Iron overload and heart fibrosis in mice deficient for both beta2-microglobulin and Rag1.

Genetic causes of hereditary hemochromatosis (HH) include mutations in the HFE gene, a ss2-microglobulin (ss2m)-associated major histocompatibility complex class I-like protein. Accordingly, mutant ss2m(-/-) mice have increased intestinal iron absorption and develop parenchymal iron overload in the liver. In humans, other genetic and environmental factors have been suggested to influence the pathology and severity of HH. Previously, an association has been reported between low numbers of lymphocytes and the severity of clinical expression of the iron overload in HH. In the present study, the effect of a total absence of lymphocytes on iron overload was investigated by crossing ss2m(-/-) mice (which develop iron overload resembling human disease) with mice deficient in recombinase activator gene 1 (Rag1), which is required for normal B and T lymphocyte development. Iron overload was more severe in ss2mRag1 double-deficient mice than in each of the single deficient mice, with iron accumulation in parenchymal cells of the liver, in acinar cells of the pancreas, and in heart myocytes. With increasing age ss2mRag1(-/-) mice develop extensive heart fibrosis, which could be prevented by reconstitution with normal hematopoietic cells. Thus, the development of iron-mediated cellular damage is substantially enhanced when a Rag1 mutation, which causes a lack of mature lymphocytes, is introduced into ss2m(-/-) mice. Mice deficient in ss2m and Rag1 thus offer a new experimental model of iron-related cardiomyopathy.

The small-cell variant of mycosis fungoides. A clinicopathological and quantitative electron microscopic study on 14 patients.

Small-cell variants of Sézary syndrome and mycosis fungoides (MF) have been described. However, in these studies the nuclear area of the small-cell variant of MF (SC-MF) as compared to histological classical MF (CL-MF) was not characterized objectively by quantitative electron microscopy. In a 14-year follow-up period, of a total of 76 patch/plaque stage MF patients seen in the Department of Dermatology of the University Hospital Utrecht, 14 (18%) had an infiltrate composed of atypical lymphocytes characterized by a distinctly smaller cell diameter and smaller, hyperchromatic, deeply indented nuclei as compared to the usual cell type of MF. The aim of the investigation was to confirm this observation objectively using quantitative electron microscopy (morphometry) and to define SC-MF as compared to CL-MF. The study was performed on the 14 patients with SC-MF, and 10 patients with clinical and histological CL-MF and 4 patients with chronic eczema. Electron micrographs of sections obtained from each biopsy were analysed by computer to produce the following data: a nuclear contour index (NCI), the mean nuclear area (MNA), the mean nuclear area of the cells above the 75th percentile (P75NA) and the percentage of cells larger than 30 microm2. The values of MNA differed significantly between patients with SC-MF and those with CL-MF (17.6 vs 23.2 microm2; P = 0.02), as did the values of P75NA (20.7 vs 27.9 microm2; P = 0.01). The NCI of the SC-MF and CL-MF patients were similar. These results are consistent with our observations that SC-MF does indeed exist.

Heterogeneity of epithelial cells in the rat thymus.

The morphological heterogeneity of the thymic epithelium has been well documented both at the light and electron microscopic level. Immunohistochemistry has revealed four broad classes of epithelial cells (EC): subcapsule/perivascular, cortical, medullary EC, and medullary Hassall's corpuscles. Ultrastructural analysis has revealed further heterogeneity. In the cortex, four EC subtypes have been described ultrastructurally: subcapsular/perivascular, "pale," "intermediate," and "dark" EC. These subtypes are also present in the medulla. Two additional EC subtypes are restricted to the medulla: an undifferentiated subtype, and a subtype displaying signs of high metabolic activity. Based on the morphological features of the epithelium, it has been hypothetized that the thymic EC subtypes represent a process of differentiation.

Effect of lamivudine on morphology and function of mitochondria in patients with chronic hepatitis B.

