Theoretical and experimental tests of a chromosomal fingerprint for densely ionizing radiation based on F ratios calculated from stable and unstable chromosome aberrations.

In the present study, F ratios for both stable chromosome aberrations, i.e. ratios of translocations to pericentric inversions, and unstable aberrations, i.e. dicentrics and centric rings, were measured using fluorescence in situ hybridization. F ratios for stable aberrations measured after exposure to low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 1.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiation were 6.5 +/- 1.5, 4.7 +/- 1.6, 9.3 +/- 2.5 and 10.4 +/- 3.0, respectively. F ratios for unstable aberrations measured after low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiations were 6.5 +/- 1.6, 6.3 +/- 2.3 and 11.1 +/- 3.7, respectively. No significant difference between the F ratios for low- and high-LET radiation was found. Further tests on the models for calculation of the F ratio proposed by Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) showed that the F ratio may not be straightforward as a practical fingerprint for densely ionizing radiation.

A rapid method for measuring pericentric inversions using fluorescence in situ hybridization (FISH).

We used three common fluorescent probes to measure pericentric inversion frequencies in 2.9 Gy 60Co gamma-irradiated human lymphocytes. For a given chromosome, the first probe is specific to one telomeric region, the second probe is specific to one subcentromeric region and the third probe is specific to the centromere. A pericentric inversion is made observable by the change in position (switching) of the fluorescent signals relative to the chromosome centromere. Our data showed equality between pericentric inversions and centric rings. The calculated whole-genome F-ratio of apparently simple translocations to pericentric inversions was 5.6.

Stability of the translocation frequency following whole-body irradiation measured in rhesus monkeys.

Chromosome translocations are persistent indicators of prior exposure to ionizing radiation and the development of 'chromosome painting' to efficiently detect translocations has resulted in a powerful biological dosimetry tool for radiation dose reconstruction. However, the actual stability of the translocation frequency with time after exposure must be measured before it can be used reliably to obtain doses for individuals exposed years or decades previously. Human chromosome painting probes were used here to measure reciprocal translocation frequencies in cells from two tissues of 8 rhesus monkeys (Macaca mulatta) irradiated almost three decades previously. Six of the monkeys were exposed in 1965 to whole-body (fully penetrating) radiation and two were unexposed controls. The primates were irradiated as juveniles to single doses of 0.56, 1.13, 2.00, or 2.25 Gy. Blood lymphocytes (and skin fibroblasts from one individual) were obtained for cytogenetic analysis in 1993, near the end of the animals' lifespans. Results show identical dose-response relationships 28 y after exposure in vivo and immediately after exposure in vitro. Because chromosome aberrations are induced with identical frequencies in vivo and in vitro, these results demonstrate that the translocation frequencies induced in 1965 have not changed significantly during the almost three decades since exposure. Finally, our emerging biodosimetry data for individual radiation workers are now confirming the utility of reciprocal translocations measured by FISH in radiation dose reconstruction.

Proximity effects for chromosome aberrations measured by FISH.

A Monte Carlo simulation computer program for radiation-produced chromosome aberrations, based on the breakage-and-reunion model, was extended to include proximity effects due to localization of chromosomes and limited range for break-break interactions. Two adjustable parameters were used. One corresponds to total dose: the other determines proximity effects by specifying the number of 'interaction regions' in a cell nucleus. The use of additional adjustable parameters was avoided by assuming randomness of break induction and aberration production. FISH chromosome painting data were obtained from 1.9 Gy 60Co gamma-rays-irradiated human lymphocytes. The data were compared with the computer simulation results, taking individual chromosome lengths into account. With about 13 interaction regions, agreement between the experiment and the simulation was good, even when detailed categories of damage were scored. An estimated average dsb-dsb interaction distance, based on 13 interaction regions, is about 1.3 micron. Monte Carlo methods give useful quantitative estimates of relative aberration yields, with a minimum of adjustable parameters and the theoretical assumptions, and indicated proximity effects. Computer simulation of FISH experiments can be adapted to any number of colours, any scoring criteria and any method of grouping aberrations into categories. Simulation allows systematic extrapolation of aberration data on painted chromosomes to whole-genome aberration frequencies.

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.

Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys.

The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.

The coexistence of renal artery stenosis and pheochromocytoma.

The coexistence of renal artery stenosis and pheochromocytoma has been recognized since 1958 and a total of 36 patients reported. This article provides an additional patient with an extra adrenal pheochromocytoma and fibrous bands constricting the left renal artery. Hypertension was confirmed to occur from both excess catecholamine production and hyperreninemia from the left kidney. Surgical removal of the functioning paraganglioma and correction of the renal artery stenosis restored the postoperative plasma catecholamine, renin, and blood pressure to normal. A literature review confirmed the coexistence of these two lesions but failed to provide a common etiology to explain the pathophysiology encountered. However, when the two diseases occur simultaneously, both must be diagnosed accurately and treated in a definitive manner.

Pyogenic hepatic abscesses.

A review of pyogenic liver abscess at the University of Alabama in Birmingham revealed 29 cases, of which 22 were single and seven were multiple. Etiology was cryptogenic in 11, secondary to biliary tract disease in seven, trauma in six, and pyelophlebitis in four, and secondary infection in a primary neoplasm in one. No single set of specific diagnostic criteria could be elucidated, but fever occurred in every patient. Leukocytosis, hypoalbuminemia, and elevated alkaline phosphatase were present in more than 80 per cent of patients. Other clinical findings and routine laboratory abnormalities were less consistently present. In suspect patients, a technetium-99 scan was positive in 18 of 19. Ten patients died, including six of seven with multiple abscesses, seven of seven with positive abscess and blood cultures, and seven of 11 in whom liver abscess was not suspected prior to operation or autopsy. Diagnosis demands a high index of suspicion, in patients in whom suggestive factors are present, and liberal use of the technetium-99 scan. Successful treatment requires exploratory laparotomy, with care of discern any underlying septic focus or the presence of multiple abscesses, and wide drainage of the abscesses, as well as vigorous supportive care and appropriate antibiotics.

Delineation of Robertsonian translocations in man by means of chromosome banding.

Robertsonian translocations from thirteen unrelated individuals were studied by G, Q and C banding, and by autoradiography. Each of three D-D translocations involved chromosomes 13 and 14, all four D-G translocations involved chromosomes 14 and 21, and five of six G-G translocations involved two chromsomes 21 while the sixth involved 21 and 22. Two centromeres were noted in a single 21-21 translocation chromosome by C banding. Autoradiogrpahy was much less useful in identifying the chromosomes involved in the thirteen translocations. G and Q banding were consistently accurate.