Radiation dose-dependent increases in inflammatory response markers in A-bomb survivors.

PURPOSE: The well-documented increases in malignant tumours in the A-bomb survivors have recently been supplemented by reports that non-cancer diseases, including cardiovascular disease, may also have increased in incidence with increasing radiation dose. Given that low-level inflammatory responses are widely accepted as a significant risk factor for such diseases, we undertook a detailed investigation of the long-term effects of ionizing radiation on the levels of the inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6) in A-bomb survivors. MATERIALS AND METHODS: Blood samples were taken from 453 participants in a long-term epidemiological cohort of A-bomb survivors. Plasma levels of CRP and IL-6 were measured using standard antibody-mediated procedures. Relationships between CRP or IL-6 levels and radiation dose were then investigated by multivariate regression analysis. Blood lymphocytes from each individual were used for immunophenotyping by flow cytometry with murine monoclonal antibodies to CD3, CD4 and CD8. RESULTS: CRP levels were significantly increased by about 31% Gy(-1) of estimated A-bomb radiation (p=0.0001). Higher CRP levels also correlated with age, male gender, body mass index and a history of myocardial infarction. After adjustments for these factors, CRP levels still appeared to have increased significantly with increasing radiation dose (about 28% increase at 1Gy, p=0.0002). IL-6 levels also appeared to have increased with radiation dose by 9.3% at 1Gy (p=0.0003) and after multiple adjustments by 9.8% at 1Gy (p=0.0007). The elevated CRP and IL-6 levels were associated with decreases in the percentages of CD4(+) helper T-cells in peripheral blood lymphocyte populations. CONCLUSIONS: Our results appear to indicate that exposure to A-bomb radiation has caused significant increases in inflammatory activity that are still demonstrable in the blood of A-bomb survivors and which may lead to increased risks of cardiovascular disease and other non-cancer diseases.

Estimation of mutation rate at human glycophorin A locus in hematopoietic stem cell progenitors.

Surveys of human mutant cells exhibit a few individuals with relatively high "outlying" values, which might be explained by rare mutations occurring during development. To estimate how commonly this occurs, mutant red cell frequencies at the glycophorin A locus in 135 neonates and 109 children and adolescents from three research centers are compared with simulations in which mutations arise from successive cycles of binary fission. The simulations predict the data most accurately when the mutation rate in stem cell precursors is about 2-4 x 10(-7) per division cycle, which is similar to previous estimates from adult stem cell divisions. If these mutation rates are accurate, and the number of stem cell divisions during adult life is as low as previously estimated, it is predicted that up to one-sixth of mutant stem cells over a lifetime arose in early life. However, these mutant stem cells would be difficult to detect in surveys because their distribution within the general population is so skewed.

Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice.

We have studied the effects of a defect in the p53 gene on spontaneous and radiation-induced somatic mutation frequencies in vivo by measuring T-cell receptor (TCR) and hypoxanthine phosphoribosyltransferase (HPRT) mutant frequencies (MFs) in p53 deficient mice both before and after exposure to X-irradiation. In the absence of irradiation, the TCR and HPRT mutant frequencies were roughly two-fold higher in p53 null (-/-) mice than in wild-type (+/+) mice. Unexpectedly, the TCR and HPRT MFs were slightly lower in heterozygote p53 (+/-) than in wild-type (+/+) mice, however. After 2 weeks 2Gy whole body irradiation the TCR and HPRT MFs were about two-fold higher in the p53 null (-/-) and p53 (+/-) mice than in the wild-type. Taken together, these findings suggest that a defect in the p53 gene may lead to TCR and HPRT mutants being recovered at higher frequencies in both irradiated and unirradiated mice, but it should be emphasized that the effects we have observed are not particularly strong, albeit that they are statistically significant. Interestingly, several of the highest TCR MF values that we observed in the course of our experiments were recorded in p53 (-/-) animals that had developed thymomas and hence appeared to be cancer prone.

Hexamethylene bisacetamide protects peritoneal mesothelial cells from glucose.