Nucleoside analogues can induce mitochondrial dysfunction leading to severe clinical syndromes. Lamivudine, a new nucleoside analogue, is an active inhibitor of hepatitis B viral replication without apparent clinical toxicity. To assess subclinical mitochondrial toxicity, we studied the morphology and function of the mitochondrial system in 15 patients treated with lamivudine. Morphology was investigated by routine histological evaluation and electron-microscopic studies of mitochondria in liver biopsy specimens. Mitochondrial function was assessed by 2-keto[1-14C] isocaproic acid decarboxylation (KICA breath test) and by measuring the activity in liver biopsy specimens of the mitochondrial enzymes encoded by nuclear and mitochondrial DNA (mt-DNA) (complex I and IV) as well as a mitochondrial and a cytosolic enzyme both encoded by nuclear DNA only (complex II and lactic dehydrogenase [LDH]). All 15 patients underwent a liver biopsy before treatment and a KICA breath test before and during treatment; 13 agreed to undergo a repeat liver biopsy during lamivudine treatment. Liver tissue with no or minimal fibrotic changes from 7 patients treated for 6 months with lamivudine was suitable for assessment of the mitochondrial enzyme activity. We observed no signs of toxicity by routine histological or electron-microscopic evaluation. KICA breath tests revealed no differences in either peak exhalation or the area under the curve from 0 to 60 minutes between healthy controls (3.0% and 19.3%), untreated patients with chronic hepatitis B (3.4% and 19.3%), and patients treated with lamivudine (3.1% and 20.6%). The activities of the mt-DNA-encoded enzymes remained normal after lamivudine therapy. Unexpectedly a significant decrease in the activity of nuclear-DNA-encoded enzymes in patients with chronic hepatitis B in comparison with normal controls was found. The mean activity of complex II dropped from 45.3 to 20.0 micromol x min(-1), that of lactic dehydrogenase from 106 to 44 micromol x min(-1) (Wilcoxon rank sum; P < .05). In conclusion, no subclinical signs of mitochondrial toxicity resulting from lamivudine therapy for 6 months were observed.

Fibronectin distribution in human bone marrow stroma: matrix assembly and tumor cell adhesion via alpha5 beta1 integrin.

Tumor cell interactions with fibronectin (FN) are important for the development of secondary tumors inside the bone marrow stroma. We studied and compared the in situ distribution of FN in paraffin-embedded human bone marrow sections and investigated the in vitro regulation of FN assemblage by bone marrow stromal cells (BMSC). Finally, the role of FN in the interaction of BMSC with tumor cells was studied. Fine elongated FN-positive cell extensions, probably of stromal cell origin, were observed as well as a limited amount of extracellular FN deposits in connective tissues around capillaries and sinusoids. In vitro studies, using the confocal laser scanning microscope, showed that BMSC produced a high amount of FN with a characteristic extracellular matrix formation in an extensive network. FN matrix formation was predominantly detected at contact sites between cultured BMSC. In in vitro cultures with low cell concentrations and in vivo with a limited number of stromal cell contacts only limited matrix was found. From previous studies it is known that the alpha5 beta1 integrin is involved in the regulation of FN assembly. Here the role of the alpha5-subunit of this integrin was investigated. By using two different monoclonal antibodies (mAb) against the alpha5-subunit (2H6 and mAb16) the assembly of endogenous FN was completely blocked, indicating that these antibodies are directed against the active epitope. Another mAb (mAb11) against the alpha5-subunit did not affect the FN assemblage. Codistribution analysis of alpha5-subunits, alpha v-subunits, actin, and FN demonstrated that the alpha5 beta1 integrin is associated with FN and not with intracellular actin. Integrins alpha(v) beta1, alpha(v) beta3, and alpha(v) beta5, also ligands of FN, did not colocalize with FN. Codistribution of alpha v with the terminal ends of actin and not with FN indicates that alpha(v)-subunits are mainly directed to vitronectin rather than to FN. The dominant role of alpha5 beta1 in FN interaction is underlined by effective blocking of tumor cell adhesion with BMSC using anti-alpha5, anti-beta1, and anti-FN antibodies. These results emphasize the important role of alpha5 integrin subunit in FN matrix assembly in human BMSC and an exclusive role of alpha5 beta1 in the anchorage and regulation of FN-mediated adhesion processes in the bone marrow.

Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I and CD34 expression.

AIMS: To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods. METHODS AND RESULTS: Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and alpha-smooth muscle actin (alpha-SMA) required no pretreatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1. CONCLUSIONS: This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1-1.5 h.

Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.

Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m-/- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.

Alterations of dendritic cells in the rat thymus without epithelial cell loss during cyclosporine treatment and recovery.

After cyclosporine treatment, dendritic cells disappear from the rat thymic medulla. The present study was undertaken to examine the ultrastructural alterations in the dendritic cell population during 14-day cyclosporine treatment and subsequent 6-week recovery. Four dendritic cell subtypes were defined ultrastructurally by a newly developed classification system. In addition, the potential effect of cyclosporine on six medullary epithelial cell subtypes was studied. During cyclosporine treatment, a prominent reduction of dendritic cells was seen at the ultrastructural level, whereas the total number of medullary epithelial cells remained largely unchanged. These findings were confirmed by immunohistochemistry. The number of mature dendritic cells declined later than the number of immature ones. A decrease in the antigen-processing capacity of remaining dendritic cells was suggested by the disappearance of Birbeck granules and the reduced number of tubulovesicular complexes. These findings support a disturbance of clonal deletion during cyclosporine treatment. The dendritic cell alterations appeared reversible 4 weeks after the restoration of the original architecture. During recovery, dendritic cells displaying lysosomal elements outnumbered those found in the normal uninvoluted thymus. This phenomenon probably reflects an enhanced turnover of cell organelles. No treatment-related effect on epithelial cell subtypes was seen.

beta1-Integrins dominate cell traffic of leukemic cells in human bone-marrow stroma.

