High concentration of gamma-aminobutyric acid in pancreatic beta cells.

The gamma-aminobutyric acid (GABA) concentration of pancreatic islets in rats treated with streptozotocin (STZ) and of human insulinoma tissue was studied. Seven hours after the administration of 65 mg/kg body weight of STZ, a distinct increase in serum insulin concentration and at the same time a decrease in blood glucose level were seen. Twenty-four hours after the injection of STZ, however, the level of serum insulin decreased much, whereas that of blood glucose increased considerably. On the other hand, the GABA concentration of the islet was reduced dramatically to about one-tenth the control level after both 7 and 24 h. The histologic investigations of the islets revealed the destruction of B cells but no changes in A and D cells 7 and 24 h after the treatment of STZ. Nerve fibers and nerve endings in the islets were preserved intact all through the study. The GABA and insulin contents of the two cases of human insulinoma were determined. One insulinoma, which was compactly occupied with B cells according to its histologic features, contained a high concentration of GABA. The other tumor, having a rather sparse distribution of B cells in it as compared with the former case, possessed a lower concentration of GABA, but it was still high compared with that of its surrounding tissues. The present observations indicate that a large amount of GABA is available in the B cells of the pancreatic islets.

Expression of fas (CD95/APO-1) antigen induced by radiation therapy for diffuse B-cell lymphoma: immunohistochemical study.

Most malignant lymphomas show relatively high degrees of radiosensitivity, in which apoptosis has been shown to play an important role. Recently, the Fas (CD95/APO-1)/Fas ligand system has been identified as a key regulator of apoptosis in some types of lymphoma cell lines. In this study, we aimed to determine whether Fas antigen expression is induced by radiotherapy for malignant lymphoma and to clarify its possible correlation with the therapeutic effect of radiation therapy. Fifty-six patients with tumors of the tongue, oropharynx, and maxillary sinus were examined; four were confirmed as malignant lymphoma, and the rest were identified as squamous cell carcinoma. After obtaining the patients' informed consent, biopsies were performed before treatment and at doses of 4, 10, and 20 Gy of radiotherapy, and specimens were preserved in liquid nitrogen until further examination. Serial sectioning of 6 micrometer was performed using a cryostat, and samples were immunohistochemically stained using the streptoavidin-biotin peroxidase method and a monoclonal antibody against Fas. Two of the four patients with malignant lymphoma showed Fas antigen expression on their tumor tissue at 4 and 10 Gy of radiotherapy. These tumors showed high radiosensitivity and disappeared at a dose of 20 Gy of radiotherapy. In samples from these two patients, DNA ladder formation was identified at 10 Gy. In 52 squamous cell carcinomas, staining for the Fas antigen showed negative or only slightly positive results. However, in one of the cases of squamous cell carcinoma, lymphocytes infiltrating into cancer tissue showed Fas antigen expression at 4 Gy of irradiation, and these lymphocytes disappeared on the tumor tissue at 10 Gy. Therefore, the high radiosensitivity of malignant lymphoma among our samples could be explained by the overexpression of Fas antigen induced by small doses of radiation therapy, and Fas ligand could be produced by infiltrating lymphocytes or may be expressed simultaneously on the lymphoma cells.

Immunohistochemical study of c-fos-positive lymphocytes infiltrated into human squamous cell carcinomas of the head and neck during radiation therapy and its clinical significance.