BACKGROUND: Peritoneal dialysis causes damage to peritoneal mesothelial cells primarily because dialysis fluids have a high glucose concentration. This study examined the abnormalities of gap junctional intercellular communication (GJIC) in human peritoneal mesothelial cells (HPMCs) exposed to relatively high levels of glucose. Also, ability of hexamethylene bisacetamide (HMBA) to up-regulate GJIC in HPMCs exposed to high levels of glucose was measured. METHODS: An assay that monitors the recovery of fluorescence after photobleaching was used to measure GJIC in primary cultured HPMCs. The cells were exposed to a low (10 mmol/L) or high (50 or 90 mmol/L) glucose level for a total of six days, and some cells were also incubated with or without HMBA (1 or 6 mmol/L) from day 4. The effects of incubation in these various environments on expression of the connexin 43 (Cx43) gene were investigated by the reverse transcription-polymerase chain reaction (to detect Cx43 mRNA) or by immunofluorescence and Western blotting (to detect Cx43 protein). To evaluate the influence of protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) on GJIC, specific inhibitors were added to cultures in a high glucose medium. RESULTS: Gap junctional intercellular communication was inhibited in a concentration- and time-dependent manner when cells were exposed to high glucose. The addition of 6 mmol/L HMBA to cultures significantly enhanced GJIC despite the presence of a high glucose concentration. High glucose also down-regulated Cx43 mRNA and protein expression, with the dose-dependent decrease of Cx43 protein at gap junctions paralleled by a decrease in the phosphorylation of this protein. As expected, treatment of cells with 6 mmol/L HMBA increased both Cx43 mRNA and protein levels despite exposure to high glucose. The addition of PKC or MAPK inhibitors to high glucose cultures did not restore GJIC, and there was no significant change of Cx43 phosphorylation in the presence of these inhibitors. CONCLUSIONS: High glucose down-regulates GJIC in human peritoneal mesothelial cells. It also decreases the levels of both Cx43 mRNA and Cx43 protein, with the latter becoming hypophosphorylated. HMBA appears to reverse all of these changes. These results are consistent with our hypothesis that HMBA protects HPMCs from the adverse effects of high glucose by reversing various processes that would otherwise lead to harmful loss of GJIC.

Possible role of natural killer cells in negative selection of mutant lymphocytes that fail to express the human leukocyte antigen-A2 allele.

Increased frequencies of cells carrying mutations at several loci have been found in the blood cells of atomic-bomb (A-bomb) survivors upon testing four or five decades after the bombing. Interestingly, though, we have been unable to demonstrate any radiation-associated increases in the frequencies of mutant blood cells in which human leukocyte antigen (HLA)-A expression has been disrupted; this is true both of preliminary tests on the T cells of a small subset of A-bomb survivors and of the much more extensive study reported here in which we screened a much larger group of survivors for HLA-A2 loss mutations in B cells and granulocytes as well as in T cells. In attempting to explain our inability to detect any increases in HLA-A2-negative cell numbers in HLA-A2 heterozygous individuals exposed to A-bomb irradiation, we decided to test the hypothesis that HLA-A mutant lymphocytes might well have been induced by radiation exposure in much the same way as every other type of mutant we encountered, but may subsequently have been eliminated by the strong negative selection associated with their almost inevitable exposure to autologous natural killer (NK) cells in the bloodstream of each of the individuals concerned. We now report that mutant B lymphocyte cell lines that have lost the ability to express the HLA-A2 antigen do indeed appear to be much more readily eliminated than their parental heterozygous counterparts during co-culture in vitro with autologous NK cells. We make this claim first because we have observed that adding autologous NK cells to in vitro cultures of HLA-A2 heterozygous B or T cell lines appeared to cause a dose-dependent decrease in the numbers of HLA-A2-negative mutants that could be detected over a period of 3 days, and second because when we used peripheral blood HLA-A2 heterozygous lymphocyte cultures from which most of the autologous NK cells had been removed we found that we were able to detect newly-arising HLA-A2 mutant T cells in substantial numbers. Taken together, these results strongly support the hypothesis that autologous NK cells are responsible for eliminating mutant lymphocytes that have lost the ability to express self-HLA class I molecules in vivo, and may well therefore explain why we have been unable to detect increased frequencies of HLA-A2 mutants in samples from any of the 164 A-bomb survivors whose HLA-A2 heterozygote status made their lymphocytes suitable for our tests.

