Multianalyte profiling of serum cytokines for detection of pancreatic cancer.

Early detection of pancreatic cancer might improve clinical outcome. Significant alterations in the levels of individual serum cytokines have been reported in pancreatic cancer. We hypothesized that a multicytokine panel could serve as biomarkers for pancreatic cancer. To evaluate the diagnostic utility of such a panel, we have utilized a novel multianalyte LabMAP profiling technology that allows simultaneous measurement of multiple markers. In this study, a panel of 31 serological markers including cytokines, chemokines, growth and angiogenic factors in combination with CA 19-9 was analyzed in sera of pancreatic cancer patients, patients with chronic pancreatitis, and matched control healthy subjects. Statistical analysis identified a multicytokine panel that was able to distinguish pancreatic cancer from healthy controls with a sensitivity of 85.7% and specificity of 92.3%, which was superior to performance of CA 19-9 alone. Importantly, a multicytokine panel allowed the discrimination of pancreatic cancer from chronic pancreatitis with high sensitivity of 98% and specificity of 96.4%. In conclusion, we demonstrated that analysis of multiple serum cytokines using a novel LabMAP technology is a promising approach for development of a diagnostic assay for pancreatic cancer.

Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation.

Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that gamma irradiation with doses of 2-50 Gy from (137)Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.

Genetic instability and hematologic disease risk in Werner syndrome patients and heterozygotes.

Werner syndrome (WRN) is an uncommon autosomal recessive disease in which progeroid features are associated with genetic instability and an elevated risk of neoplasia. We have used the glycophorin A (GPA) somatic cell mutation assay to analyze genetic instability in vivo in WRN patients and heterozygotes. GPA variant frequencies were determined for 11 WRN patients and for 10 heterozygous family members who collectively carry 10 different WRN mutations. Genetic instability as measured by GPA O/N allele loss variant frequency was significantly increased, and this increase was strongly age-dependent in WRN patients. GPA O/N allele loss variants were also significantly elevated in heterozygous family members, thus providing the first evidence for in vivo genetic instability in heterozygous carriers in an autosomal recessive genetic instability syndrome. Our results and comparable data on other human genetic instability syndromes allow an estimate of the level of genetic instability that increases the risk of human bone marrow dysfunction or neoplasia.

Biomonitoring of human genotoxicity induced by complex occupational exposures.

Sensitivity, specificity and correlations among several biomarkers for monitoring occupational exposure to complex mixtures of genotoxic agents were assessed in occupational environments in Hungarian study populations. The studies have been focused on DNA adduct formation, urinary metabolites, mutations and micronuclei induced by exposures to complex organic mixtures. In two Hungarian aluminium plants, increased DNA adduct and 1-hydroxypyrene (1-OH-PY) levels were observed in workers as compared to controls. However, no association between the biomarker levels was evident on an individual basis. In Hungarian garage mechanics, DNA adduct determinations did not show increased genotoxic exposure as compared to the controls. However, ambient air measurements, significantly enhanced 1-OH-PY levels, and slightly enhanced frequency of micronuclei indicated increased polycyclic aromatic hydrocarbon (PAH) exposure in the garages, as compared to the general environment. In a Hungarian vulcanizing plant, DNA adduct determinations and 1-OH-PY did not show significantly elevated exposure levels as compared to controls. The glycophorin A (GPA) somatic mutation assay was also negative for this occupational exposure. The results support previous observations of a lack of correlation between DNA adducts detectable by 32P-postlabelling and those measured by the PAH-DNA immunoassay in the same DNA sample. These studies also demonstrate a lack of close correlation between levels of DNA adducts and urinary 1-OH-PY in the same individual.

Impact of maternal lifestyle factors on newborn HPRT mutant frequencies and molecular spectrum--initial results from the Prenatal Exposures and Preeclampsia Prevention (PEPP) Study.

