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Biomedical subjects

J D Tucker

Publications and source records attributed to J D Tucker.

At least 19 recordsLinked to original sources
PubMed2006

Retrospective assessment of radiation exposure using biological dosimetry: chromosome painting, electron paramagnetic resonance and the glycophorin a mutation assay.

Biological monitoring of dose can contribute important, independent estimates of cumulative radiation exposure in epidemiological studies, especially in studies in which the physical dosimetry is lacking. Three biodosimeters that have been used in epidemiological studies to estimate past radiation exposure from external sources will be highlighted: chromosome painting or FISH (fluorescence in situ hybridization), the glycophorin A somatic mutation assay (GPA), and electron paramagnetic resonance (EPR) with teeth. All three biodosimeters have been applied to A-bomb survivors, Chernobyl clean-up workers, and radiation workers. Each biodosimeter has unique advantages and limitations depending upon the level and type of radiation exposure. Chromosome painting has been the most widely applied biodosimeter in epidemiological studies of past radiation exposure, and results of these studies provide evidence that dose-related translocations persist for decades. EPR tooth dosimetry has been used to validate dose models of acute and chronic radiation exposure, although the present requirement of extracted teeth has been a disadvantage. GPA has been correlated with physically based radiation dose after high-dose, acute exposures but not after low-dose, chronic exposures. Interindividual variability appears to be a limitation for both chromosome painting and GPA. Both of these techniques can be used to estimate the level of past radiation exposure to a population, whereas EPR can provide individual dose estimates of past exposure. This paper will review each of these three biodosimeters and compare their application in selected epidemiological studies.

Biological Assay↗
PubMed2003

Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA.

The fluorescence polarisation assay (FPA) is a recently described test for the serological diagnosis of Brucella infection. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. To validate the FPA, serum samples from 146 confirmed (by culture) Brucella-infected cattle were tested in conjunction with serum samples from 1947 noninfected cattle. The competitive ELISA (cELISA) was validated using these positive reference samples and 1440 negative samples, while data for the indirect ELISA (iELISA) was generated from 6957 negative samples plus the positive sera. Published diagnostic specificity (DSp) data for the complement fixation test (CFT) and serum agglutination test (SAT) was used in conjunction with the test results on the positive sera to obtain diagnostic specificity plus diagnostic sensitivity (DSn). After selection of a cutoff for the FPA and cELISA, the diagnostic specificity and sensitivity total for each test were compared. The results, with 95% confidence intervals, were: FPA (195.7+/-2.79), iELISA (195.0+/-2.70), cELISA (194.9+/-3.48), CFT (191.7+/-4.45), and SAT (180.4+/-6.33). The data presented supports the use of the FPA in diagnosis of brucellosis and questions the use of the SAT and CFT for either screening or confirmatory testing.

Agglutination Tests↗
PubMed2001

Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

Base Sequence↗
PubMed2001

Fish cytogenetics and the future of radiation biodosimetry.

Fluorescence in situ hybridisation (FISH) with whole chromosome paints has greatly facilitated the analysis of structural chromosome aberrations and has led to translocations replacing dicentrics as the aberration of choice for many applications. Major challenges remain if we are to go from translocations to an understanding of the health consequences of radiation exposure. Yet to be surmounted are the roles of individual susceptibility, time since exposure, and the effects of subjects age. Accomplishing these objectives will require automation, reduced costs, improved calibration, and extensive use of baseline samples.

Cytogenetic Analysis↗
PubMed2001

Evaluation of three somatic genetic biomarkers as indicators of low dose radiation effects in clean-up workers of the Chernobyl nuclear reactor accident.

The goals of this study were to assess three biomarkers of genetic effect for their individual and collective ability to detect and estimate radiation exposure in Russian Chernobyl clean-up workers. Work assignments were planned to limit dose to 0.25 Gy. The three biomarkers employed were chromosome translocations detectcd in lynmphocytes by florescence in situ hybridisation (FISH), and mutation at two genes, glycophorin A (GPA) in red blood cells detected by flow cytometry and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes detected by selective cell culture. Samples were Obtained from 1992 to 2000. The time between exposure at Chernobyl and sample acquisition was > or =5 years. The lymphocyte assays detected an elevation over controls in average outcomes it clean-up workers: translocation rates were 46% higher when adjusted for age and smoking and HPRT mutant frequencies were were 16% higher when adjusted for age. The G PA assay did not detect an exposure effect. The results indicate that measuring frequency of translocations by FISH is preferred for low dose radiation, retrospective biochemistry.

Adult↗
PubMed2000

The in vivo dose rate effect of chronic gamma radiation in mice: translocation and micronucleus analyses.