Arrest of human dendritic cells at the CD34-/CD4+/HLA-DR+ stage in the bone marrow of NOD/SCID-human chimeric mice.

Human dendritic cell (DC) precursors were engrafted and maintained in NOD/SCID- human chimeric mice (NOD/SCID-hu mice) implanted with human cord blood mononuclear cells, although no mature human DCs were detected in lymphoid organs of the mice. Two months after implantation, bone marrow (BM) cells of NOD/SCID-hu mice formed colonies showing DC morphology and expressing CD1a in methylcellulose culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The CD34-/CD4+/HLA-DR+ cell fraction in NOD/SCID-hu mouse BM generated CD1a(+) cells that were highly stimulatory in mixed leukocyte reactions in culture with GM-CSF and TNF-alpha. These results suggest a strong potential for NOD/SCID-hu BM to generate human DCs, although DC differentiation may be blocked at the CD34-/CD4+/HLA-DR+ stage. (Blood. 2001;97:3655-3657)

X-rays induce dose-dependent and cell cycle-independent accumulation of p21(sdi1/WAF1).

Cell cycle arrest at the G1 checkpoint is governed by a function of wild-type p53. We assessed the behavior of the sdi1 gene, which codes for a 21kDa potent inhibitor of cdk/cyclins, after X-irradiation. X-irradiation induced sdi1 mRNA accumulation and G1 arrest only in cells possessing wild-type p53. Elevation of p21(sdi1/WAF1) was preceded by p53 accumulation, which occurred despite p53 mRNA constancy in normal cells growing in the log phase. The quantity of accumulated p53 and p21(sdi1/WAF1) was radiation dose dependent. A decrease in the S phase cell population in normal cells observed after irradiation reached a minimum at less-than-maximum levels of p53 and p21(sdi1/WAF1). Furthermore, an accumulation of p53 and p21(sdi1/WAF1) was also observed when cells were synchronized in the G0, G1 and S phase and X-irradiated. These results indicated that an X-ray induced p53 and p21(sdi1/WAF1) accumulation mechanism exists throughout the cell cycle, and that the signal strength induced by X-irradiation is dose-dependent.

T-cell responses to mitogens in atomic bomb survivors: a decreased capacity to produce interleukin 2 characterizes the T cells of heavily irradiated individuals.

Significant decreases in the fraction of lymphocytes that are CD4(+) and increases in serum levels of some classes of immunoglobulin have been reported to occur in atomic bomb (A-bomb) survivors and in victims of the Chernobyl nuclear plant accident. To investigate the long-term effects of nuclear radiation on cellular immunity in more detail, we used limiting dilution assays with peripheral blood mononuclear cell preparations to analyze the T-cell responses of 251 A-bomb survivors exposed to less than 0.005 Gy and 159 survivors exposed to more than 1.5 Gy. The percentages of CD2-positive cells that were capable of proliferating in response to phytohemagglutinin (PHA) in the presence of exogenous interleukin 2 (IL2) did not differ substantially between distally exposed and more heavily exposed survivors. The heavily exposed survivors appeared to possess fewer T cells that were capable of proliferating in response to concanavalin A (Con A) or of producing interleukin 2. Assuming that CD4 T cells were the ones primarily responsible for producing IL2 in response to Con A, we were able to estimate how many cells in any given CD4 T-cell population were actually producing IL2. The results indicated that peripheral blood samples from heavily exposed survivors contained significantly fewer IL2-producing CD4 T cells than did similar samples from distally exposed survivors, indicating that significant exposure to A-bomb radiation may have a long-lasting negative effect on the capacity of CD4 T-cell populations to produce IL2.

NK-mediated elimination of mutant lymphocytes that have lost expression of MHC class I molecules.

Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.

Modelling haemopoietic stem cell division by analysis of mutant red cells.

