John Curry

Monitoring hprt mutant frequency over time in T-lymphocytes of people accidentally exposed to high doses of ionizing radiation

Modern technologies have provided the opportunity to monitor mutations in people in vivo. The subjects of this study were accidentally exposed to 137Cesium in a radiological accident that occurred in September 1987 in Goiânia, Brazil, during which more than 150 people received doses greater than 0.1 Gy and as high as 7 Gy. The objective of this study was to determine how long the hprt mutant T‐cells in the peripheral blood contribute to mutant frequency by examining the time‐course of the T‐lymphocyte response to ionizing radiation. This report describes the results obtained over a period of 2.3 to 4.5 years subsequent to the accident, from 11 subjects with doses ranging from 1 to 7 Gy, and from nine control subjects selected from the same population. The mean ln MF (±SE) of the control group was 2.5 (±0.2) + ln 10−6. The exposed group had a significantly increased mutant frequency; the mean In MF (± SE) were 3.3 (±0.3) + ln 10−6, 2.8 (±0.2) + ln 10−6, and 2.3 (±0.2) + ln 10 −6, in the years 1990–1992 respectively. Based on the decline of mutant frequency and using Bucktons models [Buckton et al. (1967): Nature 214:470–473], we demonstrated that mutant T‐cells have a short‐term memory with a half‐life of 2.1 years. This relatively short half‐life limits the effective use of the hprt assay as the method of choice to monitor past exposure. The data also demonstrate a positive correlation with age, and an inverse correlation with plating efficiency.

A fast and simple method to determine the clonal relationship among human T-cell lymphocytes

For the analysis of mutation in human T-cell lymphocytes, it is crucial to determine the clonal relationship between isolated mutants, particularly if they harbour identical mutations. Here we report an efficient method to determine the clonal relationships between these cells. The method is based on the analysis of restriction fragment length polymorphisms of a polymerase chain reaction amplified, rearranged T-cell receptor gamma-gene. As few as 600 cells are sufficient, regular agarose gels can be used for the separation of the restriction fragments, no radioactive label is required, and results can be obtained in 2 days.

Coamplification of hprt cDNA and γ T-cell receptor sequences from 6-thioguanine resitant human T-lymphocytes

Summary The nature of mutation at the HPRT locus in human T-lymphocytes in vivo is currently a subject of considerable interest. Determination of clonality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respective T-cell receptors (TCR). We have developed a polymerase chain reaction (PCR)-based method for coamplification of hprt cDNA and the rearranged γ T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transcription to produce hprt cDNA, the crude cell lysate is treated with proteinase K and subjected to a primary PCR with two sets of amplification primers, one specific for the hprt cDNA and the other for the rearranged γ TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce sufficient uantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and γ TCR gene respectively.

Molecular analysis of T‐lymphocyte HPRT‐ mutations in individuals exposed to ionizing radiation in Goiânia, Brazil

We have characterized 54 HPRT‐ point mutations in T‐lymphocytes from 17 individuals exposed to ionizing radiation of 137Cs in Goiânia, Brazil and compared this spectrum to that of 30 HPRT‐ mutants from 9 unexposed Brazilian controls. The average internal exposure of the exposed group was 205 mCi, and the average external exposure was 1.7 Gy. The average HPRT‐ mutant frequency for the exposed group was 13.3 × 10‐5, approximately a 10‐fold increase over the mutant frequency of the unexposed controls, which was 1.56 × 10‐5. The types of point mutations characterized included base substitutions, small deletions, frameshifts, in‐sertions, complex mutations, and losses of exon sequences from the mRNA. The relative frequency of the different mutation types was similar in the two studied groups. However, in our study the distribution of events within the hprt coding sequence seemed to cluster at the same regions of the gene. These observations imply that the hprt gene does not present a homogeneous target to radiation mutagenesis, and perhaps this class of information may be used to detect radiation exposure in human populations. Environ. Mol. Mutagen. 29:107‐116, 1997.

Radiation risk estimation in human populations : Lessons from the radiological accident in Brazil

The development of radiological and nuclear technologies and the deployment of nuclear weapons have made ionizing radiation one of the most studied human mutagens. Exposure to ionizing radiation produces DNA damage which can result in mutation and cancer, making the risk associated with human exposure a critical issue. In this paper we estimate the risk associated with radiation exposure for individuals exposed to 137Cs during the 1987 Goiânia radiological accident. Using combined regression slopes from both the in vivo hprt mutant frequency and micronucleus frequency data we estimated a doubling dose of 173 (+/-47) cGy for these two endpoints. This is in close agreement with the published estimates for low dose rate and chronic exposure to low-LET radiation. We obtained risk estimates of about 24-fold increase in dominant disorders in the post-exposure generation of the directly exposed population. No detectable increase was found in the population at large. The risk of carcinogenesis in the directly exposed population was found to be increased by a factor in the range of 1.4 to 1.5. The small sample size in this study requires a large element of caution with respect to risk estimates interpretation. Moreover, the doubling dose estimates prepared here are derived from lymphocytes. This somatic data may require additional considerations for both cancer and certainly germ-line events. Nevertheless, the risk of carcinogenesis and genetic harm for this population are good indicators of the potential genetic damage imposed by ionizing radiation to the Goiânia population.

