sample. Samples of standardized BPDE-I-DNA with high (36 pmol adduct/microgram DNA; 1.2 adducts/10(2) nucleotides) and low (4.5 fmol/microgram DNA; 1.5 adducts/10(6) nucleotides) modification levels were prepared and used in each laboratory. The antisera were all elicited against DNAs modified to a high extent, and it was therefore not surprising that they detected adducts in a slightly modified DNA sample with lower efficiency than those in highly modified DNA samples. The discrepancy of antibody recognition between the highly and slightly modified samples varied between 1.4- and 11.2-fold depending on the antiserum and assay. To ascertain the quantitative capability of the immunoassays, the modification level of DNA isolated from mouse keratinocytes treated with [3H]benzo[a]pyrene was determined by radioactivity and immunoassay. These results indicated that when a biological sample is assayed against a BPDE-I-DNA standard modified in the same range as the biological samples (4.5 fmol/microgram), quantitative recovery of adducts is achieved by immunoassay. These studies resulted in the realization that interlaboratory differences in immunoassay procedure can have significant consequences for data comparison and that where possible it is preferable for laboratories to use the same antisera and modified DNA standards.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Detection and characterization of human serum antibodies to polycyclic aromatic hydrocarbon diol-epoxide DNA adducts.
The presence of serum antibodies to the diol-epoxide DNA adducts of representative polycyclic aromatic hydrocarbons (PAH), chrysene, benz[a]anthracene and benzo[a]pyrene, was determined by ELISA using serum samples obtained from normal healthy individuals. Antibodies that reacted against PAH adducted-DNA, but not against PAH-adducted protein, were found in the serum of approximately 40% of the test individuals. Specificity analysis of the antibodies demonstrated that serological cross-reactions between the benzo[a]pyrene and the chrysene diol-epoxide adducts were present. Similar cross-reactivity between the benz[a]anthracene and the chrysene adducts was observed. Sera containing antibodies that were apparently specific for each of the three PAH-DNA adducts were also identified. The presence of antibodies to PAH-DNA adducts indicates both past exposure to these carcinogenic PAH and their metabolic activation to the DNA damaging metabolites. These antibodies may prove to be useful in both retrospective and prospective epidemiological studies of various diseases associated with PAH exposure.
Adult↗
DNA restriction fragment length polymorphism analysis of human bronchogenic carcinoma.
DNA restriction fragment length polymorphism (RFLP) analysis has been successfully applied both to evaluate possible human susceptibility factors as well as to identify genes involved in malignant transformation of human cells. In this report we review previous applications of RFLP analysis to evaluation of human malignancies and discuss our RFLP studies of human bronchogenic carcinoma, which are in progress. Preliminary results of our analysis of the RFLP associated with the cHa ras variable tandem repeat (VTR) indicate that rare alleles of this VTR are more frequent in patients with bronchogenic carcinoma than in controls.
Carcinoma, Bronchogenic↗
DNA restriction fragment length polymorphisms at either end of the c-raf-1 locus at 3p25.
We report the identification and characterization of two restriction fragment polymorphisms (RFLPs) associated with the human c-raf-1 protooncogene located on chromosome 3 (3p25). EcoRI digestion produces an RFLP derived from the 5'flanking sequences with a heterozygosity of 28%, and TaqI produces an RFLP representing the 3' flanking sequences with a heterozygosity of 25%. The finding of RFLPs associated with a potential oncogene may provide a method of analysis for determining genetic susceptibilities to certain cancers.
Chromosome Mapping↗