C-fos has been reported to be one of the immediate early genes in signal transduction systems after many kinds of stresses, including ionizing radiation. Changes in c-fos expression induced by radiation therapy in tumor tissues have not yet been reported. In this study, we have attempted to determine whether c-fos expression is induced by radiotherapy in human squamous cell carcinomas of the head and neck and to establish a possible correlation between c-fos expression and the therapeutic effects of radiation therapy. Twenty-seven patients with tumors of the oral cavity, oropharynx, and maxillary sinus were examined, all of which were confirmed as squamous cell carcinomas. After obtaining the patients' informed consent, biopsies were performed before treatment and at doses of 4, 10, and 20 Gy of radiotherapy, and the specimens were preserved in liquid nitrogen for further examination. Serial sectioning of 6 micrometer was performed using a cryostat, and samples were immunohistochemically stained using the streptoavidin-biotin peroxidase method and a monoclonal antibody against c-fos. Three of the 27 patients with squamous cell carcinoma showed slight expression of c-fos in their tumor cells before and/or at 4 or 10 Gy of radiotherapy. The tumors showed high radiosensitivity. Concerning tumor-infiltrating lymphocytes, the rate of moderate or remarkable grades of c-fos-positive lymphocytes before radiotherapy and at radiation doses of 4, 10, and 20 Gy was 8.0, 29.2, 4.8, and 0%, respectively. The relationship between the immunohistochemical findings and the antitumor effect at a radiation dose of 20 Gy was examined on the corresponding H&E-stained sections. In patients whose infiltration of c-fos-positive lymphocytes into tumor tissues were moderate or remarkable at 4 Gy of radiotherapy, the tumors responded significantly well to radiation therapy (P < 0.025, chi2 test), and the patients took a significantly favorable clinical course (P < 0.05, chi2 test). In a sample from one of the patients, c-fos-positive lymphocytes were identified as CD4 positive and CD8 negative. Therefore, the high radiosensitivity of squamous cell carcinomas in our samples could be explained by an overexpression of c-fos in the tumor-infiltrating lymphocytes induced by small doses of radiation therapy, and these activated lymphocytes exerted a cytotoxic effect against the cancer cells.

Evaluation of neutrophil structure and function by electron microscopy: cytochemical studies.

Methods for studying human neutrophils at the ultrastructural level by enzyme cytochemistry and immunocytochemistry are presented. The focus of these methods is on the analysis of the alkaline phosphatase-positive secretory organelle of these cells. These methods provide unique information which, when coupled with biochemical studies, provide for the most complete analysis of neutrophil structure and function.

Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry.

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).

Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH.

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.

Biochemical properties and cytochemical localization of ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase activity in rat atrial myocytes.

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3. Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).

The relaxant effect of adrenomedullin on particular smooth muscles despite a general expression of its mRNA in smooth muscle, endothelial and epithelial cells.

1. By use of the reverse transcription polymerase chain reaction (RT-PCR), we determined the expression of adrenomedullin (AM) mRNA in the various tissues of the pig. To evaluate the significance of the expression of AM mRNA, we also determined the effects of AM on the cytosolic Ca2+ concentration ([Ca2+]i) and tension development of the porcine smooth muscle strips obtained from the coronary artery, pulmonary vein, trachea, ileum and urinary bladder. 2.AM mRNA was widely expressed in the porcine tissues examined, which included myocardium (left and right ventricle and right atrium), kidney, lung, endothelial cells (aorta and aortic valve), smooth muscles (aorta, main pulmonary artery, pulmonary vein, renal artery and vein, coronary artery, ileum, trachea and urinary bladder) and epithelial cells (trachea and urinary bladder). 3. AM induced a decrease in [Ca2+]i and tension of the coronary artery, but not the pulmonary vein. AM had no effects on either the [Ca2+]i or tension of the trachea and urinary bladder strips or on the tension development of strips of ileum. 4. These results indicated that AM has a role as an autocrine and/or paracrine regulator of the coronary arterial tone. AM probably does not have an important role in the regulation of the pulmonary venous, tracheal, ileac and urinary bladder smooth muscle tone, even though AM mRNA is expressed in these tissues; the functional significance of AM in these smooth muscles remains to be determined.

The mechanism of relaxation induced by atrial natriuretic peptide in the porcine renal artery.

