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Biomedical subjects

A Weston

Publications and source records attributed to A Weston.

At least 55 records · Page 3Linked to original sources
PubMed1994

Possible link between anaphylactoid reactions to anaesthetics and chemicals in cosmetics and biocides.

Binding-inhibition studies involving human IgE antibody to suxamethonium, SUX (from a patient with a near-fatal reaction), were performed with five different quaternary ammonium compounds (QAC): three common ingredients of cosmetics and two commonly used disinfectants, cetrimide and benzalkonium. All the five QAC showed immunological cross-reaction with SUX. In addition, at high concentrations, they released histamine from basophils of normal subjects. Thus, QAC may be both sensitizers and histamine releasers.

Anaphylaxis↗
PubMed1994

Identification of 1-hydroxypyrene glucuronide as a major pyrene metabolite in human urine by synchronous fluorescence spectroscopy and gas chromatography-mass spectrometry.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) from various occupational, environmental, medicinal and dietary sources. The measurement of specific PAH metabolites, particularly 1-hydroxypyrene, in human urine treated with deconjugating enzymes (e.g. beta-glucuronidase) has been extensively used as a means of assessing recent exposure to PAHs. We have examined pyrene metabolites in human urine prior to enzymatic deconjugation in order to determine the relative proportions of conjugated and unconjugated pyrene metabolites. The analytical method utilized immunoaffinity chromatography, high performance liquid chromatography (HPLC) and the complementary techniques of synchronous fluorescence spectroscopy (SFS) and gas chromatography-mass spectrometry (GC-MS) to measure pyrene-containing metabolites. SFS analysis of immunoaffinity-purified urine samples showed fluorescence spectra characteristic of the pyrene moiety (using wavelength differences of 34 nm, 54 nm and 102 nm). These spectra are produced by several PAHs containing the pyrene moiety. HPLC analysis with fluorescence detection indicated that the major fluorescent metabolite in immunoaffinity-purified urine was much more polar than simple hydroxylated metabolites of pyrene (1-hydroxypyrene) or benzo[a]pyrene (benzo[a]pyrene-diols or -tetrols). Following digestion with beta-glucuronidase, this metabolite co-chromatographed with authentic 1-hydroxypyrene and exhibited fluorescence spectra characteristic of 1-hydroxypyrene, suggesting that the major metabolite was a glucuronide conjugate of 1-hydroxypyrene. This was subsequently confirmed by GC-MS analysis of trimethylsilyl derivatives of the major metabolite; both 1-hydroxypyrene and glucuronic acid were detected independently as derivatized products. Since 1-hydroxypyrene glucuronide is approximately 5-fold more fluorescent than 1-hydroxypyrene, it may provide a more sensitive biomarker for assessing exposure to pyrene in mixtures of PAHs.

Chromatography, High Pressure Liquid↗
PubMed1994

Determination of the allelic frequencies of an L-myc and a p53 polymorphism in human lung cancer.

The L-myc and p53 genes have been implicated in lung cancer. Both of these genes have restriction fragment length polymorphisms (RFLPs) that could account for differential expression or activity of variant forms. An EcoRI restriction site in the L-myc gene was previously reported to be a predictor of poor prognosis in Japanese lung cancer patients. There are several RFLPs in the p53 gene. In exon 4 there is a polymorphism that codes for either an arginine or proline residue at codon 72. We previously reported the frequency of DNA-RFLPs at these gene loci revealed by EcoRI and AccII respectively. Here we report results from a study comparing lung cancer cases (n = 31) with chronic obstructive pulmonary disease controls (n = 49). No association was found between these RFLPs and disease status. Previous observations that the frequencies of these RFLPs varied by race were confirmed. The p53 arginine allele was found to be more common in Caucasians (0.71) than African-Americans (0.50). The EcoRI restriction site present allele in L-myc was more frequent in African-Americans (0.71) than Caucasians (0.49). Thus, the allelic frequency for L-myc was similar in African-Americans to that reported for Japanese, and the allelic frequency for p53 was similar in Caucasians to that reported for Japanese.

