Low dose radiation response curves, networks and pathways in human lymphoblastoid cells exposed from 1 to 10cGy of acute gamma radiation.

We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose-response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of ∼80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10cGy, some with suggestive evidence that transcription was modulated at doses below 1cGy. MYC, FOS and TP53 were the major network nodes of the low-dose-response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

Highly elevated PSA and dietary PhIP intake in a prospective clinic-based study among African Americans.

African-American men die from prostate cancer (PC) nearly twice as often as white US men and consume about twice as much of the predominant US dietary heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a genotoxic rat-prostate carcinogen found primarily in well-cooked chicken and beef. To investigate the hypothesis that PhIP exposure increases PC risk, an ongoing prospective clinic-based study compared PC screening outcomes with survey-based estimates of dietary PhIP intake among 40-70-year-old African-American men with no prior PC in Oakland, CA. They completed food-frequency and meat-cooking/consumption questionnaires and had a prostate-specific antigen (PSA) test and digital-rectal exam. Results for 392 men indicated a 17 (+/-17) ng/kg day mean (+/-1 s.d.) daily intake of PhIP, about twice that of white US men of similar age. PhIP intake was attributable mostly to chicken (61%) and positively associated (R(2)=0.32, P<0.0001) with saturated fat intake. An odds ratio (95% confidence interval) of 31 (3.1-690) for highly elevated PSA > or =20 ng/ml was observed in the highest 15% vs lowest 50% of estimated daily PhIP intake (> or =30 vs < or =10 ng/kg day) among men 50+ years old (P=0.0002 for trend) and remained significant after adjustment for self-reported family history of (brother or father) PC, saturated fat intake and total energy intake. PSA measures were higher in African-American men with positive family history (P=0.007 all men, P<0.0001 highest PSA quartile). These preliminary results are consistent with a positive association between PhIP intake and highly elevated PSA, supporting the hypothesis that dietary intervention may help reduce PC risk.

Gene expression changes in mouse brain after exposure to low-dose ionizing radiation.

PURPOSE: To characterize the cellular functions associated with the altered transcript profiles of mouse brain exposed to low-dose in vivo gamma-irradiation. MATERIALS AND METHODS: Cerebral RNA was isolated at 30 min and 4 h after whole-body irradiation at 0.1 or 2 Gy, hybridized to random oligonucleotide arrays, and evaluated for time and dose-response patterns by multifactorial analyses. RESULTS: Brain irradiation modulated the expression patterns of 1574 genes, of which 855 showed more than 1.5-fold variation. about 30% of genes showed dose-dependent variations, including genes exclusively affected by 0.1 Gy. About 60% of genes showed time-dependent variation with more genes affected at 30 min than at 4 h. Early changes involved signal transduction, ion regulation and synaptic signalling. Later changes involved metabolic functions including myelin and protein synthesis. Low-dose radiation also modulated the expression of genes involved in stress response, cell-cycle control and DNA synthesis/repair. CONCLUSIONS: Doses of 0.1 Gy induced changes in gene expression that were qualitatively different from those at 2 Gy. The findings suggest that low-dose irradiation of the brain induces the expression of genes involved in protective and reparative functions, while down-modulating genes involved in neural signalling activity.

Frequency of XY sperm increases with age in fathers of boys with Klinefelter syndrome.

With increasing availability of drugs for impotence and advanced reproductive technologies for the treatment of subfertility, more men are fathering children at advanced ages. We conducted a study of the chromosomal content of sperm of healthy men aged 24-57 years to (a) determine whether father's age was associated with increasing frequencies of aneuploid sperm including XY, disomy X, disomy Y, disomy 21, and sperm diploidy, and (b) examine the association between the frequencies of disomy 21 and sex-chromosomal aneuploidies. The study group consisted of 38 fathers of boys with Klinefelter syndrome (47, XXY) recruited nationwide, and sperm aneuploidy was assessed using multicolor X-Y-21 sperm FISH ( approximately 10,000 sperm per donor). Paternal age was significantly correlated with the sex ratio of sperm (Y/X; P=.006) and with the frequency of XY sperm (P=.02), with a clear trend with age by decades (P<.006). Compared with fathers in their 20s (who had an average frequency of 7.5 XY sperm per 10,000), the frequencies of XY sperm were 10% higher among fathers in their 30s, 31% higher among those in their 40s, and 160% higher among those in their 50s (95% CI 69%-300%). However, there was no evidence for age effects on frequencies of sperm carrying nullisomy sex; disomies X, Y, or 21; or meiosis I or II diploidies. The frequencies of disomy 21 sperm were significantly associated with sex-chromosomal aneuploidy (P=.04)-in particular, with disomy X (P=.004), but disomy 21 sperm did not preferentially carry either sex chromosome. These findings suggest that older fathers produce higher frequencies of XY sperm, which may place them at higher risk of fathering boys with Klinefelter syndrome, and that age effects on sperm aneuploidy are chromosome specific.

