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J A Nicklas

Publications and source records attributed to J A Nicklas.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mitochondrial genome damage associated with cigarette smoking.

We have investigated the level of mitochondrial DNA (mtDNA) damage and deletions in bronchoalveolar lavage tissues from smokers and nonsmokers using quantitative, extra-long PCR and a "common" mtDNA deletion assay. Smokers had 5.6 times the level of mtDNA damage, 2.6 times the damage at a nuclear locus (beta-globin gene cluster), and almost 7 times the level of a 4.9-kb mtDNA deletion compared to nonsmokers, although the latter increase was not significant. Although both genomes (mitochondrial and nuclear) showed significantly increased levels of DNA damage in smokers (mtDNA P = 0.00072; beta-globin P = 0.0056), the relative differences were greatest in the mtDNA. Damage to the mtDNA may inhibit oxidative phosphorylation and, therefore, potentially cause or contribute to chronic lung disease and cancer. Consequently, the mtDNA may be a sensitive biomarker for environmentally induced genetic damage and mutation.

Adult↗
PubMed1996

Increased frequency of specific genomic deletions resulting from in vitro malathion exposure.

Malathion is a widely used pesticide with high potential for human exposure. Epidemiological studies suggest that individuals with chronic environmental exposures to pesticides have increased risks of various hematological malignancies. The genotoxic data to date have been somewhat inconclusive with regard to malathion exposure. We have used a cell cloning assay to study the genotoxicity of in vitro exposure of human T lymphocytes to malathion. We exposed cells in G0 to doses of malathion ranging from 10 to 600 microg/ml. Mutant frequencies of treated samples showed both intra- and interindividual variability and, in some cases, slight significant increases over the controls. Molecular analysis of hprt mutants resulting from both in vitro and an in vivo malathion exposure was performed by genomic multiplex PCR. In seven in vitro experiments (using cells from four different individuals) and one experiment on an individual exposed in vivo, one or more independent mutant(s) containing a partial deletion of exon 3 have been isolated from each individual. In five of the seven mutants, the deleted regions overlap extensively, revealing an area within exon 3 exceptionally prone to deletions upon exposure to malathion, This work provides the first evidence of an association between malathion exposure and specific mutations in human T lymphocytes. Additional work is necessary to determine the underlying molecular mechanism for these deletions and how this may relate to agricultural workers' increased risk of cancer.

Adult↗
PubMed1996

TCR beta PCR from crude preparations for restriction digest or sequencing.

T cell specificity is determined by the combinatorial association of specific variable (V), diversity (D), and junctional (J) regions. Clones of T cells (clonality) can occur, in the blood or in tissue, after proliferation of activated T cells. Determining clonality in mutation assays is necessary to distinguish between mutants and mutational events. We have developed a novel approach to determine clonality among T cell isolates, using restriction digests of PCR-amplified cDNA of the T cell receptor beta gene. The T cell receptor beta gene was PCR-amplified by use of a consensus primer, beginning from a cell pellet of 2,000-5,000 cells or from extracted RNA. This TCR (T cell receptor) beta chain PCR product can also be directly sequenced, allowing simple and easy identification of Vbeta and CDR3 sequence from a small number of cells. The utility of this method is demonstrated by PCR, restriction digest, and sequencing of the TCR beta cDNA from eight T cell clones isolated from 2 individuals. A clone of three identical isolates (one 3-mer) and a clone of two identical isolates (one 2-mer) were determined from restriction digests using two different enzymes. This new method is an easier and more rapid way of determining clonality than traditional methods, e.g., Southern blotting.

Base Sequence↗
PubMed1996

Hprt mutations in human T-lymphocytes reflect radioprotective effects of the aminothiol, WR-1065.