Base Sequence↗
PubMed1994

Analysis of cytochrome P450 2E1 genetic polymorphisms in relation to human lung cancer.

Human cancer risk assessment using molecular genetic techniques is a rapidly emerging field. Many studies suggest that both inherited and acquired genetic predispositions play an important role in carcinogenesis. Cytochrome P450 (CYP) 2E1 is involved in the metabolic activation of N-nitrosamines and other low molecular weight compounds. A recently described genetic polymorphism of CYP2E1 [DraI restriction fragment length polymorphism (RFLP)] has been associated with an increased risk of lung cancer in Japanese. We have assessed the allelic frequency of three RFLPs (PstI, RsaI, and DraI) in African-Americans (n = 109), Caucasian Americans (n = 153), and octogenarian Japanese (n = 42), and also in a United States case-control study of lung cancer (histologically confirmed lung cancer, n = 58; controls, n = 56; total, n = 114). The relationship of the CYP2E1 DraI polymorphism to other CYP2E1 polymorphisms (PstI and RsaI RFLP) was examined. The allelic frequency of the DraI C minor allele for all subjects was 0.09 in Caucasians, 0.09 in African-Americans, and 0.31 in Japanese. In the case-control study of lung cancer, no association of the CYP2E1 DraI genotype with lung cancer was found (odds ratio, 1.57; 95% confidence interval, 0.59-4.18). Comparison after discordant CYP2E1 genotypes suggests the presence of different haplotypes in Americans and Japanese. These results indicate that the CYP2E1 DraI RFLP is probably not a cancer risk factor in United States Caucasian or African-Americans, although statistical power is limited given the low frequency of the CYP2E1 DraI C minor alleles.

Aged↗
PubMed1993

Protein O-mannosylation in Candida albicans. Determination of the amino acid sequences of peptide acceptors for protein O-mannosyltransferase.

A protein-O-D-mannosyltransferase (PMT) assay was optimised using a microsomal membrane preparation from Candida albicans and a peptide acceptor, YNPTSV. [14C]Mannose was transferred from dolichyl phosphate [14C]mannose to the threonine or serine residues of the peptide. During the assay, the peptide was highly susceptible to proteolysis. A blocked peptide Ac-YNPTSV-NH2 was resistant to proteolysis and was apparently a better acceptor for O-mannosylation. This peptide had a Km value of 4.3 mM in the assay. A number of other peptides were tested with altered sequences. Maximum incorporation of [14C]mannose was obtained with a pentapeptide YATAV (Km 2.2 mM) which was further improved by blocking both ends: Ac-YATAV-NH2 (Km 0.25 mM). Finally, and unexpectedly, an improvement was noted if the acetyl group on the N terminus was replaced by a biotin residue. Biotin-YATAV-NH2 had a Km of 0.075 mM. The biotin residue may be important in increasing the lipophilicity of the peptide and thus aid its adhesion to the Candida membranes. The simplest peptide that could act as an efficient mannose acceptor was Ac-ATA-NH2, whilst no incorporation was observed with Ac-GTG-NH2.

Amino Acid Sequence↗
PubMed1993

Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes.

Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.

Adult↗
PubMed1993

Physical methods for the detection of carcinogen-DNA adducts in humans.

This report has attempted to summarize the principles, advantages, limitations and the source of data derived in the use of physical detection techniques for DNA-adduct measurement in human biomonitoring. Each method, although inherently chemically-specific, has advantages and limitations depending on the adduct type under study. These methods have a niche that is at least consistent with corroborative technology, and are being applied to dosimetry problems in the field.

Carcinogens↗
PubMed1993

7-Alkyldeoxyguanosine adduct detection by two-step HPLC and the 32P-postlabeling assay.