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.

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.

Minimization of heterocyclic amines and thermal inactivation of Escherichia coli in fried ground beef.

BACKGROUND: Heterocyclic amine carcinogens are formed during the cooking of a number of foods, especially well-done meats. Lower temperatures and shorter cooking times can minimize the formation of these carcinogens, yet a major food safety concern is that pathogens in the meat must be thermally inactivated. This study investigated cooking techniques that minimize heterocyclic amine formation while simultaneously destroying contaminating bacteria. METHODS: Ground beef patties were inoculated with Escherichia coli K12 bacteria and fried to internal temperatures ranging from 35 degrees C to 70 degrees C in a skillet preheated to 160 degrees C, 180 degrees C, or 200 degrees C. Each patty was then analyzed for four common heterocyclic amines and for surviving bacteria. Additionally, the frequency of turning of the beef patty during cooking was varied (a single turn or multiple turns), length of time required for each patty to reach 70 degrees C was recorded, and heterocyclic amine levels were determined. An additional pan temperature of 250 degrees C was tested for its effect on heterocyclic amine formation but not on bacterial killing. Statistical tests were two-sided. RESULTS: Colony-forming bacteria were reduced by five orders of magnitude at internal temperatures greater than 60 degrees C, regardless of cooking method, and were completely inactivated at 70 degrees C. For patties turned just once, heterocyclic amine levels increased as the cooking temperatures increased. However, levels of heterocyclic amines were statistically significantly lower with turning every minute. For each pan temperature, patties reached 70 degrees C internal temperature sooner when they were turned every minute than when they were turned just once during cooking. CONCLUSION: Lowering the pan temperature and turning the patties frequently can greatly reduce the formation of heterocyclic amines and can simultaneously achieve bacterial inactivation with little or no increase in cooking time, ensuring a product that is safe for human consumption.

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.

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.

Species and strain comparisons in the macromolecular binding of extremely low doses of [14C]benzene in rodents, using accelerator mass spectrometry.

The kinetics of macromolecular binding of a 5 micrograms/kg body wt dose of [14C]benzene was studied over 48 h in B6C3F1, DBA/2, and C57BL/6 mice and Fischer rats to determine if adduct levels reflect known differences in metabolic capacity, genotoxicity, and carcinogenic potency. Previous studies have suggested that differences in benzene toxicity among strains result from differences in metabolism. Rats and mice were administered [14C]benzene (i.p.), followed by removal of liver and bone marrow at time intervals up to 48 h postexposure. Protein and DNA were isolated and analyzed by accelerator mass spectrometry. Area under the curves for protein and DNA adducts in bone marrow were greatest in B6C3F1 mouse > DBA/2 mouse > C57BL/6 mouse > Fischer rat. These data are consistent with the hypothesis that metabolic capacity contributes to the difference in benzene's carcinogenicity among species. Additionally, these data suggest that target organ adduct levels correlate with tumorigenicity and thus may be indicative of an individuals risk.

Elevated frequencies of hypoxanthine phosphoribosyltransferase lymphocyte mutants are detected in Russian liquidators 6 to 10 years after exposure to radiation from the Chernobyl nuclear power plant accident.