Epidemiological studies have demonstrated associations between maternal tobacco smoke exposure and consumption of alcohol during pregnancy and increased risk of pediatric malignancies, particularly infant leukemias. Molecular evidence also suggests that somatic mutational events occurring during fetal hematopoiesis in utero can contribute to this process. As part of an ongoing multi-endpoint biomarker study of 2000 mothers and newborns, the HPRT T-lymphocyte cloning assay was used to determine mutant frequencies (Mf) in umbilical cord blood samples from an initial group of 60 neonates born to a sociodemographically diverse cohort of mothers characterized with respect to age, ethnicity, socioeconomic status, and cigarette smoke and alcohol exposure. Non-zero Mf (N = 47) ranged from 0.19 to 5.62 x 10(-6), median 0.70 x 10(-6), mean +/- SD 0.98 +/- 0.95 x 10(-6). No significant difference in Mf was observed between female and male newborns. Multivariable Poisson regression analysis revealed that increased HPRT Mf were significantly associated with maternal consumption of alcohol at the beginning [Relative Rate (RR) = 1.84, 95% CI = 0.99-3.40, P = 0.052) and during pregnancy (RR = 2.99, 95% CI = 1.14-7.84, P = 0.026). No independent effect of self-reported active maternal cigarette smoking, either at the beginning or throughout pregnancy, nor maternal passive exposure to cigarette smoke was observed. Although based on limited initial data, this is the first report of a positive association between maternal alcohol consumption during pregnancy and HPRT Mf in human newborns. In addition, the spectrum of mutations at the HPRT locus was determined in 33 mutant clones derived from 19 newborns of mothers with no self-reported exposure to tobacco smoke and 14 newborns of mothers exposed passively or actively to cigarette smoke. In the unexposed group, alterations leading to specific exon 2-3 deletions, presumably as a result of illegitimate V(D)J recombinase activity, were found in five of the 19 mutants (26.3%); in the passively exposed group, two exon 2-3 deletions were present among the seven mutants (28.6%); and in the actively exposed group, six of the seven mutants (85.7%) were exon 2-3 deletions. Although no overall increase in HPRT Mf was observed and the number of mutant clones examined was small, these initial results point to an increase in V(D)J recombinase-associated HPRT gene exon 2-3 deletions in cord blood T-lymphocytes in newborns of actively smoking mothers relative to unexposed mothers (P = 0.011). Together, these results add to growing molecular evidence that in utero exposures to genotoxicants result in detectable transplacental mutagenic effects in human newborns.

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults.

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2 + 3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (< 0.049 x 10(-7)). The frequency of HPRT exons 2 + 3 deletions was 0.10 x 10(-7) per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%-70%) and previous results using the T-cell cloning assay (approximately 2-3 x 10(-7) per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7 x 10(8) leukocytes analyzed), and which occurred at frequencies from < 1 x 10(-7) (undetected) to 1.6 x 10(-4) among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2 + 3 deletions in the leukocytes of the newborns, from 0.07 x 10(-7) to 0.23 x 10(-7). We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2 + 3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.

Overexpression of the human manganese superoxide dismutase (MnSOD) transgene in subclones of murine hematopoietic progenitor cell line 32D cl 3 decreases irradiation-induced apoptosis but does not alter G2/M or G1/S phase cell cycle arrest.

To determine whether overexpression of the human MnSOD transgene protected 32D cl 3 hematopoietic progenitor cells from ionizing irradiation, 32D cl 3 cells were co-electroporated with the pRK5 plasmid containing the human MnSOD transgene and SV2-neo plasmid with G418-resistant colonies selected. Two clones (1F2 and 2C6) were identified to overexpress the human MnSOD transgene by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and increased biochemical activity. Measurement of irradiation-induced damage was determined in cells removed from G418 for 1 week before irradiation. Irradiation survival curves, apoptosis tunnel assay, and Comet assay was performed. Cell cycle distribution was determined for each line at 0, 1, 3, 6, 24, and 48 hr after 500 cGy by fixing the cells in 70% ethanol, staining with propidium iodide, and analysis by flow cytometer. Biochemical MnSOD activity in U/mg protein was 2.6 for 32D cl 3 and significantly elevated to 8.4 and 6.6 (P < 0.001) U/mg protein for subclones 1F2 and 2C6, respectively. Irradiation survival curves demonstrated an increased shoulder on the irradiation survival curve for 1F2 and 2C6 cells with an n of 4.95 +/- 0.48 (P = 0.042) and 4.95 +/- 0.13 (P = 0.011), compared with 2.77 +/- 0.20 for 32D cl 3. A higher percent of 32D cl 3 cells demonstrated apoptosis at 24 and 48 hr after 1,000 cGy irradiation, compared with 1F2 and 2C6 cells (at 24 hr, 29.37% +/- 2.01% of 32D cl 3 cells were apoptotic compared with 5.21 +/- 2.61 (P = 0.018) and 5.27 +/- 2.58 (P = 0.004) for 1F2 and 2C6, respectively). Significantly more DNA strand breaks were detected by Comet assay in 32D cl 3 cells (Comet length at 600 cGy of 103.4 +/- 50.3 units, compared with 69.7 +/- 36.3 (P < 0.001) and 48.9 +/- 27.5 (P < 0.001) for 1F2 and 2C6, respectively). In contrast, irradiation-induced cell cycle arrest was similar between the cell lines with a G2/M phase arrest at 6 hr and a G1/S phase arrest at 24 and 48 hr after irradiation. While overexpression of MnSOD increases the shoulder on the irradiation survival curve of 32D cl 3 cells, decreases irradiation-induced apoptosis, and DNA strand breaks by Comet assay, irradiation-induced alterations in cell cycle distribution were not significantly altered. These 32D cl 3 subclonal lines overexpressing MnSOD provide a potentially valuable system with which to study the mechanism of irradiation-induced cell cycle arrest separate from irradiation-induced apoptosis.