The in vivo effects of chronic, ultra low dose rates of gamma radiation in mice were evaluated using fluorescence in situ hybridization and the in vivo micronucleus test. SWRxC57BL/6 mice were divided into nine exposure groups and continuously exposed to 0.5, 2.0 or 4.0cGy 137Cs per day for 30, 60 or 90 days; unexposed control mice were also included. Following exposure, blood samples were taken from each animal and the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) were determined using flow cytometry. Peripheral blood lymphocytes were cultured and analyzed by chromosome painting to determine translocation frequencies. A significant dose rate response was seen in translocations and both MPCE and MNCE. Comparisons were made between the three chronic dose rates and it was determined that there was no significant difference among translocation frequencies for each rate. However, a significant difference was found between the chronic exposures reported here and the fractionated daily exposures reported previously. Dose rate reduction effects, ranging from 3 at low doses to 14 at high doses, were found for chronic versus acute exposures. The possibility of gender effects was investigated in both micronucleus and translocation data. No gender effect was found in translocation induction, but a slight effect was suggested in micronucleus induction.

Animals↗
PubMed2000

Role of maternal exposures and newborn genotypes on newborn chromosome aberration frequencies.

Maternal exposures may induce chromosome damage and birth defects in the fetus. Polymorphic variation in genes coding for enzymes involved in metabolic activation and detoxification of environmental procarcinogens may account for some of the differences in chromosome aberration frequencies in newborns. In this study, 40 mothers completed questionnaires regarding exposures they received during their pregnancy. Umbilical cord blood samples were analyzed for chromosome aberrations. An average of 1020 metaphase cell equivalents (equal to 1020 G-banded cells) were examined from each newborn. In 26 of the newborns, genotyping analysis was performed for genes functioning in metabolic activation and detoxification (cytochrome P450 genes: CYP2D6 and CYP1A1, and phase II genes: NAT1, NAT2, GSTT1, GSTM1, GSTP1, and epoxide hydrolase). A significant association between the CYP1A1 MspI polymorphism and chromosome aberration frequencies was observed in the newborns (p=0.02), with heterozygotes showing higher aberration frequencies than the wild type homozygotes. Some large differences in chromosome aberration frequencies for other genotypes were also noted, but these were not statistically significant. Exposure to tobacco smoke in utero also appeared to increase translocation frequencies. The mean frequency of translocations per 100 cell equivalents from newborns of mothers who smoked during pregnancy was significantly higher than that of newborns whose mothers did not smoke (0.21 vs. 0.11, respectively, p=0.045).

Acetyltransferases↗
PubMed2000

The relationship between genotype and chromosome aberration frequencies in a normal adult population.

Cancer susceptibility differences may be attributed in part to genetic variation in genes involved in metabolism of environmental procarcinogens. Increased risks for some cancers have been linked to polymorphisms in certain phase I and II genes, and have been associated with genomic instability and chromosomal aberrations. Aberration frequencies in general, and stable aberration frequencies (translocations and insertions) in particular, are used as biomarkers for disease. Thus, knowledge of the genetic factors that influence the frequency of stable aberrations in a normal population is important for cancer risk determination. In this work, genotypes for a number of xenobiotic enzymes (CYPIA1, CYP2D6, GSTM1, GSTT1, GSTP1, NAT1, NAT2 and epoxide hydrolase) and stable aberration frequencies were determined for 65 normal individuals aged 19-77 years. The population was divided at age 60 years for analysis because there was a significant difference in stable aberration frequencies between these groups. Subjects with low levels (0-66th percentile) of stable aberrations were compared to those with high levels (67th percentile and above). Of all the genotypes studied, only NAT2 showed a notable difference between the high and the low stable aberration groups in the percentage of polymorphisms observed, and this was seen only in the older subjects group. All individuals in the older-high stable aberration group were NAT2 rapid acetylator smokers. NAT2 slow acetylator smokers had significantly lower stable aberration frequencies compared to the NAT2 rapid acetylator smokers. Following previous work showing an increased risk of cancer associated with high levels of aberrations (above the 66th percentile), we hypothesize that smokers with the NAT2 rapid acetylator genotype may be at an increased risk for cancer.

Adult↗
PubMed2000

Lifetime persistence and clonality of chromosome aberrations in the peripheral blood of mice acutely exposed to ionizing radiation.

As the measurement of chromosomal translocations increases in popularity for quantifying prior radiation exposure, information on the possible decline of these "stable" aberrations over time is urgently needed. We report here information about the persistence of radiation-induced chromosome aberrations in vivo over the life span of a rodent. Female C57BL/6 mice were given a single whole-body acute exposure of 0, 1, 2, 3 or 4 Gy (137)Cs gamma rays at 8 weeks of age. Chromosome aberrations were analyzed from peripheral blood samples at various intervals between 1 day and 21 months after exposure. Aberrations were detected by painting chromosomes 2 and 8. Translocations decreased dramatically during the first 3 months after irradiation, beyond which time the frequencies remained relatively constant out to 1 year, when the effects of aging and clonal expansion became significant. Both reciprocal and nonreciprocal translocations increased with age in the unexposed control animals and were involved in clones. As expected of unstable aberrations, dicentrics decreased rapidly after exposure and reached baseline levels within 3 months. These results indicate that the persistence of translocations induced by ionizing radiation is complicated by aging and clonal expansion and that these factors must be considered when quantifying translocations at long times after exposure. These results have implications for biological dosimetry in human populations.