Migration patterns of leukemic cells in bone marrow are largely regulated by cell contacts between leukemic cells and stromal cells or extra-cellular matrix. The mechanism of this interaction with bone-marrow stromal cells was studied in a human in vitro model. Migration behavior of erythroleukemia cell line K562, derived from a patient with chronic myeloid leukemia, was compared with that of the erythroleukemia cell line HEL92.1.7 and the promyelocytic leukemia cell line HL60 from acute leukemias. Interaction varied between low binding affinity (K562) to intensive cell interaction (HEL92.1.7) followed by invasion into the stromal cell monolayer. Some of the HL60 cells adhered to stromal cells, while the remainder migrated into the stromal cell monolayer. The role of adhesion molecules in these cell interactions was determined. Distinct expression of beta1-integrins ICAM-1, CD44 and VCAM-1 was detected on the different cell lines. Inhibition studies pointed to a dominant role of VLA-4- and VLA-5-mediated interactions. K562 lacked VLA-4 and a low binding affinity of the VLA-5 on these cells resulted in an absence of binding to the bone-marrow stroma. These results indicate the VLA-5/fibronectin, VLA-4/fibronectin and the VLA-4/VCAM-1 interaction pathways between leukemic cells and bone-marrow stroma.

The diagnostic value of morphometry on blood lymphocytes in erythrodermic actinic reticuloid.

BACKGROUND: As the differential diagnosis of erythrodermic actinic reticuloid vs Sézary syndrome (SS) can be very difficult, we examined the value of the nuclear contour index (NCI) on blood lymphocytes as the criterion for differential diagnoses. The NCI is defined as the nuclear parameter divided by the square root of the nuclear area. Three different parameters were studied: mean NCI, percentage of cells with an NCI of 6.5 or greater, and the highest NCI. These indexes were studied on blood lymphocyte samples obtained from 10 patients with erythrodermic actinic reticuloid and were compared with the findings in 10 patients with other benign forms of erythroderma and in seven patients suffering from SS. RESULTS: The patients with erythrodermic actinic reticuloid differed significantly from the group with SS regarding the percentage of cells with an NCI of 6.5 or greater and the highest NCI, but not when the mean NCI was considered. All three parameters revealed nonsignificant results for erythrodermic actinic reticuloid compared with other benign forms of erythroderma. The group with SS differed significantly from the patients with other benign forms of erythroderma regarding all three parameters. By combining three morphometric criteria (mean NCI, > or = 5.5; > 30% lymphoid cells with an NCI of > or = 6.5; and highest NCI, > or = 11.5), all patients with erythrodermic actinic reticuloid or other benign forms of erythroderma and six of the seven patients with SS were correctly classified. CONCLUSION: Our data indicate that assessment of the NCI on peripheral blood lymphocytes is of value in the differential diagnosis of erythrodermic actinic reticuloid vs SS.

The difference in liver pathology between sporadic and familial forms of porphyria cutanea tarda: the role of iron.

Porphyria cutanea tarda is a disorder of porphyrin metabolism, of which familial and sporadic forms have been described. Factors such as iron seem necessary for porphyria cutanea tarda to become clinically manifest. To study the relationship between iron and uroporphyrins in hepatocytes of patients with porphyria cutanea tarda, a morphological and morphometrical study was performed in 13 liver biopsies of patients with porphyria cutanea tarda (eight with sporadic porphyria cutanea tarda and five with familial porphyria cutanea tarda). In addition, possible differences in clinical and biochemical features and in histopathological findings between patients with sporadic porphyria cutanea tarda and familial porphyria cutanea tarda were investigated. Familial porphyria cutanea tarda patients presented at a younger age than sporadic porphyria cutanea tarda patients (42.4 +/- 5.3 vs. 57.3 +/- 8.6 years). Biochemical features were not different between sporadic porphyria cutanea tarda and familial porphyria cutanea tarda patients. Uroporphyrin crystals and a variable degree of liver siderosis were detected in the biopsies of all 13 patients. Uroporphyrin crystals were often found close to ferritin-like iron deposits. The morphometrical analysis showed that an increased mean area fraction of ferritin iron was associated with an increased mean area fraction of uroporphyrin crystals in hepatocytes of sporadic porphyria cutanea tarda and familial porphyria cutanea tarda patients. Moreover, the amount of uroporphyrin crystals was significantly higher in livers of familial porphyria cutanea tarda than sporadic porphyria cutanea tarda patients. These findings are consistent with the hypothesis that uroporphyria is precipitated by an iron-dependent process.(ABSTRACT TRUNCATED AT 250 WORDS)

