Developing Gender-Specific Gene Expression Biodosimetry Using a Panel of Radiation-Responsive Genes for Determining Radiation Dose in Human Peripheral Blood.

In a large-scale radiological incident, rapid and high-throughput biodosimetry would be needed. Gene expression-based biodosimetry is a promising approach to determine the dose received after radiation exposure. We previously identified 35 candidate genes as biodosimetry markers based on a systematic review. The goal of the current study was to establish and validate a specific gene expression-based radiological biodosimetry using a panel of highly radioresponsive genes in human peripheral blood for improving the accuracy of dose estimation. Human peripheral blood samples from 30 adult donors were irradiated to 0, 0.5, 1, 2, 3, 4, 6 and 8 Gy with 60Co γ rays at a dose rate of 1 Gy/min. We examined the expression patterns of candidate genes using real-time polymerase chain reaction (qRT-PCR) at 6, 12, 24 and 48 h postirradiation. Stepwise regression analysis was employed to develop the gene expression-based dosimetry models at each time point. Samples from another 10 healthy donors (blind samples) and four total-body irradiated (TBI) patients were used to validate the radiation dosimetry models. We observed significant linear dose-response relationships of CDKN1A, BAX, MDM2, XPC, PCNA, FDXR, GDF-15, DDB2, TNFRSF10B, PHPT1, ASTN2, RPS27L, BBC3, TNFSF4, POLH, CCNG1, PPM1D and GADD45A in human peripheral blood at the various time points. However, the expression levels of these genes were affected by inter-individual variations and gender. We found that the gender-dependent regression models could explain 0.85 of variance at 24 h postirradiation and could also accurately estimate the absorbed radiation doses with dose range of 0-5 Gy, in human peripheral blood samples irradiated ex vivo and from TBI patients, respectively. This study demonstrates that developing gender-specific biodosimetry based on a panel of highly radioresponsive genes may help advance the application of gene expression signature for dose estimation in the event of a radiological accident or in clinical treatment.

Effects of age and gender on the baseline and 2 Gy 60Co γ-ray-induced nucleoplasmic bridges frequencies in the peripheral blood lymphocytes of Chinese population.

Baseline nucleoplasmic bridges (NPB) vary widely in the general population from different regions in the same country or from different countries. The baseline NPB level in the normal Chinese population and the factors affecting the baseline and radiation-induced NPB levels have not been explored yet. The cytokinesis-block micronucleus cytome assay was conducted on the peripheral blood samples of 121 healthy individuals for the baseline NPB and 52 healthy individuals for the 2 Gy γ-ray-induced NPB level. The effects of age and gender on the baseline NPB and 2 Gy γ-ray-induced NPB level were evaluated. The overall baseline NPB in the peripheral blood lymphocytes of 121 healthy adults from the general population in China was 0.46 ±â€¯0.20 per 1000 binucleated (BN) cells. The overall baseline NPB in males (0.56 ±â€¯0.15 per 1000 BN cells) was higher than that in females (0.36 ±â€¯0.22 per 1000 BN cells, P < 0.05). The effect of age on the baseline NPB was not significant, except for females in the 40-year age group. The overall 2 Gy γ-ray-induced NPB frequency for male donors was lower than that for female donors (P < 0.01). No evident trend of the radiation-induced NPB level with increasing age was observed for both genders. For the baseline micronucleus (MN) and radiation-induced MN levels, the effects of gender and age were confirmed. Therefore, the gender of donors affects the baseline and radiation-induced levels of NPB and MN. In addition, the effect of the age of the donors on the baseline and radiation-induced NPB levels showed no clear pattern and needed to be further investigated.

GDF-15 gene expression alterations in human lymphoblastoid cells and peripheral blood lymphocytes following exposure to ionizing radiation.

The identification of rapid, sensitive and high­throughput biomarkers is imperative in order to identify individuals harmed by radiation accidents, and accurately evaluate the absorbed doses of radiation. DNA microarrays have previously been used to evaluate the alterations in growth/differentiation factor 15 (GDF15) gene expression in AHH­1 human lymphoblastoid cells, following exposure to γ­rays. The present study aimed to characterize the relationship between the dose of ionizing radiation and the produced effects in GDF­15 gene expression in AHH­1 cells and human peripheral blood lymphocytes (HPBLs). GDF­15 mRNA and protein expression levels following exposure to γ­rays and neutron radiation were assessed by reverse transcription­quantitative polymerase chain reaction and western blot analysis in AHH­1 cells. In addition, alterations in GDF­15 gene expression in HPBLs following ex vivo irradiation were evaluated. The present results demonstrated that GDF­15 mRNA and protein expression levels in AHH­1 cells were significantly upregulated following exposure to γ­ray doses ranging between 1 and 10 Gy, regardless of the dose rate. A total of 48 h following exposure to neutron radiation, a dose­response relationship was identified in AHH­1 cells at γ­ray doses between 0.4 and 1.6 Gy. GDF­15 mRNA levels in HPBLs were significantly upregulated following exposure to γ­ray doses between 1 and 8 Gy, within 4­48 h following irradiation. These results suggested that significant time­ and dose­dependent alterations in GDF­15 mRNA and protein expression occur in AHH­1 cells and HPBLs in the early phases following exposure to ionizing radiation. In conclusion, alterations in GDF­15 gene expression may have potential as a biomarker to evaluate radiation exposure.