Aminothiols such as WR-2721 and its active free thiol WR-1065 have previously been shown to reduce mutations resulting from ionizing radiation in exponentially growing cells. In this study, non-dividing human G0 T-lymphocytes were exposed to the aminothiol radioprotective agent, WR-1065, 30 min before or 3 h after external beam gamma-irradiation and subsequently assessed for survival and mutation induction at the hprt locus. Cytotoxicity due to gamma-irradiation was reduced only when the WR-1065 was present during irradiation. The frequency of hprt mutations, however, was reduced regardless of time of administration, although the reduction was statistically significant only when WR-1065 was added 30 min before irradiation (P < 0.01). This is the first study to demonstrate the protective effects of WR-1065 against radiation-induced mutation in a reporter gene using a human non-cycling cell. Hprt mutations arising in vivo in these cells may be useful for monitoring the radioprotective effect of aminothiols in human populations.

Antimutagenic Agents↗
PubMed1996

Future research directions for evaluating human genetic and cancer risk from environmental exposures.

The utility of biomarkers for evaluating the genotoxicity of environmental exposures is well documented. Biomarkers of both exposure and effect provide bases for assessing human-genotoxicant interactions and may be indicative of future disease risk. At present, there is little information on the predictive value of these assays for either a population or the individuals tested. This paper describes some aspects of biomarker assays, the possible use of susceptibility measures in biomonitoring protocols, and the need for evaluation of disease relevance. A population study involving epidemiologists, geneticists, toxicologists, statisticians, and physicians is proposed to determine the disease relevance of these biomarkers.

Biomarkers↗
PubMed1995

Sensitivity of somatic mutations in human umbilical cord blood to maternal environments.

To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (Mf), and glycophorin A (GPA) variant erythrocytes (Vf) of both allele-loss (phi/N) and allele-loss-and-duplication (N/N) phenotypes in umbilical cord blood. The mean hprt Mf (1.40 +/- 1.11 x 10(-6), N = 66) and GPA Vf (phi/N 4.0 +/- 2.2 x 10(-6), N = 114; N/N 2.7 +/- 2.0 x 10(-6), N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt Mf was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 +/- 0.41 x 10(-6), N = 45, P < 0.01; t test, P < 0.01, Mann-Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt Mf (0.70 +/- 0.49 x 10(-6)) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann-Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated Mf revealed a significant decrease in the relative contribution of gross structural mutations to the overall Mf (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, chi 2 test), suggesting that the higher Mf resulted from an elevated level of "point" mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt Mf or GPA Vf.

Adult↗
PubMed1995

Analysis of point mutations in the hprt gene of cancer patients treated with radioimmunoglobulin therapy.

The mutagenic impact of various environmental and therapeutic agents can now be directly assayed in humans by the T-lymphocyte cloning assay. We have previously reported that following radioimmunoglobulin therapy, cancer patients exhibited increased mutant frequency of the hprt locus and an increased yield of large intergenic deletions compared to unexposed controls. Here we report the results of the analysis of 26 independent hprt mutations in nine cancer patients who underwent radioimmunoglobulin therapy. The majority of mutations (52%) had lost exon sequences from the mRNA. The remaining mutations were 20% small deletions and frameshifts and 28% base substitutions. The type of mutations observed were similar to those seen in unexposed controls. The site distribution of the mutations, however, indicates that some sequence contexts may be more sensitive to radiation mutagenesis than others.

Adult↗
PubMed1995

Breakpoints and junctional regions of intragenic deletions in the HPRT gene in human T-Cells.

DNA sequences of the deletion breakpoints of 24 human T-lymphocyte hprt gene mutations are reported. These independent deletions ranged in size from 18 to 15655 base pairs. Seven of the 21 in vivo mutations arose in normal adults, three in normal children, eight in radioimmunotherapy patients and three in platinum chemotherapy patients. One in vitro mutation was isolated after 93cGy radon exposure and two after 300cGy gamma radiation. The breakpoints were found to be non-random and a cluster of small deletions in exon 6 is reported. Ten of the mutations had 2-5bp direct repeats at the breakpoints. There was no excess of "deletion-associated" motifs over that expected by chance. Some breakpoints do occur at consensus topoisomerase II cleavage sites and the centromeric end of a Donehower sequence occurs exactly at a telomeric breakpoint. Three mutants had breakpoints at hairpins expected by the model of Glickman and Ripley.