Nasal T-cell lymphoma: a clinicopathological and immunophenotypic analysis of 13 cases.

Thirteen cases of nasal lymphomas with T-cell or natural killer (NK)-cell phenotype were studied, with attention to clinical presentation and follow-up, the presence of Epstein-Barr virus (EBV) using in situ hybridization (EBER), the immunophenotype, and the presence of cytotoxic granules. All but two patients presented with stage I disease. In three cases local progression resulted in involvement of the central nervous system. When dissemination occurred, this was predominantly to extranodal localizations, in two cases to the skin. Response to therapy was highly variable, but patients treated with radiotherapy with or without additional chemotherapy had a better prognosis than patients treated with initial chemotherapy alone. All lymphomas were associated with EBV, and most cases showed cytotoxic features, ten of which were CD56 positive. In eight cases a T-cell origin was proven, but in five cases a possible NK-cell origin could not be excluded. No clinical differences were seen between true T-cell lymphomas and possible NK-cell neoplasms. Nasal T-cell lymphomas should be considered as a distinct clinicopathological entity, strongly associated with EBV, and with cytotoxic features in most cases. No prognostic parameters were detected to predict dissemination and response to therapy.

Exogenous protoporphyrin inhibits Hep G2 cell proliferation, increases the intracellular hydrogen peroxide concentration and causes ultrastructural alterations.

Ultrastructural hepatocellular abnormalities in early stages of erythropoietic protoporphyria lead to hepatobiliary changes that cause overt liver disease in 5-10% of patients, not infrequently progressing to fulminant hepatic failure. This cannot be attributed solely to protoporphyrin crystal deposition in the liver. Hepatic redox systems have therefore been postulated as an equivalent for the photoreaction of protoporphyrin. We studied the dark effects of protoporphyrin and hematoporphyrin on HL60 and Hep G2 cells. Cell proliferation and intracellular H2O2 concentrations were assessed and related to the ultrastructural morphology. The incubation with protoporphyrin and hematoporphyrin resulted in a dose- and time-dependent inhibition of proliferation indices of Hep G2 cells. Flow cytometric analyses of intracellular H2O2 concentrations demonstrated a dose-dependent increase in both cell lines upon incubation with protoporphyrin. Hep G2 cells displayed ultrastructural alteration of the endoplasmatic reticulum and plasma membranes. Also 'cell blebbing' occurred. We conclude that exogenous protoporphyrin increases the intracellular H2O2 concentration and exerts a cytotoxic dark effect. This may contribute to the liver injury observed in erythropoietic protoporphyria.

Interaction of tumour cells with cultured stromal cells from human bone marrow.

Adhesion of tumour cells to cultured bone marrow stromal cells has been studied in an in vitro model system. Stromal cells were isolated from bone marrow aspirates. Immunohistochemical and electron microscopical analysis revealed a uniform cell monolayer of myofibroblastic cells, expressing fibroblast antigens and smooth muscle actin. Cell interactions with tumour cells lines showed different patterns. The K562 cells bound in low numbers to stromal cells. HEL-DR- and HL60 cells adhered to stromal cells showing an enlarged cell contact area (spreading) attenuated by distinct contact sites and they invaded the monolayer. Adhesion molecules, important for cell contacts, were detected on tumor cells. Different VLA antigens were detected on tumour cells, but on stromal cells only VLA-5 and CD29 were found. In vitro inhibition studies with mAbs against adhesion molecules indicated two major pathways for binding of tumour cells to stromal cells: VCAM-1/VLA-4 and fibronectin/VLA-5. Variation in inhibition of mAbs to VLA-4 and VCAM-1 indicated the existence of critical epitopes in the adhesion of tumour cells.

Accumulation of iron in erythroblasts of patients with erythropoietic protoporphyria.