Data describing the number of human red cells mutated at the glycophorin A locus, measured flow cytometrically, are reported for 752 adults and 49 neonates. The variance increases with age more rapidly than the approximately linear increase in mean. It is postulated that this discrepancy is explained by the known property of asymmetric stem cell division, so that the division of a single mutant stem cell may result in zero, one or two progeny stem cells. A mathematical analysis allows description of this process with three parameters: stem cell number, mean division rate and mutation rate per division. The values of these parameters can not be deduced from the data presented here. However, estimates of either stem cell number or mutation rate from other sources enable deduction of the two other parameters. The mean number of divisions per stem cell per lifetime was estimated to be about 70. This analysis therefore implies that the rate at which blood cell telomeres shorten with age acts as a direct measure of stem cell turnover. Furthermore, it is argued that this low figure implies that mutations occurring during early life, including organogenesis, are relatively important in initiating stem cell-derived malignancy. Finally, the number of human stem cell divisions per lifetime is similar to shorter-lived mammals, suggesting this number is important in the ageing process.

Preferential induction of RET/PTC1 rearrangement by X-ray irradiation.

Ionizing radiation is a well known risk factor of thyroid cancer development, but the mechanism of radiation induced carcinogenesis is not clear. The RET/PTC oncogene, an activated form of the RET proto-oncogene, is frequently observed in papillary thyroid carcinoma (PTC); RET/PTC1, -2 and -3 are known to be the three major forms. High frequencies of RET/PTC rearrangements have been observed in radiation-associated PTC, such as those appearing post-Chernobyl or post-radiotherapy, but the rearrangement types differ between these two populations. We investigated whether a specific type of RET/PTC rearrangement was induced by X-rays in vivo and in vitro. In human normal thyroid tissues transplanted in scid mice, the RET/PTC1 rearrangement was predominantly detected throughout the observation period (up to 60 days) after X-ray exposure of 50 Gy. On the other hand, RET/PTC3 was detected only 7 days after X-irradiation, and no transcript of RET/PTC2 was detected. These results are supported by the results of an in vitro study. The RET/PTC1 rearrangement was preferentially induced in a dose-dependent manner by X-rays within a high dose range (10, 50 and 100 Gy) in four cell lines. On the other hand, RET/PTC3 was induced at a much lower frequency, and no induction of RET/PTC2 was observed. These results suggest that the preferential induction of the RET/PTC1 rearrangement may play an important role in the early steps of thyroid carcinogenesis induced by acute X-irradiation.

Decreased proportion of CD4 T cells in the blood of atomic bomb survivors with myocardial infarction.

Epidemiological studies of the atomic bomb survivors have suggested dose-related increases in mortality from diseases other than cancer. Cardiovascular disease is one such noncancer disease for which increases in both mortality and incidence have been found to be associated with radiation dose. Immunological studies have revealed long-term impairment of T-cell-mediated immunity, especially involving deficiencies of CD4 helper T cells, in atomic bomb survivors. In the present study, we investigated whether decreases in CD4 T cells were associated with myocardial infarction in atomic bomb survivors. Of 1,006 survivors examined to determine the proportion of CD4 T cells in peripheral blood lymphocytes, 18 persons had a history of myocardial infarction. The proportion of CD4 T cells was significantly decreased with increased radiation dose [corrected]. Further, the prevalence of myocardial infarction was significantly greater in individuals with a lower proportion of CD4 T cells. These results suggest that myocardial infarction in atomic bomb survivors may be associated with defects in CD4 helper T cells.

Mutant p53: epigenetic mutator of the T-cell receptor via induction of methylation.

The mechanism and effects of epigenetic alterations in human carcinogenesis are not well understood, except that cancers often have alterations in the methylation status of their genomes. Additionally, human cancers, including aggressive T-cell leukemias and lymphomas, have a high frequency of p53 mutations, particularly missense mutations, which raises the possibility of gain-of-new-function proteins, but the new proteins' oncogenic functions are mechanistically ill-defined. To investigate the mechanisms behind the high prevalence of p53 tumor suppressor gene mutations in aggressive or relapsed T-cell leukemias, we transfected Jurkat cells null for p53 protein with a temperature-sensitive p53 mutant. We showed that this mutant p53 abrogated expression of the T-cell antigen receptor (TCR) by affecting the methylation of an at least 20-kb region of DNA, 5'to the TCR beta-chain gene enhancer region, which includes TCRbetaC1 and betaC2. Expression of the TCR is restored when the temperature is reduced to 32 degrees C, at which temperature the mutant p53 regains wild-type function. The TCR, a common site of dysfunction in T-cell malignancies, is the principal signal transduction moiety controlling both T-cell activation and activation-induced apoptosis. These results suggest a new role for mutant p53-as an epigenetic mutator, bridging p53, methylation, and transcriptional silencing-and suggest novel mechanisms in immunosuppression and cancer progression.

