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PubMed1993

Using three-color chromosome painting to test chromosome aberration models.

Ionizing radiation induces DNA double-strand breaks (DSB), which interact pairwise to produce chromosome aberrations. There have long been two main competing theories of such pairwise DSB-DSB interactions. The "classical" theory asserts that an unrepaired DSB makes two ends that separate within the cell nucleus, with each end subsequently able to join any similar (nontelomeric) end. The "exchange" theory asserts that at a DSB the chromatin does not separate completely; rather the DSB ends remain associated until repair, or an illegitimate recombination involving another DSB, occurs. The DSB-DSB interaction mechanism was tested by using three-color fluorescence in situ hybridization to paint chromosomes and observe "three-color triplets": three broken and misrejoined chromosomes having cyclically permuted colors. We observed 18 "three-color triplets" in 2000 cells after 2.25 Gy of gamma-irradiation. On the exchange model in its standard form such three-color triplets cannot occur, so this model is inconsistent with the observations. On the classical model, formalized as a discrete time Markov chain embedded at the transitions of a continuous time Markov chain, the frequency of occurrence of three-color triplets can be computed by Monte Carlo simulations. The number of three-color triplets predicted mathematically by the classical model was found to be slightly larger than the observed number. Thus our data, together with our computer simulations, exclude the standard form of the exchange model but are compatible with the classical model. The results are also compatible with other, more complicated models.

Chromosome Aberrations↗
PubMed2006

Application of the inverse Monte Carlo treatment planning system IKO for an inhomogeneous dose prescription in the sense of dose painting.

Biological imaging (PET SPECTJfMRI, MRS, etc.) is able to provide tri-dimensional biological information, i.e. proliferation, cell density, hypoxia or choline/citrate ratio. The implementation of this information in a treatment plan can be utilised to escalate the dose in target subvolumes. For this purpose, a treatment planning system has to be able to realise an inhomogeneous dose prescription with sufficient spatial resolution. The present study investigated to which extent the inverse Monte Carlo treatment planning system IKO (inverse kernel optimization), developed at our department, can modulate an inhomogeneous dose prescription. As a qualifier to describe this ability, we defined in analogy to imaging a modulation transfer function for treatment planning systems. In addition two clinical cases, a prostate case and a head-and-neck case, were set up with different dose prescriptions in different subtargets. The modulation transfer function revealed that IKO is able to modulate structures larger than 1.3 cm with sharp dose gradients. Also, IKO is able to modulate several subtargets inside a prostate with different escalated doses. The dose-volume histograms of the head-and-neck case showed a good dose coverage of the target volumes, as well as a good protection of the organs at risk according to the dose constraints. As a result, IKO is able to realise a heterogeneous dose prescription in the sense of "dose painting".

Algorithms↗
PubMed1995

Combined FISH painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes.

We present a simple method that allows scoring of FISH-painted chromosomes exclusively in the first division human lymphocytes. It consists of a combination of FISH with chromosome-specific libraries and a differential sister chromatid staining after BrdU treatment of lymphocyte cultures. The method allows a precise quantification of induced chromosome damage for human biodosimetry.

Cell Cycle↗
PubMed1996

Quantification of FISH-painted chromosome aberrations after domestic radon exposure.

Chromosome painting (target chromosomes 1, 4, 12) was performed in peripheral lymphocytes from 25 occupants of nine houses with indoor radon concentrations of 210-3000 Bqm-3. Compared to a control group, the mean frequency of symmetrical translocations of the radon group was slightly but not significantly (p < 0.10) increased. A similar tendency became apparent for a comparison of two groups of subjects with cumulative radon exposures above and below 2800 Bqm-3 y. It is concluded that FISH-based measurements of stable symmetrical translocations should reflect the cumulative radon exposure to haematopoietic compartments such as the red bone marrow rather than to mature blood lymphocytes. Since, however, radon-derived bone marrow doses are low and control frequencies of translocations are very high (about 10-fold higher than the value for conventionally scored dicentrics), the observed relative increase (1.5-fold) of the translocation frequency in blood lymphocytes is too small to discriminate chronic radon exposure from background.