7-Alkyldeoxyguanosine DNA adducts may be a marker for some N-nitroso compound exposures and subsequent human cancer risk. A sensitive and highly specific assay for the detection of 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-methyldGp) and 7-ethyl-2'-deoxyguanosine-3'-monophosphate (7-ethyldGp) has been developed by combining two different HPLC purification steps with the 32P-postlabeling assay. We previously reported that ion-pair reverse-phase (IP) chromatography coupled with the 32P-postlabeling assay detects 7-methyldGp in human lung, but have found that other nucleotides and unknown adducts co-elute. Thus, weak anion exchange (AE) HPLC was added in tandem with IP HPLC prior to the 32P-postlabeling assay. 2'-Deoxyguanosine-3'-monophosphate (dGp) is incorporated into the assay as an internal standard for the assessment of enzyme labeling efficiency and adduct recovery. The methodology was validated using radiolabeled DNA and liquid scintillation counting, which accounts for adduct loss from enzymatic digestion to detection. Levels of 7-ethyldGp also were correlated with accelerator mass spectrometry. The overall adduct recovery with this method was 58% for 7-methyldGp and 98% for 7-ethyldGp. The detection limit for both assays using 100 micrograms of DNA was one adduct in 10(8) unmodified dGp. 7-MethyldGp and 7-ethyldGp levels were determined in ten human lung samples at levels of 1.4-5.4 and 0.6-3.1 adducts per 10(7) dGp respectively, and in five human lymphocyte samples at levels of 5.0-8.3 and 0.3-1.4 adducts per 10(7) dGp respectively. Combining the two HPLC purification steps and the 32P-postlabeling assay attains chemical specificity, retains sufficient quantitative sensitivity and should be useful in human biomonitoring studies.

Animals↗
PubMed1993

Detection of metabolites of polycyclic aromatic hydrocarbons in human urine.

A non-invasive assay has been developed for the recovery of r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[alpha]-pyrene (BP-7,10/8,9-tetrol) from human urine. This tetrol is excreted as a metabolite of benzo[alpha]pyrene (BP) in a process catalyzed by cytochrome P450 enzymes and epoxide hydrolases. Urine was hydrolysed to release activated benzo[alpha]-pyrene-diol-epoxides covalently bound to macromolecular species or conjugated tetrols. The relatively non-polar organic molecules from urine hydrolysates were collected on octadecasilane chromatography columns (Sep-Paks). Materials eluted in solvent (80% CH3OH), were further purified on immunoaffinity columns with antibodies raised against anti-N2-[10(7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo [alpha]pyrenyl)]-guanosine. HPLC was then used to isolate BP-7,10/8,9-tetrol, which was quantitated by synchronous fluorescence spectroscopy (SFS). This assay detected 0.24-3.12 pmol BP-7,10/8,9-tetrol per ml urine (limit of detection 0.01 pmol/ml, given 10 ml urine), in four study subjects. Reproducibility was assessed by adding tritium labeled BP-7,10/8,9-tetrol (1500 fmol) to a urine sample previously identified to contain the tetrol at levels below the limit of detection of the fluorescence assay; a recovery of > 30% of the added radioactivity was achieved (510 +/- 64 fmol, mean +/- SD, n = 3). Because HPLC alone was not sufficient to isolate materials for quantitation by SFS directly from human urine, immunoaffinity chromatography was found to be a necessary preparatory step in BP-7,10/8,9-tetrol isolation. These data demonstrate the presence of tetrahydrotetrol metabolites of BP in human urine and suggest that measurement of BP-7,10/8,9-tetrol and other polycyclic aromatic hydrocarbon-tetrols may prove to be valuable dosimeters of human internal exposure to polycyclic aromatic hydrocarbons.

Benzo(a)pyrene↗
PubMed1993

Standardization of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon-DNA adducts.

A modified method for the 32P-postlabeling assay that permits standardized quantitation for specific polycyclic aromatic hydrocarbon (PAH)-DNA adducts is described. This method has been designed to test the components of the 32P-postlabeling assay for use in the chemically specific detection of individual adducts with the ultimate goal of testing the biological significance of PAH-DNA adducts in humans. The approach relies upon high performance liquid chromatography (HPLC), concomitant labeling of 2'-deoxyguanosine (dGp) as an internal standard and thin layer chromatography (TLC), which identifies unmodified nucleotides along with PAH-DNA adducts on the same TLC plate. This method assesses labeling efficiency, detects the presence of unknown inhibitors, assesses the adequacy of digestion (when combined with HPLC) and allows for the development of calibration curves for directly determined molar ratios (adduct:internal standard). Chemically synthesized adduct standards and quantitative 32P-postlabeling data have been corroborated by UV spectroscopy, fluorescence spectroscopy and liquid scintillation counting where radiolabeled materials were available. Labeling efficiencies of the PAH-DNA adducts were found to be up to 100-fold less than expected and depended upon both the adduct and adduct levels (lower levels being less efficiently detected). The presence of unmodified nucleotides resulted in a 1.5-fold lower labeling efficiency. Mixtures of selected PAH-DNA adducts did not affect the labeling efficiencies of each other. The data suggest that previous 32P-postlabeling assay studies for PAH-DNA adducts may have underestimated adduct levels due to variations in labeling efficiency.