1. The mechanisms underlying the relaxation of the porcine renal artery induced by atrial natriuretic peptide (ANP) were investigated, using front-surface fluorimetry with fura-2 and receptor-coupled permeabilization by alpha-toxin. 2. ANP decreased the cytosolic Ca2+ concentration ([Ca2+]i) and tension during the contraction induced by a high external K+ solution, in a concentration-dependent manner. This ANP-induced decrease in [Ca2+]i during the contraction induced by high K+ solution was composed of two phases, an initial rapid phase, followed by a maintenance phase. The initial rapid decrease in [Ca2+]i, but not the maintained decrease in [Ca2+]i, was inhibited when the tissue was treated with thapsigargin, a selective Ca2+ pump inhibitor of the sarcoplasmic reticulum. When the tissues were treated with thapsigargin and external Ca2+ was replaced by Ba2+, which cannot be transported by the Ca2+ pump, ANP did not induce a decrease in [Ba2+]i, even though the elevation of tension induced by Ba2+ was strongly inhibited. 3. In the absence of extracellular Ca2+, ANP inhibited the release of Ca2+ from the intracellular store induced by noradrenaline (NA). 4. The [Ca2+]i (abscissa scale)-tension (ordinate scale) relationship observed during the contraction induced by various concentrations of high external K+ solution was shifted downwards by the addition of 10(-8) M ANP, indicating that, at any given [Ca2+]i, the tension generated by high K+ solution was considerably inhibited by the addition of 10(-8) M ANP. The [Ca2+]i-tension curve of the contraction obtained by the cumulative application of external Ca2+ (0-3.75 mM) during depolarization with 118 mM K+ solution was shifted to the left by 3 x 10(-7) M NA. This NA-induced [Ca2+]i-tension relationship was shifted to the right by 10(-8) M ANP, indicating that the ANP-induced reduction of Ca(2+)-sensitivity operates during the contraction induced by NA. 5. In alpha-toxin-permeabilized preparations, ANP induced relaxation of tissues precontracted with a mixture of 3 x 10(-7) M Ca2+, 10(-5) M guanosine 5'-triphosphate (GTP) and 10(-6) M NA. Thus a component of ANP-induced relaxation took place by way of a reduction in the Ca2+ sensitivity of the myofilaments, independent of changes in [Ca2+]i. 6. These results indicate that ANP induces relaxation of the porcine renal artery by: (1) reducing [Ca2+]i mainly via the activation of the Ca2+ pumps located on the sarcoplasmic reticulum and sarcolemma, as well as via inhibition of agoinist-induced release of Ca2+ from the intracellular store; and (2) decreasing the Ca(2+)-sensitivity of the contractile elements.

Expression of mucinous ovarian-cancer antigen in hybrid-cells derived by fusing a malignantly transformed bloom-syndrome cell-line (bs-shi-4m ovc-mu) and mouse L-cell-line.

The expression of mucinous ovarian cancer (OVC) antigen by hybrid cell lines derived by fusing a malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M OVC-MU) and mouse L cells has been studied. Cell surfaces of 16 hybrids which grew 12-40 days post-fusion have been analysed by using a number of sera from mucinous OVC patients and indirect membrane immunofluorescence (IF). Three hybrids which appeared at a late stage (30-40 days in 24-well culture plates) were clearly positive in almost 100% of the hybrid cell population, while all of those which appeared earlier (in 12 days) were completely negative. Cytogenetic analysis showed that these three hybrids with positive mucinous OVC antigen contained human chromosome 22 or 22q-, though no metaphases from antigen negative population had 22 or 22q-. Therefore, we assign the gene for mucinous OVC antigen to the chromosome 22. Identification of chromosome 22 in hybrid cells was confirmed by the fluorescence in situ hybridization (FISH) analysis using chromosome 22 painting probe.

Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes.

We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.

The fate of the luminal asymmetric unit membrane of the superficial cell of the rat transitional epithelium.