Up-regulation of gap junctional intercellular communication by hexamethylene bisacetamide in cultured human peritoneal mesothelial cells.

Gap junctional intercellular communication (GJIC) is believed to be an important means of regulating cell growth and the malignant potential of tumors. This study examined the effect of hexamethylene bisacetamide (HMBA), a hybrid polar compound and a potent differentiation inducer, on GJIC in cultured primary human peritoneal mesothelial cells. The redistribution of fluorescence after photobleaching was used to detect GJIC. After the incubation of confluent cell cultures with 3 or 6 mM HMBA for 3 and 6 days, GJIC was significantly increased in a concentration-dependent manner compared with cultures without HMBA. Western blotting showed that connexin 43 (Cx43), the major functional protein of gap junctions in peritoneal mesothelial cells, was present in unphosphorylated and phosphorylated forms in control cell cultures. The addition of HMBA to cultures induced a significant increase of total Cx43 protein because of an increase of the phosphorylated forms. Immunofluorescence studies showed that HMBA increased the intensity of fluorescence for Cx43 at cell membrane borders. Quantitative reverse transcription and PCR analysis revealed that the addition of HMBA to cultures resulted in the concentration-dependent up-regulation of mRNA for Cx43. These results indicate that HMBA induces the enhancement of GJIC in peritoneal mesothelial cells through both the up-regulation of Cx43 messages and an increase of post-translational phosphorylation. HMBA may contribute to the maintenance of cellular homeostasis through the up-regulation of GJIC.

In vivo somatic mutations in Werner's syndrome.

The frequencies of mutant erythrocytes with loss of heterozygosity at the glycophorin A (GPA) locus and mutant CD4+ T cells lacking surface expression of the T-cell receptor alphabeta (TCR)/CD3 complex were measured by flow cytometry for Japanese Werner's syndrome (WRN) patients. The hemizygous and homozygous GPA mutant frequencies (GPA Mfs) and the TCR/CD3-defective mutant frequency (TCR Mf) in WRN patients were found to be significantly higher than those in normal controls in the same age range. However, because these Mfs in the patients are only about twice those in controls, it is difficult to conclude that the WRN gene mutations cause instability of somatic genes. This contrasts markedly with Bloom's syndrome (BLM) patients, whose GPA and TCR Mfs were previously reported to increase about 50- and 15-fold, respectively. The difference in Mfs is one aspect of the large variation in the phenotype observed between WRN and BLM patients, suggesting a different role of the responsible genes, both of which belong to the RecQ DNA helicase gene family, in the control of somatic mutagenesis.

[Long-term effects of chronic radiation exposure on the level of somatic mutations in peripheral blood cells]. / Vliianie khronicheskogo radiatsionnogo vozdeistviia na uroven' somaticheskikh mutatsii v kletkakh perifericheskoi krovi liud ei v otdalennye sroki.

The level of comatic mutations was studied at GPA locus and in the TCR system at late times since the onset of radiation exposure in persons exposed to chronic radiation accidents at the Mayak Production Association in the period 1949-1952. Individuals with predominantly external gamma-exposure and those with predominantly internal (mainly due to 90Sr) exposures have been identified among the exposed population. The average dose to red bone marrow made up 121.5 cSv with individual values ranging from 11.0 to 462.7 cSv. An increased frequency of TCR-mutant lymphocytes was noted for exposed subjects. The level of GPA-variant erythrocytes of different types in the study group did not differ from the respective value registered in control. The analysis of TCR status showed no dependence of mutant cell frequency on exposure dose. The assumption is made that the rate of exposure dose and the nature of its formation (chronic exposure) exert a decisive influence on the level of somatic mutations in peripheral blood cells in humans.