Adenosine Triphosphate↗
PubMed1993

Detection of polycyclic aromatic hydrocarbon-DNA adducts in human lung.

Synchronous fluorescence spectroscopy has been combined with immunoaffinity chromatography (IAC) and HPLC to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts and measure r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adducts in human tissues and cells. A monoclonal antibody (8E11) that recognizes a range of PAH-DNA adducts, but not chemically unrelated adducts, was used to prepare IAC columns. Samples of DNA (25 from human lung and 8 positive and negative controls) were hydrolyzed enzymically and subjected to IAC. Adducts captured by the antibodies and eluted in NaOH (50 mM) were analyzed for fluorescent properties. The spectral fluorescence excitation-emission matrices suggested the presence of mixtures of PAH-DNA adducts in some of the eluates. The eluates were subsequently hydrolyzed with acid (HCl, 0.1 N, 3 hr) and reanalyzed by synchronous fluorescence spectroscopy using a wavelength differential of 34 nm. In 6 of the 25 human lung DNA samples, materials with HPLC retention times identical to benzo[a]pyrene-7,10/8,9-tetrahydrotetrol were found to have fluorescence characteristics indistinguishable from pyrene. Comparisons with appropriate standards indicated that BPDE-DNA adduct levels were between 1 and 40 adducts in 10(8) unmodified nucleotides. No correlation was observed between lung DNA-adduct levels and measures of recent smoking (serum cotinine), but tissue samples taken from different portions of the same lungs showed variation in the DNA adduct levels detected. This finding complicates interpretation of the data and has important implications for the design of future experiments.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Detection of oxidative damage by 32P-postlabelling: 8-hydroxydeoxyguanosine as a marker of exposure.

Human exposure to reactive oxygen species is unavoidable and has been implicated in the etiology of a number of human diseases. This exposure results in the formation of various modified DNA bases: the promutagenic lesion 8-hydroxydeoxyguanosine (8OHdG), in particular, is a major product. We have developed an assay using ion-pair HPLC and 32P-postlabelling to quantify 8OHdG in human DNA with high specificity and sensitivity. An internal standard is used to account for variations in labelling efficiency. Chemically synthesized 8OHdG 3'-monophosphate and 5'-monophosphate standards were used to optimize the HPLC-32P-postlabelling and TLC separative steps, respectively. The assay was validated using known ratios of 8OHdG to normal nucleotides. The limit of detection is in the range of one 8OHdG residue per 10(6)-10(7) dG residues. Using this procedure, 8OHdG levels of 16-35 8OHdG adducts per 10(5) dG residues have been found in leukocytes isolated from patients who received 180-200 cGy of ionizing radiation. These levels were 2-4-fold greater than those found in an unexposed individual. Since 8OHdG may be formed during DNA extraction and digestion, current procedures for measuring background levels are discussed.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Combined micropreparative techniques with synchronous fluorescence spectroscopy or 32P-postlabelling assay for carcinogen-DNA adduct determination.