Adult↗
PubMed1995

Physical mapping of the human hprt chromosomal region (Xq26).

The human hprt chromosomal region (Xq26) was physical-mapped using pulsed field gel electrophoresis (PFGE). This work involved: (i) the recovery of three new genomic DNA markers (DXS1327, DXS1328, and DXS1329), (ii) the ordering of new markers relative to 11 previously available hprt-linked markers by deletion mapping, and (iii) the completion of human T-lymphocyte PFGE Southern blots using the 14 Xq26 markers. A contiguous 1.5-Mb physical map of the region telomeric to hprt was determined. As this map identifies clusters of in vivo unmethylated rare-cutter restriction sites, potential CpG islands are revealed.

Adult↗
PubMed1995

Pulsed field analysis of hprt T-cell large deletions: telomeric region breakpoint spectrum.

In order to determine a large deletion breakpoint spectrum, 25 independent hprt T-lymphocyte mutants with deletions extending from hprt into the telomeric or centromeric flanking chromosomal region were analyzed by pulsed field gel electrophoresis (PFGE). PFGE was used to determine deletion sizes which allowed localization of breakpoints external to hprt to specific chromosomal positions in mutants containing an intra-hprt breakpoint. A breakpoint spectrum based on 19 large deletion mutants is reported for the Xq26 chromosomal region telomeric to hprt. A potential cluster of breakpoints (4/19) was observed approximately 60 kb from hprt. In addition, maximum recoverable deletion size was at least 3.5 Mb. Three of the 25 mutants analyzed appeared to be complex deletion events.

Chromosome Mapping↗
PubMed1994

Determination of hprt mutant frequencies in T-lymphocytes from a healthy pediatric population: statistical comparison between newborn, children and adult mutant frequencies, cloning efficiency and age.

Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning assay in a healthy pediatric population. Assays were performed on 49 subjects (29 males and 20 females) ranging in age from 0.08 to 15.2 years. A statistical analysis of the thioguanine-resistant (TGr) mutant frequency (MF), unselected cloning efficiency (CE) and age was performed using data obtained in this study and those previously obtained in our laboratory on 66 newborn umbilical cord blood samples and 230 adult blood samples. For statistical comparisons pediatric subjects were divided into 4 groups. Group I included cord blood samples (age 0 years); Group II were subjects between 0 and 5 years; Group III were between 6 and 11 years and Group IV were between 12 and 17 years. The ln MF of Groups I and II were significantly lower than Groups III and IV (p < 0.05). The mean ln MF for each of Groups I-IV was significantly lower than the adult value. The cloning efficiency for Group I was significantly lower than that for Groups II-IV and adults. The relationships among the ln MF, unselected CE and age were expressed by the equations: ln (MF) = 0.945 -2.453 CE (p < 0.001) and ln (MF) = 0.114 + 0.063 age (p 0.004). The slope coefficients for unselected CE and age were significantly different from adults (p < 0.05). Regression analysis of combined data from Groups I-IV and adults were performed using both age and unselected CE as well as terms to reflect differences in their relationships with ln MF in adults and children. The results showed that the intercept and the age coefficients differ significantly for children and adults after adjustment for CE and yielded the following equations: ln (MF) = 0.548 -1.676 CE + 0.075 age, (Groups I-IV) and ln (MF) = 2.263 -1.676 CE + 0.014 age (adults). An alternative statistical model using ln (age ), ln (MF) = 0.381 -1.767 CE + 0.673 ln (age + 1), (p < 0.001), describes the rapid increase in MF with age that levels off in late adolescence. These findings demonstrate the changing influence of age on mutant frequency in the pediatric population as compared to the adult populations. These studies also illustrate that the increase in background somatic mutant frequencies at the hprt locus in T-lymphocytes is not linear from birth to adolescence and is significantly different from that seen in the adult population.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1993

Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations.