Aging↗
PubMed1999

Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B.

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.

Animals↗
PubMed1999

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

Angelman Syndrome↗
PubMed1999

Requirement for the Xrcc1 DNA base excision repair gene during early mouse development.

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of the X-ray cross-complementing (Xrcc)-1 gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings produced Xrcc1-/- embryos at early developmental stages, but not Xrcc1-/- late-stage fetuses or pups. Histology showed that mutant (Xrcc1-/-) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5-7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos. Xrcc1-/- cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage. Xrcc1 mutant embryos that were also made homozygous for a null mutation in Trp53 underwent developmental arrest after only slightly further development, thus revealing a Trp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.

Animals↗
PubMed1999

Frequency of spontaneous chromosome aberrations in mice: effects of age.

In this paper, we present data on chromosome aberration frequencies in mice which served as unexposed controls in a variety of radiation and chemical toxicology experiments conducted in our laboratory in recent years. All chromosome aberration data were obtained by chromosome painting. In peripheral blood lymphocytes from 102 animals, the frequencies of translocations and insertions increased significantly with age. No increase with age was seen for dicentrics or acentric fragments. When the data were analyzed by strain, the age-related increase in translocation frequencies was observed only in the 71 homozygous C57BL/6 mice and not in any of the three heterozygous strains. Very few aberrations of any type were observed in 62 bone marrow samples, and no effect of age was seen for any aberration type in this tissue. These results are similar to those observed in unexposed humans, and suggest that the increase in translocations is not the result of accumulated damage from chronic 'background' environmental exposures but instead may be due to biological processes associated with aging.

Aging↗
PubMed1999

The distribution of chromosome damage, non-reciprocal translocations and clonal aberrations in lymphocytes from Chernobyl clean-up workers.

In this paper we determined whether the frequencies of translocations and insertions are proportional to chromosome size in peripheral blood lymphocytes from Chernobyl nuclear accident clean-up workers and healthy unexposed control subjects. The frequency of aberrations among chromosomes 1, 2 and 4 in both groups was found to be significantly different from the distribution expected on the basis of chromosome size, although the difference was only marginally significant in controls. We also determined whether differences exist in aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as one event, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results which yielded an interpretable arithmetic relationship between frequency measurements using the two systems. Approximately 34% of all translocations were observed to be non-reciprocal, and cells bearing clones of abnormal cells were observed in 6 of 198 subjects (3.0%). Our results demonstrate that clones of abnormal cells and the presence of non-reciprocal translocations contribute to the non-proportional distribution of radiation-induced and spontaneous cytogenetic damage.

Chromosome Breakage↗
PubMed1999

PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes.

Whole-chromosome painting probes (WCPs) and chromosome-arm painting probes (CAPs) are an integral part of the cytogenetic analysis of chromosome abnormalities. While these are routinely made by chromosome microdissection, multiple copies of the dissected region have been necessary to achieve a library sufficiently complex to provide adequate painting. Performing multiple dissections of chromosomes or chromosome regions is time consuming and occasionally impossible, such as when working with species whose banded karyotype is not well defined. We have developed a method whereby chromosome paints can be reliably generated by dissecting single chromosomes. The technique consists of performing degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) in situ on the chromosomes, prior to dissection. Enough amplification occurs to enable a single dissected chromosome to be used to create a painting probe sufficiently complex for use in fluorescence in situ hybridization (FISH). The amplification products remain localized on the chromosomes; this allows region-specific chromosome paints to be made. We detail this novel technique and show whole-chromosome, arm-specific, and contiguous region-specific probes for human and rat, each created from single dissected fragments of chromatin.

Animals↗
PubMed1999

Estimation of the target stem-cell population size in chronic myeloid leukemogenesis.

Estimation of the number of hematopoietic stem cells capable of causing chronic myeloid leukemia (CML) is relevant to the development of biologically based risk models of radiation-induced CML. Through a comparison of the age structure of CML incidence data from the Surveillance, Epidemiology, and End Results (SEER) Program and the age structure of chromosomal translocations found in healthy subjects, the number of CML target stem cells is estimated for individuals above 20 years of age. The estimation involves three steps. First, CML incidence among adults is fit to an exponentially increasing function of age. Next, assuming a relatively short waiting time distribution between BCR-ABL induction and the appearance of CML, an exponential age function with rate constants fixed to the values found for CML is fitted to the translocation data. Finally, assuming that translocations are equally likely to occur between any two points in the genome, the parameter estimates found in the first two steps are used to estimate the number of target stem cells for CML. The population-averaged estimates of this number are found to be 1.86x10(8) for men and 1.21x10(8) for women; the 95% confidence intervals of these estimates are (1.34x10(8), 2. 50x10(8)) and (0.84x10(8), 1.83x10(8)), respectively.

Adolescent↗