Dose response of multiple parameters for calyculin A-induced premature chromosome condensation in human peripheral blood lymphocytes exposed to high doses of cobalt-60 gamma-rays.

Many studies have investigated exposure biomarkers for high dose radiation. However, no systematic study on which biomarkers can be used in dose estimation through premature chromosome condensation (PCC) analysis has been conducted. The present study aims to screen the high-dose radiation exposure indicator in calyculin A-induced PCC. The dose response of multiple biological endpoints, including G2/A-PCC (G2/M and M/A-PCC) index, PCC ring (PCC-R), ratio of the longest/shortest length (L/L ratio), and length and width ratio of the longest chromosome (L/B ratio), were investigated in calyculin A-induced G2/A-PCC spreads in human peripheral blood lymphocytes exposed to 0-20Gy (dose-rate of 1Gy/min) cobalt-60 gamma-rays. The G2/A-PCC index was decreased with enhanced absorbed doses of 4-20Gy gamma-rays. The G2/A PCC-R at 0-12Gy gamma-rays conformed to Poisson distribution. Three types of PCC-R were scored according to their shape and their solidity or hollowness. The frequencies of hollow PCC-R and PCC-R including or excluding solid ring in G2/A-PCC spreads were enhanced with increased doses. The length and width of the longest chromosome, as well as the length of the shortest chromosome in each G2/M-PCC or M/A-PCC spread, were measured. All L/L or L/B ratios in G2/M-PCC or M/A-PCC spread increased with enhanced doses. A blind test with two new irradiated doses was conducted to validate which biomarker could be used in dose estimation. Results showed that hollow PCC-R and PCC-R including solid ring can be utilized for accurate dose estimation, and that hollow PCC-R was optimal for practical application.

Dose-effect relationships of nucleoplasmic bridges and complex nuclear anomalies in human peripheral lymphocytes exposed to 60Co γ-rays at a relatively low dose.

The dose effect between nucleoplasmic bridges (NPB) and relatively low doses of ionising radiation remains unknown. Accordingly, this study investigated the NPB frequencies in human peripheral blood lymphocytes exposed to low-dose (60)Co γ-rays. Complex anomalies, including fused nuclei (FUS), horse-shoe nuclei (HS) and circular nuclei (CIR), which possibly originated from multiple NPBs, were also scored. Human peripheral blood samples were collected from three healthy males and irradiated with 0-1 and 0-0.4 Gy (60)Co γ-rays. A cytokinesis-block micronucleus cytome assay was then conducted to analyse NPB, PFHC (NPB plus three complex nuclear anomalies) and micronucleus (MN) in binucleated cells. All dose-response curves followed the linear model for both NPB frequency and PFHC cell frequency. The dose-response curves between NPB frequency and absorbed dose at 0-1 and 0-0.4 Gy were y = 0.0037x + 0.0005 (R (2) = 0.979, P < 0.05) and y = 0.0043x + 0.0004 (R (2) = 0.941, P < 0.05), respectively. The dose-response curves between PFHC cell frequency and absorbed dose at 0-1 and 0-0.4 Gy were y = 0.0044x + 0.0007 (R (2) = 0.982, P < 0.05) and y = 0.0059x + 0.0005 (R (2) = 0.969, P < 0.05), respectively. The statistical significance of differences between the irradiated groups (0-0.4 Gy) and background levels of NPB, PFHC and MN were also analysed. The lowest analysable doses of NPB, PFHC and MN were 0.12, 0.08 and 0.08 Gy, respectively. In conclusion, NPBs and PFHC positively correlated with the absorbed radiation at a relatively low dose.

Dose-effect of ionizing radiation-induced PIG3 gene expression alteration in human lymphoblastoid AHH-1 cells and human peripheral blood lymphocytes.