This study was conducted to determine whether the frequency of hypoxanthine phosphoribosyltransferase (HPRT) deficient lymphocyte mutants would detect an effect of radiation exposure in a population of Russians who were exposed to low levels of radiation while working in 1986 and 1987 as liquidators cleaning up after the Chernobyl nuclear power reactor accident. The HPRT lymphocyte cloning assay was performed on peripheral blood lymphocytes collected between 1992 and 1996 from 142 liquidators and 66 Russian controls, and between 1989 and 1993 from 231 American controls. Russian and American controls were not significantly different for either cloning efficiency or mutant frequency (MF); inclusion of both sets of controls in the analysis increased the ability to detect a Chernobyl exposure effect in the liquidators. After adjusting for age and smoking, the results revealed no significant difference in cloning efficiency of Chernobyl liquidators relative to Russian controls but a significant, 24% increase in liquidator HPRT mutant frequency over Russian controls (90% confidence interval was 7% to 45% increase). The analytical method also accounted for differences in precision of the individual estimates of log CE and log MF and accommodated for outliers. The increase in HPRT mutant frequency of liquidators is an attribute of the exposed population as a whole rather than of individuals. These results demonstrate that, under appropriate circumstances, the HPRT specific locus mutation assay of peripheral blood lymphocytes can be used to detect a semi-acute, low dose radiation exposure of a population, even 6 to 10 years after the exposure.

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators.

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991-1992 Liquidators than in Russian Controls (chi 2 = 10.5, p = 0.001) and in 1993-1994 Liquidators (chi 2 = 8.3, p = 0.004), and was marginally elevated relative to 1995-1996 Liquidators (chi 2 = 3.3, p = 0.07). This type of mutations has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991-1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p = 0.009), not with increased MF in 1991-1992 Liquidators (p = 0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p = 0.0001), and also was elevated in Liquidators sampled in 1991-1992 (72%, p = 0.0076), 1993-1994 (22%, p = 0.037), and 1995-1996 (62%, p = 0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.

Covalent binding of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline to albumin and hemoglobin at environmentally relevant doses. Comparison of human subjects and F344 rats.

Covalent binding of the food-borne heterocyclic amine 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to albumin and hemoglobin (Hb), 3.5-6.0 hr after oral administration of a single dose of either 21.3 or 228.0 microg of [14C]MeIQx (304 and 3257 ng/kg of body weight, respectively, based on a 70-kg subject weight), was studied in human volunteers using accelerator mass spectrometry. Human protein adduct levels were compared with data obtained for male F344 rats 4.5 hr after oral administration of 0.94-11,420 ng/kg of body weight [14C]MeIQx. Dose-dependent levels of MeIQx-albumin and MeIQx-Hb adducts were detected in both humans and rats. In each case, the regression coefficient (slope) of the dose-response curve was approximately 1. The highest levels of adduct formation per unit dose of MeIQx occurred with human albumin, followed by rat albumin, human Hb, and rat Hb (in that order). Although the human subjects were elderly and underwent colon resection surgery during the study period, the results indicate that formation of albumin and Hb adducts is dose dependent and that a trend exists for higher adduct levels per unit dose in humans, compared with F344 rats. Furthermore, MeIQx-albumin adducts are likely to provide a more sensitive marker of exposure to MeIQx than are MeIQx-Hb adducts.

The accumulation of chromosome aberrations and Dlb-1 mutations in mice with highly fractionated exposure to gamma radiation.

The dichotomy between the doses at which experimental measurements of genetic effects can be made and the doses to which people are exposed is often different by two or more orders of magnitude. This presents a significant problem when determining the effects of low doses of radiation or chemicals. The solution has usually involved extrapolating the data by curve-fitting or by applying theoretical considerations. Both approaches are unsatisfactory due to uncertainties of the assumptions used in each process. The alternative solution has been to increase the sample size enormously at the lower doses. This is impractical beyond a certain point due to the variation in the spontaneous frequency and the need to quadruple the sample size for a doubling of precision. The development of new methods for measuring stable genetic effects, however, permits a simple and effective approach to this problem: if the genetic events being detected have no effect on survival, i.e., are selectively neutral, then the effects of multiple independent treatments will be additive. If the independent treatments are identical, then the effect of each is easily calculated by dividing the total effect by the number of treatments. Here we report a limited test of this approach using mice. Chromosome aberrations induced in lymphocytes and Dlb-1 mutations induced in the small intestine were measured after daily doses of 0.64, 1.85 or 5.5 cGy 137Cs gamma rays administered for 21, 42 or 63 days. The dose response curve for chromosome translocations obtained in this way, combined with the data from single larger acute doses, shows no evidence for a threshold over a 500-fold dose range. Dlb-1 mutations were increased at each dose and time but the results do not permit reliable extrapolations. The results suggest that translocations might be useful for quantifying the effect of doses below 0.05 cGy and that the effect of dose rate and dose fractionation at much lower doses than reported here could be investigated.