Adolescent↗
PubMed1996

Chromosome translocations in thyroid tissues from Belarussian children exposed to radioiodine from the Chernobyl accident, measured by FISH-painting.

Chromosome painting of chromosomes 1, 4 and 12 was performed on metaphase preparations of cultured thyroid cells to analyse the frequency of radiation-induced stable chromosome translocations in papillary thyroid carcinomas from 40 Belarussian children exposed to radioiodine from the Chernobyl accident, and from 31 reference case. As expected, we found the highest translocation frequencies in secondary thyroid tumours after radiotherapy, but there were also high frequencies in tumour tissues as well as in non-tumourous tissues from childhood papillary carcinoma samples from Belarus. Among the Belarussian tumours the cases from the Gomel region exhibited the highest frequency of translocations and five cases lie within the range of frequencies observed in secondary thyroid tumours after radiotherapy. The findings support the assumption that radiation was the principal cause of the tumours in Belarus, but they indicate also that only a minority of the Belarus cases, which have developed papillary carcinomas, were exposed to very high doses of radioiodine.

Adolescent↗
PubMed1996

Detection and evaluation of chromosomal aberrations induced by high doses of gamma-irradiation using immunogold-silver painting of prematurely condensed chromosomes.

We developed a method for detecting chromosomal aberrations after irradiation with high doses of gamma-rays. At high dose levels, few of the irradiated cells are able to enter mitosis so that it is difficult to obtain enough mitotic chromosomes using a conventional colcemid block. Therefore, okadaic acid was used to condense prematurely the chromosomes of interphase cells. Even after irradiation with doses > 40 Gy, okadaic acid was able to force sufficient numbers of cells to condense their chromosomes. Segments of chromosomes were then detected by chromosome painting using a gold-conjugated antibody followed by silver enhancement. This simple method allows highly damaged chromosomes to be detected with great sensitivity.

Cell Cycle↗
PubMed1997

Retrospective biodosimetry of Chernobyl clean-up workers using chromosome painting and conventional chromosome analysis.

Blood samples of 52 Chernobyl clean-up workers were analysed by fluorescence in situ hybridization (FISH) using whole-chromosome painting probes for chromosomes 1, 4 and 12, simultaneously with a pancentromeric probe and by conventional chromosome analysis, for radiation-induced symmetrical translocations and dicentrics in T-lymphocytes. Based on FISH measurements of translocations, individual biodosimetry estimates between 0.32 and 1.0 Gy were obtained from 18 cases. Pooled data for the total group of 52 workers provided a collective biodosimetry estimate of 0.23 Gy. For a group of 34 workers with documented doses, the mean dose estimate of 0.25 Gy compared well with the mean documented dose of 0.26 Gy. However, no correlation between individual translocation frequencies (FG) and documented doses could be found. A statistical analysis of the expected dose-response suggests exposures to higher doses than documented for a substantial fraction of workers with ascribed doses < 0.2 Sv. For subjects working repeatedly at the reactor site between 1986 and 1995 the mean translocation frequency was significantly higher than for those working only in 1986. A comparison of dicentric frequencies obtained by conventional scoring and by FISH measurements showed no significant difference, although only two of 52 cases revealed significantly higher yields than the mean control level. Based on conventionally scored dicentric frequencies, a collective biodosimetry estimate of 0.23 Gy could be derived only of the group of persons working at Chernobyl exclusively in 1986 for which a documented average dose of 0.19 Gy was reported.

Adult↗
PubMed1998

Analysis of symmetrical translocations for retrospective biodosimetry in radiation workers of the Mayak nuclear-industrial complex (Southern Urals) using FISH-chromosome painting.