PURPOSE: To identify new ionizing radiation (IR)-sensitive genes and observe the dose-effect of gene expression alteration (GEA) induced by IR. MATERIALS AND METHODS: Microarray was used to screen the differentially expressed genes in human lymphoblastoid cells (AHH-1) using three doses of (60)Co γ-rays (0.5-8 Gy at 1 Gy/min). Given that p53-inducible gene 3 (PIG3) was consistently upregulated, the GEA of PIG3 in AHH-1 cells and human peripheral blood lymphocytes (HPBL) induced by γ-rays (1 Gy/min) was measured at messenger RNA (mRNA) and protein levels. The GEA of PIG3 in AHH-1 cells exposed to neutron radiation (californium-252, 0.073 Gy/min) was also quantified. RESULTS: PIG3 was one of the seven differentially expressed genes found in the microarray analysis. The PIG3 mRNA and protein levels in AHH-1 cells were significantly increased from 1-10 Gy of γ-rays 8-72 h or 8-168 h after exposure, respectively. The enhancement was also observed in AHH-1 cells from 0.4-1.6 Gy of neutrons 48 h post-irradiation. The PIG3 mRNA levels (mRNA copy numbers) in HPBL were significantly increased from 1-8 Gy of γ-rays within 4-24 h post-irradiation, but the highest increase in signal-to-noise responsiveness is approximately two-fold, which was less than that of AHH-1 (approximately 20-fold). CONCLUSIONS: IR can upregulate the PIG3 gene expression in AHH-1 and HPBL in the early phase after exposure; however, the IR induced expression levels of PIG3 are greater in AHH-1 than HPBL.

Characteristics of nucleoplasmic bridges induced by 60Co γ-rays in human peripheral blood lymphocytes.

Few studies have shown that the yields of ionising-radiation-induced nucleoplasmic bridges (NPBs) in human cells are dose dependent. However, a dose-response curve between the NPB frequency and the absorbed dose of ionising radiation has not yet been established. This study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by cobalt-60 (60Co) γ-rays and to establish a dose-response curve. Human peripheral blood samples were collected from three healthy males, and some of these samples were irradiated with 0-6 Gy 60Co γ-rays. A cytokinesis-block micronucleus cytome assay was then carried out to analyse NPBs and micronuclei (MN) in binucleated cells. The remaining blood samples were irradiated with 0, 2 and 5 Gy of γ-rays, and unstable chromosome aberrations (dicentric chromosome, ring chromosome and acentric chromosome fragment) were analysed. The correlation between NPBs and dicentric plus ring chromosome (dic+r) induced by the same γ-ray dose was also analysed. Results showed that the NPB yields among the three subjects at each dose level were not significantly different. NPBs in binucleated cells at all γ-ray doses conformed to Poisson distribution. The dose-response curve of the γ-ray-induced NPB frequencies followed the linear-quadratic model y = (1.39×10-3)x 2 + (4.94×10-3)x. A positive correlation was observed between the frequencies of NPB and dic+r, as well as between the frequencies of MN and acentric fragments. Therefore, NPB is an important biomarker of early chromosome damage event induced by ionising radiation.

Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation.

The present study aims to evaluate the use of the fluorescence in situ hybridization (FISH) translocation assay for retrospective dose estimation of acute accidental exposure to radiation in the past. Reciprocal translocation analysis by FISH with three whole-chromosome probes was performed on normal peripheral blood samples. Samples were irradiated with 0-5Gy (60)Co γ-rays in vitro, and dose-effect curves were established. FISH-based translocation analyses for six accident victims were then performed, and biological doses were estimated retrospectively by comparison with the dose-effect curves. Reconstructed doses by FISH were compared with estimated doses obtained by analysis of di-centrics performed soon after exposure, or with dose estimates from tooth-enamel electron paramagnetic resonance (EPR) data obtained at the same time as the FISH analysis. Follow-up FISH analyses for an adolescent victim were performed. Results showed that dose-effect curves established in the present study follow a linear-quadratic model, regardless of the background translocation frequency. Estimated doses according to two dose-effect curves for all six victims were similar. FISH dose estimations of three adult victims exposed to accidental radiation less than a decade prior to analysis (3, 6, or 7 years ago) were consistent with those estimated with tooth-enamel EPR measurements or analyses of di-centrics. Estimated doses of two other adult victims exposed to radiation over a decade prior to analysis (16 or 33 years ago) were underestimated and two to three times lower than the values obtained from analysis of di-centrics or tooth-enamel EPR. Follow-up analyses of the adolescent victim showed that doses estimated by FISH analysis decrease rapidly over time. Therefore, the accuracy of dose estimates by FISH is acceptable only when analysis is performed less than 7 years after exposure. Measurements carried out more than a decade after exposure through FISH analysis resulted in underestimation of the biological doses compared with values obtained through analysis of di-centrics and tooth-enamel EPR.

Cytogenetic analysis in 16-year follow-up study of a mother and fetus exposed in a radiation accident in Xinzhou, China.