Methods for the detection of carcinogen-DNA adducts require both sensitivity and specificity. The 32P-postlabelling assay is highly sensitive but lacks adduct specificity. The strategy reported herein combines micropreparative techniques including HPLC and immunoaffinity chromatography (IAC) to enhance chemical specificity. The resultant assays have retained sensitivity for human DNA analysis. Combined HPLC and 32P-postlabelling has detected 7-methyldeoxyguanosine in human lung samples, while combined IAC and 32P-postlabelling has detected O6-methyldeoxyguanosine adducts in stomach tissue. The limits of detection are one adduct in 10(7) and 10(8) unmodified deoxyguanosine (dG), respectively. IAC was combined with 32P-postlabelling to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human lung. The detection limit was one adduct in 10(7) dG. Our laboratory has also employed synchronous fluorescence spectroscopy (SFS) for the detection of adducts formed from benzo[a]pyrene in human lung. Complex fluorescence matrices suggest the presence of other PAH-DNA adducts. Both the SFS assay and the 32P-postlabelling assay were applied to a series of human samples and a high correlation was found for adduct levels. The development of such assays using synthetic standards, internal standards, determination of calibration curves and validation with corroborating methods is required for chemically specific and sensitive adduct detection.

Benzo(a)pyrene↗
PubMed1993

Functional studies of a germ-line polymorphism at codon 47 within the p53 gene.

A rare germ-line polymorphism in codon 47 of the p53 gene replaces the wild-type proline (CCG) with a serine (TCG). Restriction analysis of 101 human samples revealed the frequency of the rare allele to be 0% (n = 69) in Caucasians and 4.7% (3/64, n = 32) among African-Americans. To investigate the consequence of this amino acid substitution, a cDNA construct (p53 mut47ser) containing the mutation was introduced into a lung adenocarcinoma cell line (Calu-6) that does not express p53. A growth suppression similar to that obtained after introduction of a wild-type p53 cDNA construct was observed, in contrast to the result obtained by introduction of p53 mut143ala. Furthermore, expression of neither p53 mut47ser nor wild-type p53 was tolerated by growing cells. In transient expression assays, both mut47ser and wild-type p53 activated the expression of a reporter gene linked to a p53 binding sequence (PG13-CAT) and inhibited the expression of the luciferase gene under the control of the Rous sarcoma virus promoter (RSVluc). In the same assay, mut143ala did not activate the expression of PG13-CAT and produced only a slight inhibitory effect on RSVluc. These findings indicate that the p53 variant with a serine at codon 47 should be considered as a rare germ-line polymorphism that does not alter the growth-suppression activity of p53.

Alleles↗
PubMed1992

Cytochrome P450IIE1 genetic polymorphisms, racial variation, and lung cancer risk.

Cytochrome P450IIE1 is responsible for the activation of carcinogenic N-nitrosamines, benzene, urethane, and other low-molecular-weight compounds. Restriction fragment length polymorphisms (PstI and RsaI restriction enzymes) have been identified in the cytochrome P450IIE1 transcription regulatory region that may affect expression. This study describes the PstI and RsaI polymorphisms in different racial populations and in a case-control study of lung cancer. The allelic frequencies were markedly different in Japanese, African-Americans, and Caucasians: the PstI rare allele was present at a frequency of 2% in Caucasians, 5% in African-Americans, and 24% in Japanese (P < 0.05). For the RsaI rare allele, frequencies were 2% in Caucasians, 2% in African-Americans, and 27% in Japanese (P < 0.05). The assay was also applied to 128 individuals enrolled in a case-control study of lung cancer. Although limited in statistical power, the data indicate no evidence for an association in the aggregate of cytochrome P450IIE1 PstI [for which the odds ratio was 0.7 (95% confidence interval (C.I.) = 0.2-2.8)] or RsaI [for which the odds ratio was 0.9 (95% C.I. = 0.2-5.4)] restriction fragment length polymorphisms with lung cancer in this U.S. population. When analyzed by race, the lung cancer odds ratio for the PstI mutant allele in African-Americans was 0.19 (95% C.I. = 0.03-1.38), and in Caucasians it was 4.13 (95% C.I. = 0.34-48.8). For the RsaI mutant allele, the odds ratios were 0.20 (95% C.I. = 0.02-2.43) and 4.28 (95% C.I. = 0.35-50.6), respectively. The ethnic differences of these restriction fragment length polymorphisms might be related to genetic susceptibilities for lung cancer among Caucasians and for gastric or esophageal cancer among Japanese.

Alleles↗