PURPOSE: Frequencies of symmetrical translocations were determined by fluorescence in situ hybridization (FISH) for retrospective biodosimetry in workers occupationally exposed to external gamma-rays and internal plutonium at the Mayak nuclear-industrial complex (Southern Urals, Russia). MATERIALS AND METHODS: Chromosome analyses were carried out on peripheral lymphocytes from 75 Mayak workers who had received their main exposures between 1948 and 1963. Cumulative external gamma-ray doses between 0.02 and 9.91 Sv and plutonium burdens ranging between 0.26 and 18.5 kBq are reported. As controls, 33 unexposed persons from non-contaminated areas of the Southern Urals were used. Whole-chromosome painting probes for chromosomes 1, 4 and 12 were used simultaneously with a pancentromeric probe. RESULTS: Compared with the control group, a significantly elevated translocation frequency was found for the total study group and for either of two subsets with (48 subjects) and without (27 subjects) plutonium incorporation. The dicentric frequency was not significantly different from the control level. In the pooled data set, translocation frequencies showed a significant dependence on cumulative external gamma-ray doses. Plutonium uptake had no substantial influence. Individual dose estimates for 21 cases exhibiting at least five translocations ranged between 0.5 and 1.8 Gy, which is substantially lower than the workers' registered personal doses. CONCLUSION: At 35-40 years after protracted exposure to low-dose rate external gamma-rays, the postulated lifetime stability of translocations cannot be confirmed. Apparently, the natural loss of translocation-bearing peripheral lymphocytes cannot be fully compensated so that a temporal decline even of transmissible aberrations takes place. As a consequence, individual retrospective biodosimetry estimates cannot be obtained reliably from the remaining fraction of translocations.

Aged↗
PubMed1998

Relationship between the DNA content of human chromosomes and their involvement in radiation-induced structural aberrations, analysed by painting.

PURPOSE: To study the relationship between the DNA content of human chromosomes and their involvement in radiation-induced structural chromosome aberrations. MATERIAL AND METHODS: Human lymphocytes were cultured after exposure to 5 Gy of X-rays. FISH-painting was performed for all human chromosomes. RESULTS: The results indicate that with the exception of chromosome 20, there was a good fit between the DNA content and the number of exchange-type aberrations and the number of breaks. However, there was a significant tendency for short chromosomes to be more affected than expected and for long chromosomes to be less affected than expected. A better fit was observed when, instead of DNA content, the surface area of the chromosome territories obtained from a spherical model was taken into consideration. CONCLUSIONS: The tendency for short chromosomes to be more involved and long chromosomes to be less involved in exchange-type aberrations could be related not only to their DNA content but also to their interphase territory surface area.

Chromosome Aberrations↗
PubMed1976

Dermal carcinogenicity study by mouse-skin painting with 2,4-toluendediamine alone or in representative hair dye formulations.

The chronic toxicity of 2,4-toluenediamine (2,4-TDA) alone or in conbination with a hair dye complex (2,5-toluenediamine, p-phenylenediamine, and resorcinol) was studied in Swiss-Webster mice of both sexes by a skin-painting technique. The predominant neoplasms seen in these mice were primary pulmonary adenomas and adenocarcinomas. Skin neoplasms were seen in most groups of mice, including untreated control mice. Statistical analysis of the incidences of skin neoplasms among the various groups of mice did not show any significant differences. The 2,4-TDA alone or mixed with the hair dye complex did not produce any abnormal proliferation and maturation of the squamous epithelium of the skin. The 2,4-TDA under our experimental conditions was found to be nontoxic and noncarcinogenic to the skin of mice.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Particle settling after lead-based paint abatement work and clearance waiting period.

This study investigated the evolution of airborne particle concentration and size distribution following abatement work in a controlled environment utilizing direct real-time particle monitoring and used it to project potential lead loadings as those particles settle. An 860 ft3 environmental test chamber with sophisticated ventilation and air purifying systems was built. Wooden doors with lead-based paint were dry sanded or scraped to generate the highest feasible airborne lead concentrations. Size-fractional airborne particle concentrations decreased exponentially with time in all tests, even with no air exchange, consistent with the stirred model of constantly mixed air, which predicts longer settling than for tranquil settling. Very low levels of air mixing generated by temperature gradients and initial room air turbulence affected particle settling. About 90% of airborne lead mass settled within 1 hour after active abatement, before final cleaning began. During the second waiting period of 1 hour, which followed cleaning of the floor, additional dust settled so that the additional potential lead loading from remaining airborne lead was less than 20 microg/ft2. For this worst case scenario, the underestimate of the lead loading done by the clearance sampling did not exceed about 30%. For more realistic conditions, the underestimates are projected to be much lower than the new 40 microg/ft2 Housing and Urban Development (HUD) clearance standards for floor dust lead. These results were obtained for the first waiting period (between the end of active abatement and the beginning of cleaning) of 1 hour, as recommended by HUD guidelines. Thus, this study demonstrates no need to increase either the first or second waiting period.