In November 1992, a radiation accident occurred in Xinzhou, due to the collection by a farmer of an unused (60)Co source; 37 individuals were exposed to ionizing radiation. Three individuals died and the farmer's 19-weeks-pregnant wife suffered acute radiation symptoms. Conventional chromosome analysis, cytokinesis-block micronuclei (CBMN) assay and fluorescence in situ hybridization (FISH) painting with three pairs of whole chromosome probes were used to analyze chromosomal aberrations for the pregnant female and her baby during the 16 years following the accident. The yields of dicentrics and rings (dic+r) continually declined between 41 days and 16 years after the accident. The frequency of binucleated MN also decreased over time for both mother and daughter. Sixteen years after exposure, the yields of dic+r and binucleated MN decreased to normal levels, but the reciprocal translocation frequencies remained elevated, for both mother and daughter. FISH results showed a decreasing yield of translocations with time. Based on the changes in maternal translocation frequency, the daughter's dose at the time of exposure was estimated as 1.82 (1.35-2.54)Gy. This was consistent with the clinical manifestations of severe mental retardation and low IQ score. FISH-based translocation analysis can be used for follow-up studies on accidental exposure and, after correction, for retrospective dose estimation for individuals prenatally exposed to radiation.

Identification of two novel mitochondrial DNA deletions induced by ionizing radiation.

OBJECTIVE: We identify ionizing radiation-induced mitochondrial DNA (mtDNA) deletions in human lymphocytes and their distribution in normal populations. METHODS: Long-range polymerase chain reactions (PCR) using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy (60)Co γ-rays. Limited-condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long-range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy (60)Co γ-rays, and real-time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated. RESULTS: Two mtDNA deletions, a 7455-bp deletion (nt475-nt7929 in heavy strand) and a 9225-bp deletion (nt7714 -nt369 in heavy strand), occurring between two 8-bp direct repeats, were identified in lymphoblastoid cells using long-range PCR, limited-condition PCR and sequencing. These results were also observed for (60)Co γ-rays irradiated human peripheral blood cells. CONCLUSION: Two novel mtDNA deletions, a 7455-bp deletion and a 9225-bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors' age, but was independent of gender.

Role of dissection of secondary branches of splenic pedicle in portal hypertension cases undergoing splenectomy.

BACKGROUND: It is well known that conventional splenectomy, which requires careful handling and ligation of tissue of the splenic hilum, can easily cause complications such as splenic fever and pancreatic fistula. Here, we use the technique of dissection of the secondary branches of the splenic pedicle to handle the hilum in the portal hypertension patients who are subjected to splenectomy. METHODS: We retrospectively compared and analyzed the complications, postoperative hospital stay, operative time, and occurrence of hemorrhage in 121 patients with portal hypertension undergoing splenectomy and devascularization of the gastric cardia from January 1999 to December 2007. The selected cases consisted of 51 patients undergoing conventional splenectomy and 70 patients undergoing dissection of secondary branches of the splenic pedicle. In addition, we analyzed the relationship between size of the spleen and occurrence of complications. RESULTS: The incidence of pancreatic fistula and splenic fever (0/70 and 9/70) was lower in patients undergoing dissection of secondary branches of the splenic pedicle as compared with that of the conventional group (5/51 and 18/51 respectively). In addition, there was no significant difference in operative time and volume of blood loss between two groups. The spleen thickness of those patients who had pancreatic fistula and splenic fever was significantly greater than those without complications. CONCLUSIONS: These results indicate that dissection of secondary branches of the splenic pedicle in portal hypertension patients undergoing splenectomy can decrease the incidence of splenic fever and pancreatic fistula, and shorten the postoperative hospital stay, especially in the patients with a large spleen. So dissection of secondary branches of the splenic pedicle is a valuable technique for splenectomy.

Detection of chromosome aberrations in Chinese children with autism using G-banding and BAC FISH.

OBJECTIVE: To detect the characteristic chromosomal changes in Chinese children with infantile autism. METHODS: Chromosome aberrations in 68 cases of infantile autism were analyzed by high-resolution G-banding and fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones. RESULTS: Chromosomal changes were detected in 4 cases by high-resolution G-banding: one case with t(4;6)(q23-24;p21), one case with longer p arm of chromosome 21 (21p+), and two cases with pericentric inversion of chromosome 9 (inv(9)) which was confirmed by C-banding. BAC FISH analysis was performed to confirm these observations and changes in chromosomes 2, 7 and 15, which are often found in autistic children. There could exist the translocation of t(4;6) (q25-26;p21.1). Chromosome changes often reported previously in chromosomes 2, 7 and 15 were not detected in this study. Inv(9) and 21p+ were not confirmed with present BAC clones. CONCLUSION: Chromosomal changes were detected in four cases of infantile autism, with a detectability of 5.9% , far lower than that (10% to 48%) reported in literature. The breakpoint of translocation could be detected more accurately using BAC FISH method.