The fate of the luminal asymmetric unit membrane (AUM) of the superficial cell of the transitional epithelium of the rat urinary bladder was electron microscopically and cytochemically investigated using exogenously administered horseradish peroxidase (HRP) as tracer. HRP-positive discoidal vesicles were formed by the folding of the AUM of the luminal surface plasma membrane. With the passage of time, these vesicles changed their shapes and were finally transformed into lysosomes by the following possible routes: 1) by becoming multivesicular bodies (MVBs); 2) by directly fusing with lysosomes; and 3) by becoming autophagic vacuoles. Another possibility would be reutilization.

Ultrastructural and immunocytochemical study of the leptomeres in the mouse cardiac muscle fibre.

The three-dimensional configuration and chemical composition of leptomeres in the mouse ventricular cardiac muscle fibre were electron microscopically and immunocytochemically investigated using semithin sections and specific antibodies against actin, the intermediate filament proteins desmin and vimentin, and the actin-binding proteins alpha-actinin, filamin and vinculin. The leptomeres appeared columnar in shape and periodically segmented by electron-dense disc-like septa. The electron-lucent areas between these septa were composed of fine interlinked filaments running obliquely to the major axis of the leptomere. Actin was localized in the electron-dense lines of the leptomeres but not in the fine filaments. No reaction was, however, detected for desmin, vimentin, vinculin, filamin and alpha-actinin. The present results suggest, therefore, that the leptomeres may not have a contractile function.

Ultracytochemical study of trimetaphosphatase activity during acrosomal formation in the mouse testis.

The localization of Trimetaphosphatase (TMPase) activity during the acrosomal formation in the mouse testis was enzyme cytochemically investigated by the cerium-salt method. In addition to the lysosomes of the Sertoli cells and the spermatogenic cells in the seminiferous tubules, positive TMPase activity was detected in the Golgi complex and in the acrosomal vesicles of the spermatids, as well as in the acrosomes of both spermatids and spermatozoa. In the Golgi complex of the spermatids, TMPase activity was observed in the first one or two lamellae of the trans-face and in the small vesicles in the vicinity of the Golgi complex. TMPase positive reaction was also detected in the acrosomes of the spermatozoa in the lumina of both the seminiferous tubules and the epididymal duct. The localization of this enzyme activity was compared with that of acid phosphatase (ACPase), as detected by the cerium-based method, using beta-glycerophosphate as substrate: ACPase activity was completely absent from the Golgi complex, small vesicles, acrosomal vesicle and acrosome throughout the entire process of acrosomal formation. TMPase is thought to become one of the acrosomal components, and may be involved in the acrosomal reaction during fertilization.

Fibronectin expression in cancer tissues from patients undergoing radiation therapy.

Fibronectin expression and distribution were examined in cancer tissues from 19 patients with cancer of the head and neck regions. Samples taken before and after irradiation of approximately 10 Gy, 20 Gy or 30 Gy were analyzed by the avidin-biotin-horseradish peroxidase method using mouse monoclonal antibodies against human fibronectin. The results were correlated with the patient's prognosis after radiation therapy. No remarkable changes in the fibronectin expression or distribution were found between tissue specimens taken before and after each dose of irradiation. The prognosis, however, varied according to the degree of expression and the distribution pattern of fibronectin. Seven patients in which the cancer tissue was encircled by a thick fibronectin network are still alive without recurrence 4.5-6 years after treatment, whereas 6 patients in which fibronectin was only faintly expressed or focally distributed died or developed recurrence soon after treatment. The present findings demonstrate that fibronectin expression and distribution in cancer tissue are intimately related to the patient's prognosis, and that the analysis of these two parameters is applicable as a predictive assay in radiotherapy of cancer of the head and neck regions.

Fine structure of the acinar and duct cell components in the parotid and submandibular salivary glands of the rat: a TEM, SEM, and HRSEM study.