Somatic cell mutant frequencies at the hprt locus of the X-chromosome were measured with the T-lymphocyte cloning technique in healthy human populations. A statistical analysis was performed of assays from 232 individuals (77 males and 155 females) ranging in age from 19 to 80 years. Data from 4 donor groups were compiled: (a) 132 participants in a study of identical and fraternal twins; (b) 17 health care workers studied as part of an assessment of the risks of handling chemotherapeutic drugs; (c) 62 women with benign breast masses; and (d) 21 normal laboratory and office personnel. The relationship between age and mutant frequency (MF) was expressed by the equation: ln MF = 1.46 + 0.018 age (P < 0.001). Thus, MF increased by about 2% per year. Increases in cloning efficiency (CE) reduced the MF, as shown in the equation: ln MF = 2.91 - 1.32 CE (P < 0.001). CE was significantly related to age (CE = 0.47 - 0.002 age, P = 0.038), and the interdependent relationship between MF, age and CE expressed by the equation: ln MF = 1.99 - 1.13 CE + 0.016 age was significant at the P < 0.001 level. There was no statistically significant effect of donor gender or smoking history on MF in our population, but CE was significantly lower in males (P < 0.001). These findings confirm the importance of age and CE as factors which influence the thioguanine-resistant MF in circulating T-lymphocytes from normal adults.

Adult↗
PubMed1993

Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH-DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH-DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH-DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Aberrations↗
PubMed1993

Somatic cell gene mutations in humans: biomarkers for genotoxicity.

Somatic cell gene mutations arising in vivo in humans provide biomarkers for genotoxicity. Four assays, each measuring changes in a different "recorder" gene, are available for detecting mutations of the hemoglobin (Hb) and glycophorin A (gpa) genes in red blood cells and the hypoxanthine-guanine phosphoribosyltransferase (hprt) and HLA genes in T-lymphocytes. Mean adult background mutant frequencies have been established; i.e., approximately 4 x 10(-8) (Hb), 5-10 x 10(-6) (hprt), 10-20 x 10(-6) (gpa) and 30 x 10(-6) (HLA). All the assays have now been used in studies of individuals exposed to physical and/or chemical genotoxic agents, and all have shown elevated values following exposures; examples are presented. In addition to quantitation, the lymphocyte assays allow molecular analyses of in vivo mutations, the definition of background and induced mutational spectra, and the search for unique changes for characterizing specific mutagens. The HPRT system currently has the largest database in this regard. Approximately 15% of adult background hprt mutations are due to gross structural alterations (primarily deletions) having random breakpoints; 85% result from "point" changes detected only by sequencing. In contrast, a specific intragenic deletion due to DNA cleavage at specific sites characterizes fetal hprt mutations, implicating a developmental mistake in their genesis. (This kind of developmental mistake in other genes is frequently observed in lymphoid malignancies.) Mutational spectra are just beginning to be defined for induced hprt mutations, e.g., ionizing radiation produces large deletions.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocytes↗
PubMed1993

In vivo mutations in human blood cells: biomarkers for molecular epidemiology.

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) < hprt (5 x 10(-6)) = GPA (10 x 10(-6)) < HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1992

V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.

Adult↗
PubMed1991

V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes.

Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human T-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only approximately 15% of hypoxanthine-guanine phosphoribosyltransferase mutations have structural alterations on Southern blots, while in the fetus 75% of mutations show alterations of which one-half are deletions of exons 2 and 3. We have now sequenced the breakpoint sites for these specific deletions in 18 mutant lymphocyte clones isolated from 13 normal newborns. Three classes of deletions were found. Each class had the same intron 1 breakpoint but a different intron 3 breakpoint. These mutations have all the signatures of a V(D)J recombinase-mediated event (a 5' consensus heptamer, 3' consensus heptamer and nonamer, nibbling, non-germline-encoded nucleotides, P-nucleotides). At the 3' breakpoint of the most common class (comprising 83% of the mutants) a perfect heptamer can be created by postulating a hairpin loop which could attain a Z-DNA configuration. This feature may indicate recombinase preference for certain DNA structures. These results implicate the V(D)J recombinase in illegitimate events causing mutation in this housekeeping gene during T-cell development. Inactivation of genes involved in the control of growth and differentiation (e.g., tumor suppressor genes) by this mechanism may have important implications for cancer development.

Base Sequence↗