Air Pollutants, Occupational↗
PubMed1996

XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure.

The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.

Cell Cycle↗
PubMedDate not supplied

Lactic acid buffering by bone and shell in anoxic softshell and painted turtles.

We tested two hypotheses: first, that the inferior anoxia tolerance of the softshell turtle, Apalone spinifera, compared to the western painted turtle, Chrysemys picta bellii, is related to its less mineralized shell, and second, that turtle bone, like its shell, stores lactate during prolonged anoxia. Lactate concentrations of blood, hindlimb bone, and shell were measured on normoxic Apalone and Chrysemys and after anoxic submergence at 10 degrees C for 2 and 9 d, respectively. Blood and shell concentrations of Ca(2+), Mg(2+), Na(+), K(+), and inorganic phosphate (P(i); for shell only) were also measured. Because a preliminary study indicated lactate distribution in Chrysemys throughout its skeleton during anoxia at 20 degrees C, we used hindlimb bones as representative skeletal samples. Apalone shell, though a similar percentage of body mass as Chrysemys shell, had higher water content (76.9% vs. 27.9%) and only 20%-25% as much Ca(2+), Mg(2+), CO(2), and P(i). When incubated at constant pH of 6.0 or 6.5, Apalone shell powder released only 25% as much buffer per gram wet weight as Chrysemys shell. In addition, plasma [Ca(2+)] and [Mg(2+)] increased less in Apalone during anoxia at an equivalent plasma lactate concentration. Lactate concentrations increased in the shell and skeletal bone in both species. Despite less mineralization, Apalone shell took up lactate comparably to Chrysemys. In conclusion, a weaker compensatory response to lactic acidosis in Apalone correlates with lower shell mineralization and buffer release and may partially account for the poorer anoxia tolerance of this species.

Acidosis, Lactic↗
PubMedDate not supplied

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle.

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).

Animals↗
PubMedDate not supplied

Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale variation in response to environmental stress.

We integrated field and laboratory studies in an investigation of water balance, energy use, and mechanisms of cold-hardiness in hatchling painted turtles (Chrysemys picta) indigenous to west-central Nebraska (Chrysemys picta bellii) and northern Indiana (Chrysemys picta marginata) during the winters of 1999-2000 and 2000-2001. We examined 184 nests, 80 of which provided the hatchlings (n=580) and/or samples of soil used in laboratory analyses. Whereas winter 1999-2000 was relatively dry and mild, the following winter was wet and cold; serendipitously, the contrast illuminated a marked plasticity in physiological response to environmental stress. Physiological and cold-hardiness responses of turtles also varied between study locales, largely owing to differences in precipitation and edaphics and the lower prevailing and minimum nest temperatures (to -13.2 degrees C) encountered by Nebraska turtles. In Nebraska, winter mortality occurred within 12.5% (1999-2000) and 42.3% (2000-2001) of the sampled nests; no turtles died in the Indiana nests. Laboratory studies of the mechanisms of cold-hardiness used by hatchling C. picta showed that resistance to inoculative freezing and capacity for freeze tolerance increased as winter approached. However, the level of inoculation resistance strongly depended on the physical characteristics of nest soil, as well as its moisture content, which varied seasonally. Risk of inoculative freezing (and mortality) was greatest in midwinter when nest temperatures were lowest and soil moisture and activity of constituent organic ice nuclei were highest. Water balance in overwintering hatchlings was closely linked to dynamics of precipitation and soil moisture, whereas energy use and the size of the energy reserve available to hatchlings in spring depended on the winter thermal regime. Acute chilling resulted in hyperglycemia and hyperlactemia, which persisted throughout winter; this response may be cryoprotective. Some physiological characteristics and cold-hardiness attributes varied between years, between study sites, among nests at the same site, and among siblings sharing nests. Such variation may reflect adaptive phenotypic plasticity, maternal or paternal influence on an individual's response to environmental challenge, or a combination of these factors. Some evidence suggests that life-history traits, such as clutch size and body size, have been shaped by constraints imposed by the harsh winter environment.