Human in vivo somatic mutation measured at two loci: individuals with stably elevated background erythrocyte glycophorin A (gpa) variant frequencies exhibit normal T-lymphocyte hprt mutant frequencies.

A survey of glycophorin A (gpa) in vivo somatic cell mutation in a population of 394 healthy people from 8 to 77 years of age (mean age +/- SD 41 +/- 15 years) revealed a subset of 37 individuals with stably elevated allele-loss and/or allele-loss with duplication variant erythrocyte frequencies (Vf) exceeding 30 x 10(-6). These 37 individuals with gpa outlier Vf are significantly older (P < 0.001) than the remainder of the larger study population from which they were drawn reflecting a highly significant increase in the prevalence of these individuals in the population beyond age 40 years. A study of hpt mutant frequencies (Mf) in the peripheral blood T-lymphocytes of 27 of these individuals, together with 15 matched control individuals with unremarkable gpa Vf, was undertaken to determine if these subjects also displayed elevated mutation frequencies at this independent locus indicative of globally elevated somatic mutation. The hprt Mf in these 27 subjects (geometric mean 11.5 x 10(-6)(dispersion interval 5.8 x 10(-6) to 22.8 x 10(-6)) was not significantly different from that observed in the 15 controls (geometric mean 12.1 x 10(-6)(dispersion interval 5.7 x 10(-6) to 25.5 x 10(-6)). These Mf are higher than typically reported values reflecting the older age distribution of these individuals (arithmetic mean age +/- SD 53 +/- 12 and 50 +/- 16 years for the subjects and controls, respectively). Taken together, these data suggest that several genetic mechanisms may be responsible for producing the gpa outlier Vf observed in these subjects. The observation that hprt Mf were not increased indicates that the majority did not arise by a genome-wide increased rate of somatic mutation detectable at both loci. The fixation and subsequent expansion of 'jackpot' mutations at the gpa locus occurring early in embryonic/fetal development also does not appear to be a predominant mechanism. Some cases may result from a stable over-representation of gpa variant cells, perhaps associated with a marked age-dependent decrease in the number of contributing erythroid stem cells in the bone marrow. The subset that displays elevated allele-loss with duplication Vf involving both gpa alleles may represent individuals with increased rates of somatic recombination. Elevations arising by this mechanism are not detected in the hprt assay, but could be confirmed using a autosomal locus in vivo somatic cell mutation endpoint such as the hla-a assay. Of primary biological significance, these results demonstrate that genetics/stochastic processes leading to the loss of heterozygosity of somatic cells occur ubiquitously in humans and in some individuals this level of somatic mosaicism can approach a frequency of 10(-3) at the gpa locus in erythroid lineage cells.

Glycophorin A as a biological dosimeter for radiation dose to the bone marrow from iodine-131.

The frequency of peripheral blood erythrocyte variants exhibiting allelic loss of glycophorin A (N/M antigen) has been used previously as a biological dosimeter to assess somatic mutations in bone marrow cells from external whole-body irradiation. The aim of the present study was to determine whether this marker could be used as a measure of bone marrow genotoxicity induced by 131I in the treatment of thyroid cancer. Flow cytometry of immunolabeled erythrocytes was performed to enumerate glycophorin A variants before and after eight therapy doses of 131I administered to five patients with differentiated thyroid carcinoma. Bone marrow radiation exposure from each dose was calculated from the integrated retention of 131I in the whole body and in the blood. In addition, the accumulated dose to the bone marrow received from earlier 131I therapy was calculated for each patient. Regression analysis was performed on the frequency of two glycophorin A variant cell types (N/O and N/N) as a function of accumulated dose to the bone marrow. Frequency of N/O variant cells showed a significant dose-related increase with a slope of 10.9 x 10(-6) per sievert. This dose effect is about one-half that previously observed after whole-body external irradiation at high dose rate. This decreased response could be explained by the low dose rate of the radiation to the bone marrow from 131I.

Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay.

The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method.

Thyroid nodularity and cancer among Chernobyl cleanup workers from Estonia.

Thyroid examinations, including palpation, ultrasound and, selectively, fine-needle aspiration biopsy, were conducted on nearly 2,000 Chernobyl cleanup workers from Estonia to evaluate the occurrence of thyroid cancer and nodular thyroid disease among men with protracted exposure to ionizing radiation. The examinations were conducted in four cities in Estonia during March-April 1995, 9 years after the reactor accident. The study population was selected from a predefined cohort of 4,833 cleanup workers from Estonia under surveillance for cancer incidence. These men had been sent to Chernobyl between 1986 and 1991 to entomb the damaged reactor, remove radioactive debris and perform related cleanup activities. A total of 2,997 men were invited for thyroid screening and 1,984 (66%) were examined. Estimates of radiation dose from external sources were obtained from military or other institutional records, and details about service dates and types of work performed while at Chernobyl were obtained from a self-administered questionnaire. Blood samples were collected for assay of chromosomal translocations in circulating lymphocytes and loss of expression of the glycophorin A (GPA) gene in erythrocytes. The primary outcome measure was the presence or absence of thyroid nodules as determined by the ultrasound examination. Of the screened workers, 1,247 (63%) were sent to Chernobyl in 1986, including 603 (30%) sent in April or May, soon after the accident. Workers served at Chernobyl for an average of 3 months. The average age was 32 years at the time of arrival at Chernobyl and 40 years at the time of thyroid examination. The mean documented radiation dose from external sources was 10.8 cGy. Biological indicators of exposure showed low correlations with documented dose, but did not indicate that the mean dose for the population was higher than the average documented dose. Ultrasound examinations revealed thyroid nodules in 201 individuals (10.2%). The prevalence of nodules increased with age at examination, but no significant associations were observed with recorded dose, date of first duty at Chernobyl, duration of service at Chernobyl, building the sarcophagus or working on the roof of neighboring buildings or close to the damaged reactor. Nodularity showed a nonsignificant (p(1) = 0.10) positive association with the proportion of lymphocytes with chromosome translocations, but associations with the frequency of variant erythrocytes in the GPA assay were weak and unstable (p(1) > or = 0.46). The majority of fine-needle biopsies taken on 77 study participants indicated benign nodular disease. However, two cases of papillary carcinoma and three benign follicular neoplasms were identified and referred for treatment. Both men with thyroid cancer had been sent to Chernobyl in May of 1986, when the potential for exposure to radioactive iodines was greatest. Chernobyl cleanup workers from Estonia did not experience a markedly increased risk of nodular thyroid disease associated with exposure to external radiation. Possible reasons for the apparent absence of effect include low radiation doses, the protracted nature of the exposure, errors in dose measurement, low sensitivity of the adult thyroid gland or the insufficient passage of time for a radiation effect to be expressed.

Radiobiological evaluation of immigrants from the vicinity of Chernobyl.

Eighty individuals (55 adults and 25 children) who were residents of four cities (Kiev, Mozyr, Gomel and Bobrujsk) located 100-200 km from Chernobyl at the time of the accident in 1986 were tested after immigrating to the US from 1989-1991. A whole-body counter was employed to quantitate radiocesium content. In addition, two biological measures of radiation effects, namely, chromosomal integrity using the micronucleus assay and somatic mutation analysis of erythrocytes at the glycophorin A (GPA) locus, were applied to this group. Radiocesium activity in the body ranged from 0 to 56.8 Bq/kg with a mean and standard deviation of 5.0 +/- 8.2 and a median value of 2.0 Bq/kg. Mean radiocesium content by groups was highest in adult males (9.0 +/- 11.7; range 0.21-56.8 Bq/kg) followed by adult females (3.3 +/- 4.5; range 0-21.3 Bq/kg), male children (3.0 +/- 5.7; range 0-20.2 Bq/kg) and lowest in female children (1.6 +/- 3.5; range 0-12.7 Bq/kg). Individuals with the highest radiocesium content in each group belonged to one family that lived in Mozyr (100 km from Chernobyl) until emigrating in 1989. The frequency of lymphocyte micronuclei and erythrocyte GPA allele-loss (O/N) somatic mutations were both significantly correlated with radiocesium content (r=0.57, p=0.002; r=0.75, p=0.002, respectively). The micronucleus frequency also correlated with the estimated internal absorbed dose from radiocesium in a subset of 20 immigrants for whom this calculation was possible (r=0.71, p=0.0005). Altogether, the biomonitoring data indicate that some subjects had radiation doses sufficient to produce gene and chromosomal mutations in blood cells, although these effects cannot be attributed solely to radiocesium exposure.