Parenchymal and stromal components of the rat parotid and submandibular glands were examined by conventional and high resolution scanning electron microscopy (HRSEM). Freeze-fractured specimens were subjected to HCl and NaOH extraction procedures to better differentiate connective tissue and cellular components. In addition, the internal three-dimensional morphology of the secretory acinar cells and duct cells was revealed by maceration with a dilute osmium tetroxide solution to selectively remove some of the cytoplasmic components. SEM and HRSEM examination of the HCl-treated samples of both glands revealed a fine filamentous network immediately surrounding each acinus. Coarser bundles of collagen that linked neighboring acini were also observed. NaOH-extracted samples selectively removed the cellular components and showed more clearly the three-dimensional structure of the connective-tissue stroma. A dense-collagenous network surrounded each lobule while more internal regions consisted of a honeycomb-like pattern of evacuated spaces previously occupied by secretory acini. These spaces were smoothened in appearance and often interconnected. Apically-located secretory granules and profiles of the rough endoplasmic reticulum and Golgi apparatus in perinuclear regions were encountered in the acinar and duct cells of macerated samples by HRSEM. In addition, a phenylephrine-induced experimental condition performed in some rats resulted in a significant increase in secretory granule size and density of the serous cells.

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.

Na-K-ATPase activity in the guinea pig stria vascularis in experimentally-induced endolymphatic hydrops.

The effect of endolymphatic hydrops on the Na-K-ATPase activity in the guinea pig stria vascularis was electron microscopically and enzyme cytochemically investigated one year after experimental induction. The morphological observations revealed intercellular dropsy in the basal infoldings of the marginal cells, and shrinkage and disappearance of intermediate cells. Moreover, shrinkage of the marginal cells, especially of the basal infoldings, was occasionally observed. In spite of these morphological alterations, the Na-K-ATPase activity was still detected on the plasma membrane of the basal infoldings of most marginal cells. No remarkable differences were found among the cochlear turns of the specimens examined. However, no reaction product was detected on the basolateral plasma membrane of severely degenerated marginal cells. The present results indicate that the Na-K-ATPase of the plasma membrane of the basal infoldings of the marginal cells plays an important role in the maintenance of the unique ion concentration of the endolymph even in the endolymphatic hydropic condition, and that the Na-K-ATPase activity is attenuated in severely atrophic cells.

An ultracytochemical study on the dynamics of alkaline phosphatase-positive granules in rat neutrophils.

Alkaline phosphatase (ALPase) activity was examined by cerium-based ultracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 ng/ml PMA or 10(-7) M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.

Induction of DNA fragmentation by total-body irradiation in murine liver.

Total-body irradiation (TBI) is an accepted modality to treat patients with disseminated tumors. The influence of the treatment on normal tissues is evaluated using mice by measuring the rate of the induction and distribution of apoptosis, as well as DNA fragmentation which occurs in the murine liver within hours of irradiation. Unanesthetized female C3H/He mice were exposed to gamma-ray TBI of 2, 7, and 20 gray (Gy) delivered from 60Co at a dose rate of 114 cGy/min. Frozen sections of livers which were excised from the animals at various times after irradiation were stained by hematoxylin-eosin (H-E) to count numbers of apoptotic cells, or were examined to detect DNA fragmentation. The percentages of apoptotic cells and length of the period during which the maximum levels of the percentages were exhibited showed a dose-dependent increase in the sections stained with H-E. No positive cells for 3'-OH ends of fragmented DNA were found in the liver before TBI, whereas positive cells were observed immediately after irradiation without dose-dependency, these positive cells returned to nearly basal levels after several hours. Positive cells were observed prior to showing apoptosis, suggesting that DNA fragmentation occurs immediately after TBI independent of apoptosis. The difference in the time courses between induction of DNA fragmentation and of apoptosis was not observed in other organs or in the samples treated with the detergent. These results suggested that the 3'-OH ends newly generated by TBI were masked by a detergent-soluble DNA-binding molecule which might be preferentially present in the murine liver.