Lifespan of human memory T-cells in the absence of T-cell receptor expression.

To evaluate the intrinsic lifespan of human memory T-cells in the absence of T-cell receptor signaling, we used radiation-induced mutant CD4+ T-cells lacking surface expression of TCR/CD3 complex as an in vivo cell marker. We analyzed the long-term kinetics of TCR/CD3 - mutant T-cells among CD4+ CD45RA+ naive and CD4+ CD45RA- memory T-cell fractions in peripheral blood of gynecological cancer patients receiving radiotherapy. Both the proportion and number of these mutant T-cells decayed exponentially with time following radiotherapy. The estimated half-life of mutant memory T-cells was 2 to 3 years and did not differ from that of mutant naive T-cells. These results indicate that the lifespan of mature CD4+ T-cells is limited regardless of their memory or naive phenotype in the absence of TCR/CD3 expression. This finding may suggest that continued T-cell receptor signaling is required for lifetime maintenance of human memory T-cells.

Flow cytometry measurements of subsets of T, B and NK cells in peripheral blood lymphocytes of atomic bomb survivors.

Previous studies of blood cells from atomic bomb survivors have shown that frequencies of chromosome aberrations and somatic mutations are elevated in heavily exposed survivors and that T-cell functions and the number of mature T cells are decreased in the survivors who were exposed to radiation as adults. Current progress in flow cytometry allows a sophisticated analysis of various subsets of T, B and NK cells. In the present study, proportions of such subsets in peripheral blood lymphocytes from atomic bomb survivors (159 survivors estimated to be exposed to > or =1.5 Gy) and 252 controls were measured using multiple combinations of monoclonal antibodies to lymphocyte differentiation antigens to investigate whether the previous radiation exposure had altered the composition of the subsets. Among T-cell subsets, the proportion of CD4+ T-cell subsets was decreased significantly in the heavily exposed survivors; this tendency was apparent for the CD4+CD45RA+ naive T-cell subset. However, there were no significant differences in the proportions of CD8+ T-cell subsets between the exposed survivors and controls. As for the B-cell subsets, the proportion of both CD5+ and CD5 B cells as well as CD23+ and CD23- B cells increased in the heavily exposed survivors. Further, no effect of radiation was found in the proportion of NK-cell subsets. These results strongly suggest that previous radiation exposure altered the composition of T and B cells in the peripheral blood of atomic bomb survivors, and they raise the possibility that atomic bomb radiation may have affected the developmental processes of T and B cells.

Radiation sensitivity of human hair follicles in SCID-hu mice.

We developed an experimental model for studying the growth and epilation of the human hair follicle by implanting human scalp tissue onto immunodeficient C.B-17 scid/scid mice. The skin grafts showed continuous growth of black human hairs for at least 1 year and maintained the normal histological structure of a human hair follicle and other tissues associated with the skin. Using this in vivo model, we evaluated the effect of irradiation on the function of human hair follicles. Localized X irradiation (1 to 6 Gy) induced hair loss dose-dependently and synchronously in the third week after irradiation. The hairs undergoing epilation showed a gradual decrease in width toward the root. The minimum width at the thinnest portion of the surviving hair 4 weeks after irradiation suggested that epilation resulted from the breaking of hairs when the hair width decreased to less than 20 microm. After the highest-dose irradiation, the normal structure of the hair bulb was totally abrogated, and long and narrow epithelial tissues associated with regressed papillary cells remained. The surviving epithelia were morphologically similar to the outer epithelial sheath of the follicle associated with palisadic basal cell layers. In the third week some cells in the basal layers of the surviving epithelium in each follicle expressed proliferating cell nuclear antigen. By about 9 weeks after irradiation, the complete structure of the follicle regenerated, with hair growth activity even in the grafts irradiated at the highest dose, although about 30% of the hairs did not regrow. These findings suggest that follicular stem cells that survive high-dose exposure in the sheath-like epithelial tissue can reproduce the complete follicle structure. This animal model can be used to assess the effects of radiation exposure on human skin and to identify and characterize human follicular stem cells.