Similar mutant frequencies observed between pairs of monozygotic twins

The relative contribution of both genetic and environmental factors to spontaneous mutation frequency in humans is unknown. We have investigated the contribution of genetic factors to this phenomenon by determining the in vivo mutant frequency at the hypoxanthine‐guanine phosphoribosyltransferase (hprt) locus in circulating T‐lymphocytes obtained from pairs of monozygotic twins. hprt mutant frequencies were determined three times over fourteen days in six sets of monozygotic male twins (mean age 30) taking part in a Russian Space Program inclined bed rest experiment. Blood samples were obtained prior to, during, and immediately following the experiment. Mononuclear cells were separated, frozen, and flown to Canada for analysis using the hprt T‐lymphocyte clonal assay. There is no evidence within this data set to demonstrate that the period of inclined bed rest to simulate the effects of weightlessness had any effect on the observed mutant frequency. However, the average mutant frequency for the six sets of Russian twins was found to be three times higher than that of Western counterparts. More surprisingly, the spontaneous mutant frequency of monzygotic twins was found to be much more similar within pairs than between pairs of twins. These data suggest that the contribution of genetics in the determination of mutation frequency is substantial. However, whether high concordance within twin pairs reflects shared environmental experience as well as common genetic factors is not entirely clear. More data will be required to distinguish genetic from environmental factors and to determine the degree to which mutant frequency is genetically determined. Hum Mutat 9:445–451, 1997.

Increased hprt mutant frequencies in Brazilian children accidentally exposed to ionizing radiation

We have examined the effects of ionizing radiation on somatic mutations in vivo, using the hprt clonal assay. The study was performed on blood samples obtained from children exposed during a radiological accident that happened in 1987, in Goiânia, Brazil. The group of children exposed to ionizing radiation includes six males and four females ranging in age from 6 to 14 years at the time of exposure. The radiation doses ranged from 15 to 70 cGy. A Brazilian control group, not exposed to ionizing radiation, was also analyzed under similar conditions. The mean hprt mutant frequency for the exposed group was 4.6 times higher than the control group, although the cloning efficiency from the exposed group was significantly reduced. Linear regression analysis of the mutant frequency and ionizing radiation dose did not show a significant relationship between these two parameters. However, a reliable inverse relationship was demonstrated when the regression analysis was performed with nonselective cloning efficiency and ionizing radiation dose. It was demonstrated that nonselective cloning efficiency diminishes as ionizing radiation dose increases. To correct mutant frequencies for clonal events, the clonal relationship between the hprt mutant clones was examined by T‐cell receptor analysis. The majority of the mutants analyzed represented individual clones, thus validating the observed mutant frequencies.

A note on the relevance of human population genetic variation and molecular epidemiology to assessing radiation health risk for space travellers

We discuss the relevance to space medicine of studies concerning human genetic variation and consequent variable disease susceptibility or sensitivity between individuals. The size of astronaut and cosmonaut populations is both presently and cumulatively small, and despite the launch of the International Space Station, unlikely to increase by orders of magnitude within the foreseeable future. In addition, astronauts-cosmonauts constitute unrepresentative samples of their national populations. While the context of exposure for the astronaut-cosmonaut group is one unlikely to be replicated elsewhere than in space, aspects of specific exposures may be simulated by events such as occupational radiation exposure or radiation therapy. Hence, population-based studies of genetic susceptibility or sensitivity to disease, especially where it is precipitated by events that may simulate consequences of the space environment, likely will prove of value in assessing long-term health risks.

Moloney murine leukemia reverse transcriptase suspect in the production of multiple misincorporations during hprt cDNA synthesis

Our laboratory has characterized several hundred mutant hprt cDNAs produced using Moloney murine leukemia reverse transcriptase to convert mRNA to cDNA. During the characterization of these mutants we have detected six T-lymphocyte mutants that demonstrate multiple G:C --> A:T transitions along the hprt cDNA coding sequence. Attempts to repeat the mRNA to cDNA conversion and subsequent characterization have demonstrated that the multiple transitions are likely artifacts. We suggest that reverse transcriptase is directly responsible for these multiple base substitutions and as such, that multiple mutations be viewed as suspect requiring confirmation at the genomic level.

Variable aberrant cDNAs in single diphtheria toxin-resistant human fibroblasts.

We treated transformed human fibroblasts with diphtheria toxin (DT) and isolated 40 single cells that were toxin resistant but unable to propagate. In 13 of them toxin resistance was associated with the presence of one or more aberrant transcripts of the structural gene for elongation factor 2 (EF-2). cDNA obtained from these transcripts had 164-447 bp-long deletions. Each of these deletions was associated with 2-8 base pairs-long repeats at its breakpoints. Only 10 out of 16 cDNA deletions were associated with presumed exon junctions. A role is suggested for errors in transcription in producing the aberrant transcripts which gave rise to the deletion-bearing cDNA species.