Persistence of radiation-induced translocations in human peripheral blood determined by chromosome painting.

We have investigated the persistence of translocations and other types of chromosome damage with time using human peripheral blood acutely exposed in vitro to 137Cs gamma rays at doses ranging from 0.5 to 4 Gy. Freshly drawn blood from one donor was irradiated and metaphase chromosomes were prepared 2 to 7 days after exposure. Chromosomes 1, 2 and 4 were painted red-orange and chromosomes 3, 5 and 6 were painted green by fluorescence in situ hybridization (FISH) using "semi-directly" labeled whole-chromosome painting probes. This type of labeling combines direct and indirect labeling and showed significant advantages over both these other methods. All types of structural chromosome aberrations were classified by the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) system. The yields of dicentric chromosomes, acentric fragments and ring chromosomes diminished with time as expected. Translocations exhibited greater persistence but showed a clear and statistically significant reduction in frequency at all doses. The mathematical model suggested that the translocation frequencies would reach a plateau of approximately 4, 15, 51, 106 and 179 translocations per 100 cell equivalents after irradiation with 0.5, 1, 2, 3 and 4 Gy, respectively. When translocations were classified by the conventional system, an analysis of the distribution of translocations and dicentrics per cell indicated that both types of exchanges were Poisson-distributed 48 h postirradiation. However, cells bearing translocations have a higher possibility of having dicentrics than cells without translocations. These findings suggest that dicentrics may contribute to a decline of translocation frequencies with time, and that some translocations are not completely persistent. The results obtained here using human blood exposed in vitro may influence the use of translocations as a retrospective biodosimeter of exposure to ionizing radiation in humans.

The limits of random fingerprinting.

Various random fingerprinting methods are sometimes used to detect overlap between pairs of clones as a first step toward producing a minimal tiling path of clones for subsequent mapping and sequencing efforts. This paper evaluates and compares various statistical procedures for detecting pairwise overlap between clones when the fingerprints arise from any random process meeting simple, plausible assumptions about the relationship between overlap and the resulting fingerprint. Examples of such random processes include, but are not limited to, large-scale hybridization procedures designed to prepare tiling paths of clones for subsequent large-scale genomic sequencing. Our goals are to assess how well random fingerprinting can possibly detect overlap, to assess the effects of inevitable fingerprinting errors on statistical detection, to determine how one can make the best use of the data random fingerprinting provides, and to evaluate how well simple, heuristic techniques for overlap detection compare to more complex, likelihood-based approaches. The paper provides a quantitative assessment of the ability of any random fingerprinting procedure to detect various proportions of clonal overlap and shows the extent to which a small amount of experimental error will vitiate the performance of such techniques. The paper outlines a simple approximation method for constructing Bayesian overlap detectors, while concluding that detectors constructed from linear combinations of fingerprint data can be designed that will perform nearly as well as more complex, likelihood-based approaches.

The four county study of childhood cancer: clusters in context.

Observations of childhood 'cancer clusters' in small communities in central California prompted us to examine the distribution of childhood cancer in communities throughout the region to see if the overall cancer rate or the distribution of 'cancer clusters' was unusual for agricultural towns where pesticide exposure might be elevated. The distribution of rates was evaluated using a variety of methods: comparison of rates to the regional average, evaluation of the empirical observed versus expected Poisson distribution of events, and multivariate modelling using Poisson regression. These analyses suggest that there were no previously undiscovered communities with excess rates, although the index community which prompted the initial investigation does stand out as unusual. We discuss the impact of a range of forces of morbidity on the likelihood of 'cancer clusters' and the distributions of observed and expected numbers of cancers in a population of locales.

A 30-Mb metric fluorescence in situ hybridization map of human chromosome 19q.

A high-resolution metric physical map of chromosome 19q has been constructed by fluorescence in situ hybridization. The map locates 136 cosmid reference points that span 30 Mb. The reference points are sequentially ordered from centromere to telomere, and the distance between neighboring cosmids is known from 240 partially overlapping, redundant estimates of genomic distances in kilobases separating pairs of cosmids. The average spacing between cosmid reference points is 220 kb, with over 75% of intervals less than 300 kb. Eighty-four genes and polymorphic markers have been assigned to mapped cosmids. The information on order and genomic distances separating pairs of cosmids, both key elements for building physical maps, has furthered the construction and integration of the genetic and physical maps of chromosome 19.