Glycophorin A somatic cell mutation frequencies in Finnish reinforced plastics workers exposed to styrene.

We have used the glycophorin A (GPA) in vivo somatic cell mutation assay to assess the genotoxic potential of styrene exposure in 47 reinforced plastics workers occupationally exposed to styrene and 47 unexposed controls matched for age, gender, and active smoking status. GPA variant erythrocyte frequencies (Vf), reflecting GPA allele loss (phi/N) and allele loss and duplication (N/N) somatic mutations arising in vivo in the erythroid progenitor cells of individuals of GPA M/N heterozygous genotype, were flow cytometrically determined in peripheral blood samples from these subjects. Measurements of styrene exposure of the workers at the time of blood sampling showed a mean 8-h time-weighted average (TWA8-h) styrene concentration of 155 mg/m3 (37 ppm) in the breathing zone. Mean urinary concentrations of the styrene metabolites mandelic acid (MA) and mandelic acid plus phenyl glyoxylic acid (MA+PGA) were 4.4 mmol/liter (after workshift) and 2.1 mmol/liter (next morning), respectively. Multivariate analysis of covariance on log-transformed GPA Vf data with models allowing adjustment for age, gender, smoking status, and styrene exposure showed that N/N Vf were nearly significantly increased among all of the exposed workers (adjusted geometric mean, 6.3 per million versus 5.0 in the controls; P = 0.058) and were statistically significantly elevated (adjusted geometric mean, 6.8 versus 5.0 in the controls; P = 0.036) among workers classified into a high-exposure group according to personal TWA8-h concentration of styrene in the breathing zone of > or = 85 mg/m3 (20 ppm; Finnish threshold limit value). Women in this high exposure group showed especially elevated N/N Vf (adjusted geometric mean 8.5 versus 5.3 in control women; P = 0.020); this elevation was also significant if urinary MA+PGA of > or = 1.2 mmol/liter was used as the basis of classification (adjusted geometric mean, 8.3; P = 0.030). The occupational exposure could not be shown to influence phi/N Vf. Cigarette smoking was associated with significantly elevated GPA Vf among active smokers (P = 0.042 for phi/N and P = 0.020 for N/N) and among active and ex-smokers combined (P = 0.014 for N/N). Its influence on phi/N Vf was especially clear among active smokers in the control group (P = 0.005). An effect of smoking, nearly statistically significant, was also observed for the phi/N Vf of control ex-smokers (P = 0.055) and of all active and ex-smokers combined (P = 0.050). Thus, the two characterized chemical exposures experienced by this group of workers and controls appear to produce differential effects on the two independent classes of GPA variants enumerated in the assay. This result suggests that the genotoxicity of these agents is mediated, at least in part, by different genetic mechanisms. Styrene exposure is associated with a specific increase in GPA N/N Vf; these allele loss and duplication variants reflect predominantly somatic recombination mechanisms in erythroid progenitor cells. Tobacco smoke exposure in active and ex-smokers is also associated not only with an increase in N/N Vf but also with an increase in phi/N Vf, reflecting the induction of GPA gene-inactivating mutations, including point mutations and deletions. This finding is consistent with a broad mechanistic spectrum of tobacco smoke genotoxicity associated with this complex mixture of chemical mutagens. Finally, there was no detectable effect of age on phi/N Vf; however, a highly significant (P = 0.0002) increase in N/N Vf with age, even after adjustment for other variables, was observed.

Direct immunofluorescence labeling provides an improved method for the glycophorin A somatic cell mutation assay.

