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.

Intracellular recordings from thermosensitive preoptic neurons.

Intracellular recordings were made from locally thermosensitive preoptic neurons in the green sunfish, Lepomis cyanellus. Stable resting potentials, action potentials, and spontaneous synaptic activity were observed over approximately 4 degrees to 5 degrees C changes in local brain temperature. A small percentage of the warm-sensitive neurons showed exponential firing-rate responses to temperature. These cells discharged rhythmically, lacked visible synaptic input, and showed slowly depolarizing potentials leading to action potentials. Other linear and nonlinear warm-sensitive and cold-sensitive neurons showed spontaneous excitatory and inhibitory synaptic potentials giving rise to action potentials. Cells that appear to be endogenously active may be true thermodetectors, and other thermosensitive neuronal activity may be synaptically mediated.

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.

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.

Sequence diversity and chromosomal distribution of "young" Alu repeats.

Members of the recently inserted human-specific (HS)/predicted variant (PV) subfamily of Alu elements were sequenced. A number of these Alu elements share greater than 98% sequence identity with the subfamily consensus sequence, and they are flanked by perfect 5' and 3' direct repeats ranging in size from 6 to 15 nucleotides (nt). Based on the low number of random mutations, the estimated average age of these elements was calculated to be 1.5 million years (Myr). All the young Alu subfamily members were restricted to the human genome, as judged by polymerase chain reaction (PCR) amplification of human and non-human primate DNA samples using the unique flanking sequences specific for each Alu element. The chromosomal locations of several Alu elements belonging to the young subfamilies, designated as HS/PV and Sb2, were determined by PCR amplification of DNA samples from human/rodent somatic cell hybrid panels. A statistical analysis of the chromosomal distribution pattern showed that the recently inserted Alu elements appear to integrate randomly in the human genome.

Effects of fasting and refeeding on blood pressure are determined by nutritional state, not by body weight change.

It is commonly assumed that caloric restriction is effective in lowering blood pressure because of the accompanying weight loss and reversal of obesity. However, clinical trials of caloric restriction reporting the greatest falls in blood pressure were those that produced the most rapid weight loss on diets allowing the fewest calories, but the amount of weight loss was unrelated to antihypertensive effect. In obese rats undergoing a supplemented fast, blood pressure fell almost immediately but then stabilized despite continuing weight loss. The depressor effect of fasting was reversed within 2 days of refeeding. Body weight change was no longer correlated with blood pressure change after nutritional state was controlled for. Nutritional state (fed, fasted, refed), but not body weight, has important effects on blood pressure.

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.

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.

Altered angiotensin II sensitivity of neurons in the organum vasculosum lamina terminalis region of the spontaneously hypertensive rat.

Using in vitro hypothalamic brain slices, differences in angiotensin II (AII) sensitivity of neurons in the organum vasculosum lamina terminalis (OVLT) region were compared between spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY). AII, the AII competitive antagonist saralasin, and L-glutamate were micropressure-applied onto OVLT neurons. AII excitation of SHR neurons was blocked or antagonized by simultaneous application of saralasin, evoked at significantly lower thresholds and displayed exaggerated periods of postactivity compared to OVLT neurons in preparations taken from WKY controls. Neuronal responses to L-glutamate were similar between the two rat strains. Differences in neuronal sensitivity to AII may be causally linked to hypertension in SHR.

Development of angiotensin II-sensitive OVLT neurons in SHR and WKY rats.

Angiotensin II (AII) sensitivity of neurons in the region of the organum vasculosum laminae terminalis (OVLT) was examined electrophysiologically using in vitro hypothalamic brain slices taken from 4-, 9- and 14-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Micropressure application of AII, its competitive antagonist saralasin, and L-glutamate revealed that neurons in this region of SHR were significantly more sensitive to AII than cells in age-matched WKY preparations. Neuronal sensitivity to L-glutamate was similar between SHR and WKY rats at all ages. Following electrophysiological study, hypothalamic and cortical brain slices were assayed for 125I-labelled AII binding. AII receptor binding in the hypothalamic slices from SHR was elevated significantly above binding in WKY hypothalamic slices at 4, 9, and 14 weeks of age. In contrast, AII binding in cortical slices taken from SHR and WKY rats was similar. These data suggest that altered neuronal AII-sensitivity is not a consequence of hypertension development in SHR and may contribute to its development.

Altered brainstem structure of spontaneously hypertensive (SHR) rats.

Computerized morphometric analysis of soma cross-sectional areas of single neurons in selected brainstem nuclei revealed that significant structural differences exist between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Neuronal sizes were significantly reduced in 5 of 9 brainstem regions of SHR's compared to WKY rats. Differences in cell densities were also found.

Altered CNS neuroanatomical organization of spontaneously hypertensive (SHR) rats.

Compared to Wistar-Kyoto (WKY) normotensive control rats, spontaneously hypertensive (SHR) rats have significantly reduced brain weights (-10.6%) and brain volumes (-11.8%). Computerized morphometric analysis of soma cross-sectional areas of single neurons in 12 selected hypothalamic regions revealed significant differences between SHR and WKY animals. Neurons from the periventricular, medial and lateral preoptic nuclei and ventromedial hypothalamus show significantly increased soma cross-sectional areas in SHR animals when compared to normotensive controls. Cells located in the two circumventricular organs, organ vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), also showed significantly greater cross-sectional areas in the SHR. In contrast, neurons in the paraventricular and arcuate nuclei and dorsomedial hypothalamus were significantly smaller in spontaneously hypertensive rats when compared to normotensive controls. Only neurons in supraoptic nucleus, lateral and anterior hypothalamus have equivalent cross-sectional areas in WKY and SHR animals. Differences also exist in the number of cells in certain nuclei in SHR animals. Cell densities in periventricular preoptic nucleus, paraventricular nucleus, arcuate nucleus, ventromedial and anterior hypothalamus, organ vasculosum lamina terminalis and subfornical organ were reduced in SHR animals compared to WKY controls. Because of decreased brain weight and volume along with observed morphometric differences in individual neuronal soma size and cell densities, it is suggested that the SHR brain differs significantly from normotensive control rats. The differences may underlie some of the abnormalities in cardiovascular and endocrine regulation associated with neurogenic hypertension.

A slice chamber for intracellular and extracellular recording during continuous perfusion.

The design of a tissue slice perfusion system is described, and examples are given showing the stability of this system for intracellular and extracellular recordings during changes in perfusion media. The stability of this system is attributed to several features. Mini-drips serve to cushion transient changes in flow rate when switching from one medium to another. Solenoid valves are used to quickly switch perfusion media with minimal mechanical movement. A finely-controlled adjustable flow valve provides a uniform flow rate for all media. Constant tissue temperature is maintained by media perfusion through a thermoelectric Peltier assembly. In addition, a filter paper wick insures that the perfusate is constantly removed without movement in the tissue slices. With this design, the slices are supported on a net at the interface between the perfusion medium and a humidified, oxygenated atmosphere. This arrangement appears to be conducive to tissue viability and facilitates the placement of microelectrodes in the slices.

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.

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.

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.

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.

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.