We have studied the iron metabolism in nine patients with erythropoietic protoporphyria (EPP) and three patients with sideroblastic anaemia (SA). All, except one EPP patient were iron deficient. The SA patients had a secondary haemochromatosis. The bone marrow aspirates of patients with SA and also three patients with EPP had a high incidence of ring sideroblasts. Ultrastructural examination of the bone marrow consistently showed finely dispersed electron-dense deposits localized in mitochondria of erythroblasts in all patients with EPP and SA. Mitochondrial electron energy-loss spectroscopy (EELS) indicated identical iron compounds in erythroblasts of all EPP and SA patients. These findings indicate that the mitochondrial iron utilization is disturbed in EPP and SA. The observation of mitochondrial iron deposition in erythroblasts in EPP and SA suggests that this failure is not of pathognomonic value for diagnosis of SA, but is apparently the result of an inefficient haem synthesis, in EPP due to a defective ferrochelatase. The mitochondrial iron deposition does not depend on the iron status (iron overload or iron deficiency) of the EPP patient.

Involvement of the omental lymphoid organ in the induction of peritoneal immunity against tumor cells.

The omental lymphoid organ (OLO) is a part of the greater omentum composed of vascularized spots containing lymphocytes, plasma cells and macrophages located in a regular pattern between fat cells. To gain insight in the involvement of the OLO in the induction of immunity against tumor cells in the peritoneal cavity, we studied the penetration of tumor cells into the OLO after intraperitoneal, subcutaneous and intravenous injection. Furthermore we analyzed the tumoricidal activity of macrophages isolated from the OLO. Our results indicate that the OLO is only infiltrated by tumor cells directly from the peritoneal cavity, but not by subcutaneously or intravenously injected tumor cells unless they have reached the peritoneal cavity. Bromodeoxy-uridine labelled tumor cells can be detected in the OLO within 10 min after i.p. injection. The penetration is facilitated by the induction of fenestrations between the mesothelial cells (lining the OLO) after intraperitoneal injection of the tumor cells. These fenestrations can also be seen after nonspecific stimulation of the peritoneal cavity. Macrophages isolated from the OLO of mice immunized against syngeneic as well as allogeneic tumor cells express a significant cytotoxicity, which (at least in the syngeneic situation) precedes the cytotoxicity of the macrophages isolated from the peritoneal cavity. In conclusion, our data support the hypothesis that immune reactions against intraperitoneally injected tumor cells are initiated in the OLO leading to 'peritoneal immunity' against these tumor cells.

The cortical epithelium of the rat thymus after in vivo exposure to bis(tri-n-butyltin)oxide (TBTO). An (immuno)histological and ultrastructural study.

Bis(tri-n-butyltin)oxide (TBTO) induces cortical atrophy in the rat thymus. We studied the potential involvement of the cortical epithelium in TBTO-induced thymotoxicity by (immuno) histology and electron microscopy. Juvenile male Wistar rats were orally intubated once with either 30 or 90 mg/kg TBTO and sacrificed 4 or 10 days later. A dose-dependent thymic atrophy occurred. Anti-keratin labelling showed epithelial cell aggregation in some animals at 10 days after exposure to 90 mg/kg TBTO, when recovery of the thymus was apparent. At the ultrastructural level, a relative shift was observed from "pale" to darker epithelial cell subtypes at the 30 mg/kg dose level, both at day 4 and day 10 after intubation. This phenomenon was not observed after exposure to 90 mg/kg TBTO. Both the altered keratin distribution and the increased electron density of the epithelium probably represent non-specific phenomena. The present morphological observations support the concept that TBTO affects the thymus via its action on lymphoid cells rather than on the epithelial compartment.

Ultrastructure of the cortical epithelium of the rat thymus after in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known for inducing cortical atrophy in the rat thymus. The present study was conducted to provide ultrastructural evidence for the cortical epithelium to be a target for TCDD in vivo. Juvenile male Wistar rats were orally intubated once with either 50 or 150 micrograms/kg TCDD and killed 4 or 10 days thereafter. Major changes were found in the cortical thymic epithelium. First, a relative shift occurred from "pale" to darker cortical epithelial cell types, as judged by their nuclear and cytoplasmic electron density. This effect was most prominent at 10 days after exposure to 150 micrograms/kg TCDD. The increased electron density of the cortical epithelium indicates an altered state of cellular differentiation. Secondly, at the 150 micrograms/kg dose level focal epithelial cell aggregates were seen both at day 4 and day 10 after administration. This aggregation may either be compound induced or represent a secondary event to the collapse of the thymic stroma. Thirdly, increased vacuolation of cortical epithelial cells was apparent. This effect is interpreted as a consequence rather than a cause of thymocyte depletion from the cortex. This study indicates that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Electron microscopy reveals that the differentiation of epithelial cells is altered. In addition, epithelial cell aggregates are formed.