The glycophorin A (GPA) somatic cell mutation assay is being applied widely as an in vivo biomarker in molecular epidemiologic studies of human populations. The assay uses two-color immunolabeling and flow cytometry of peripheral blood samples to enumerate allele-loss variant erythrocytes that appear as a result of mutations at the GPA locus in bone marrow erythroid cells. We have developed an improved version of the assay in which both anti-GPA monoclonal antibodies are directly conjugated with distinguishable fluorophores, fluorescein and phycoerythrin. Parallel analyses of 77 blood samples using the existing BR6 assay and our new DB6 assay demonstrate that the DB6 assay produces cleaner bivariate flow histograms with generally lower variant cell frequencies and lower coefficients of variation on replicate analyses of individual blood samples. With the BR6 assay, an artifact was shown to exist that results in enumeration of high frequencies of variant erythrocytes in a small fraction of samples that have been subjected to poor shipping and/or storage conditions. Using DB6, these same samples display acceptable histograms and low frequency of variant cells. High speed cell sorting followed by immuno analysis indicates that the BR6 artifact results from inhibited binding of the very high molecular weight antibody plus secondary reagent, which is used for the BR6 assay. We therefore recommend that DB6 be adopted as standard protocol for the GPA assay, and to assist other researchers in standardization and comparison, we are making available a set of calibrated, fixed blood samples.

Elevated frequency of glycophorin A mutations in erythrocytes from Chernobyl accident victims.

In 1986, when an explosion accident occurred at the Chernobyl, Ukraine nuclear power plant, a large number of people were exposed to significant amounts of ionizing radiation. During the time between 1986 and 1992, peripheral blood samples were obtained from 102 people who either were on site during the emergency or were brought to Chernobyl shortly thereafter to assist in the cleanup of radioactive contaminants and isolate the damaged reactor from the environment. These blood samples plus samples from 13 unexposed Soviet individuals were analyzed by flow cytometry using the allele-loss somatic mutation assay for glycophorin A. Results of these assays show that the frequency of N/O variant red cells increased in proportion to the estimated radiation exposure of each individual. The radiation dose-response function derived from this population closely resembles that determined previously for atomic bomb survivors whose blood samples were obtained and analyzed 40 years after their exposure. This suggests comparable mutation induction per unit dose for these two populations and long-term persistence of the mutational damage. In addition, measurements on multiple blood samples from each of 10 donors taken over a 7-year period showed no significant changes in N/O variant cell frequencies, confirming the persistence of radiation-induced somatic mutations in long-lived bone marrow stem cells.

Familial clustering of breast cancer: possible interaction between DNA repair proficiency and radiation exposure in the development of breast cancer.

The capacity of peripheral blood lymphocytes to repair X-ray-induced DNA damage, manifest as chromatid damage 30-90 min after G2-phase X-irradiation, was measured among available members of a family exhibiting a cluster of breast-cancer cases occurring in one generation. The cancer patients had been exposed to repeated chest fluoroscopic examinations during early childhood and adolescence. The development of breast cancer was correlated with DNA repair proficiency and history of radiation exposure. The results of the family study provide preliminary support for the hypothesis that a deficiency in repair of X-irradiation DNA damage may be a susceptibility factor for the development of breast cancer. This hypothesis, however, requires confirmation in a larger study. Studying the combined effect of susceptibility factors and environmental exposures may enhance our knowledge of the etiology of breast cancer and provide leads for effective prevention strategies aimed at reducing exposures or altering susceptibility to unavoidable exposures.

Molecular characterization of glycophorin A transcripts in human erythroid cells using RT-PCR, allele-specific restriction, and sequencing.

Glycophorin A (GPA) is an erythroid-lineage-specific membrane sialoglycoprotein which occurs in two allelic forms, M and N, which form the antigens of the MN blood group. Purified cDNAs and RNAs isolated from peripheral blood and erythroleukemia cell lines, HEL and K562, were used to develop an RT-PCR technique for amplifying GPA gene transcripts (GYPA). The relative expression of transcripts from the M and N alleles was determined using restriction analysis of these amplified products with four allele-specific restriction endonucleases. The use of this method permits the sensitive identification of GYPA transcripts in these cells and confirms GPA protein expression in the erythroleukemia cell lines and the MN phenotypes of individuals determined by immunolabeling with GPA allele-specific monoclonal antibodies. A novel restriction pattern was obtained using peripheral blood RNA from two individuals with a rare inherited variant allele, GPA Mg. Sequencing of the cDNA obtained using this method revealed a single C to A transversion in the fourth codon in the mature GYPA N coding sequence is responsible for the difference between GYPA Mg and GYPA N.

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.