Adaptation, Physiological↗
PubMedDate not supplied

Accumulation of lactate by frozen painted turtles (Chrysemys picta) and its relationship to freeze tolerance.

Hatchling painted turtles (Chrysemys picta) survived freezing at -2 degrees C for 4 d, few recovered from freezing lasting 6 d, and none survived being frozen for 8 d. Whole-body glucose and lactate were low in animals that had not been subjected to cold and ice but increased precipitously in animals that were frozen for 2 d. Both metabolites continued to increase, but at a somewhat lower rate, in animals frozen for 4, 6, or 8 d. The increase in whole-body lactate reflects a reliance by frozen hatchlings on anaerobiosis, whereas the increase in glucose presumably results from mobilization of glycogen reserves to support anaerobic metabolism. Mortality of frozen hatchlings is correlated with the increase in whole-body lactate. Factors that may contribute to the observed correlation include a compromised capacity for individual organs to cope with the lactic acidosis that accompanies anaerobic metabolism and organ-specific depletion of energy reserves. Individual organs must rely on buffering and glucose reserves available in situ because blood of frozen hatchlings does not circulate. Thus, buffer from the shell cannot be transported to other organs, lactate cannot be sequestered in the shell, and glucose mobilized from liver glycogen is not available to supplement glucose reserves of other tissues. This integrated suite of physiological disruptions may limit tolerance of freezing to conditions with little or no ecological relevance.

Acidosis, Lactic↗
PubMed2005

Age and season impact resource allocation to eggs and nesting behavior in the painted turtle.

Theory predicts that in long-lived organisms females should invest less energy in reproduction and more in growth and self-maintenance early in life, with this balance shifting as females age and the relative value of each reproductive event increases. We investigated this potential trade-off by characterizing within-population variation in resource allocation to eggs by female painted turtles (Chrysemys picta) and relating this variation to their nesting ecology and life history. We examined lipid and protein allocation to yolks, accounting for both relative female age and seasonal effects (first vs. second clutches within a female). Older females appear to increase their investment in reproduction by producing larger eggs, but these eggs are not disproportionately more lipid or protein rich than the smaller eggs from younger females. Within the nesting season, first clutches have more lipid and protein than second clutches. We also found that younger females nest closer to the water than older females. Our results indicate that trade-offs involving resource allocation and nesting behavior do occur both seasonally and with age, suggesting ontogenetic variation in life-history strategies in this long-lived organism.

Age Factors↗
PubMed1995

Chromosome aberrations produced by ionizing radiation: Monte Carlo simulations and chromosome painting data.

Monte Carlo simulations are used to analyze the reshuffling of chromosome segments which occurs when DNA is damaged by ionizing radiation. Programs are based on either Sax's classic breakage-and-reunion model or Revell's exchange model for chromosome aberrations. The simulations quantify the predictions of the two models in complete detail, using only one adjustable parameter which corresponds to total radiation dose. While testing subroutines, new analytic results on the chromosome/arm/break method of classifying aberrations were obtained. The model predictions were tested by using three-color fluorescence in situ hybridization (FISH) 'chromosome painting' on human lymphocyte cells irradiated with gamma-rays. Some of the per-cell aberration frequencies were observed to be intermediate between the predictions of the two models. This result indicates proximity 'effects', due to localization of chromosome interactions in space and time. Predictions based on chromosome arm lengths were found to be in good agreement with experiment. Monte Carlo simulations are a powerful, flexible way to compare models of chromosome aberration production with experiments quantitatively, using a minimum of theoretical presumptions.

Algorithms↗