Nicotinamide prevents the development of diabetes in the cyclophosphamide-induced NOD mouse model by reducing beta-cell apoptosis.

The development of diabetes in non-obese diabetic (NOD) mice, which normally takes between 3 and 7 months, can be accelerated by cyclophosphamide (CY) injections, with rapid progression to diabetes within only 2-3 weeks. This insulin-dependent diabetes mellitus (IDDM) can be prevented or delayed in CY-treated NOD mice by nicotinamide (NA). The present study was undertaken to determine the mode of cell death responsible for the development of IDDM in CY-treated male NOD mice and to investigate the effect of NA on beta-cell death. Apoptotic beta cells were present within the islets of Langerhans in haematoxylin and eosin-stained sections of the pancreata harvested from 3- and 12-week-old male NOD mice, from 8 h until 14 days after a single intraperitoneal injection of CY (150 mg/kg body weight). The maximum amount of beta-cell apoptosis in 3-week-old animals occurred 1-2 days after CY treatment (20 apoptotic cells per 100 islets), after which time levels of apoptosis declined steadily throughout the 14-day period studied. The incidence of beta-cell apoptosis in 12-week-old male NOD mice occurred in two peaks; the first was recorded 8-24 h after CY treatment (30 apoptotic cells/100 islets), while the second, at 7 days (36 apoptotic cells per 100 islets), coincided with increased insulitis. Administration of NA 15 min before CY treatment, and thereafter daily, substantially reduced the amount of apoptosis and effectively eliminated (4 apoptotic cells per 100 islets) the second wave of beta-cell apoptosis seen at day 7 in 12-week-old animals given CY alone. These results show that apoptosis is the mode of beta-cell death responsible for the development of CY-induced IDDM and that prevention of IDDM by NA is associated with a reduction in beta-cell apoptosis.

Poly(ADP-ribose)polymerase activation determines strain sensitivity to streptozotocin-induced beta cell death in inbred mice.

Streptozotocin (STZ) is believed to induce pancreatic beta cell death in mice by depleting the cell of NAD+NADH. The drug is known to cause a greater depletion of beta cell NAD+NADH in C57bl/6J mice than in Balb/c mice. To investigate the basis for this strain difference, we compared the effects of streptozotocin on poly(ADP-ribose)polymerase (PARP) activation - the major site of NAD consumption, and on mitochondrial activity - the major site of NAD production.%A significant strain difference was demonstrated in STZ-induced PARP activation (fmol NAD incorporated/min/microgram DNA+/-s.e.m.: Balb/c control 2.28+/-0.14, Balb STZ 3.11+/-0.25; C57bl/6J control 2.57+/-0.29, C57bl/6J STZ 4.17+/-0.24). In comparison, no strain difference could be demonstrated in hydrogen-peroxide-induced PARP activation. No strain differences could be detected in the activity of STZ-treated islet mitochondria as measured by determining ATP production (pmol/microgram protein/h+/-s. e.m.: Balb/c control 0.20+/-0.02, Balb/c STZ 0.15+/-0.02; C57bl/6J control 0.23+/- 0.03, C57bl/6J STZ 0.15+/-0.02) or by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction (change in optical density/mg protein+/-s.e.m.: Balb/c control 10.19+/-0.62, Balb/c STZ 6.01+/-1.17; C57bl/6J control 6. 15+/-0.98, C57bl/6J STZ 5.81+/-0.96).% The strain difference in STZ-induced NAD depletion appears to be due to a difference in NAD consumption and not a difference in a mitochondrial process involved in replacing decreasing NAD concentrations. It is unlikely that a strain difference in the enzymic activity of PARP is responsible for strain differences in the effects of STZ, as no strain differences in hydrogen-peroxide-induced PARP activation could be detected. Thus the greater PARP activation, NAD depletion and beta cell death observed in C57bl/6J islets may be due to greater levels of DNA damage or differences in the DNA excision repair processes.

Radiation-induced apoptosis and gene expression in neonatal kidney and testis with and without protein synthesis inhibition.

PURPOSE: To analyse the incidence of radiation-induced apoptosis, expression of two apoptosis-related genes, Bcl-2 and p53, and post-radiation levels of cell proliferation in the neonatal rat (4-5 days old) kidney and testis. MATERIALS AND METHODS: Apoptosis was quantified in control or treated kidney or testis at 2, 4, 6, 8 and 24h after 5 Gy of whole body X-irradiation (n=4 per group). Morphology (light and electron microscopy) and DNA gel electrophoresis were used to assess apoptosis. Temporal and spatial expression of Bcl-2 or p53 were analysed using immunohistochemistry. Administration of cycloheximide (1.5mg/kg) was used to determine whether new protein synthesis had a role in induction of apoptosis. Tritiated thymidine uptake and autoradiography were used to indicate alterations in cell proliferation (radiolabel administered 1 h prior to tissue collection) or S-phase cells undergoing radiation-induced apoptosis (radiolabel administered 1 h prior to irradiation). RESULTS: Apoptosis peaked at 4 h in the testis and 6 h in the kidney and was significantly higher in the renal nephrogenic zone than in the testis (p<0.05). Mitosis was almost completely negated after irradiation in both tissues. A higher proportion (almost fivefold) of the apoptotic cells died in S phase in the kidney than in the testis. Cycloheximide negated induction of apoptosis in the kidney, and markedly decreased apoptosis in the testis. Bcl-2 expression was highest in the differentiated zone of control kidneys and increased after irradiation in the nephrogenic zone, particularly near foci of apoptosis in developing nephrons. In the control testis, Sertoli cells had moderate expression of Bcl-2. After irradiation, there was complete absence of Bcl-2 expression in apoptotic Sertoli cells, with surviving cells increasing Bcl-2 expression. Irradiated kidney had more intense nuclear p53 expression compared with controls. In the testis, p53 that was present in controls continued to be expressed in surviving cells but not apoptotic cells in radiation-treated animals. CONCLUSIONS: Unique differences can be identified between the incidence and biomolecular control of radiation-induced apoptosis in the normal neonatal kidney and testis. These results may find application for minimizing damage to these normal neonatal tissues in the development of, for example, cancer treatment regimens.

Differential metabolite accumulation may be the cause of strain differences in sensitivity to streptozotocin-induced beta cell death in inbred mice.

Inbred strains of mice vary in their sensitivity to the diabetogenic effects of streptozotocin (STZ). To investigate the basis for this strain difference we exposed islet cells from two strains of mice that differ in sensitivity to the drug. We examined them morphologically and measured islet NAD + NADH content, streptozotocin metabolite accumulation, glucose transport capacity, Glut2 levels and medium nitrite accumulation. C57bl/6J mice were more sensitive to STZ than Balb/c mice as judged by the extent of pancreatic insulin depletion and beta cell death, in vivo and in vitro. The mode of cell death was necrosis. After a 30-min in vitro exposure to the drug the more sensitive C57bl/6J islets contained higher levels of streptozotocin metabolites and less NAD + NADH than the more resistant Balb/c islets. The lack of any strain differences in 3-O-methyl glucose transport, Glut2 levels and medium nitrite accumulation suggested that STZ transport and nitric oxide metabolism were not responsible for differences in STZ sensitivity and metabolite accumulation. Thus the strain differences in STZ sensitivity appears to be due to intracellular events within the beta cell occurring after STZ transport and before NAD + NADH depletion. STZ metabolite accumulation appears to be associated with STZ sensitivity. Further studies are warranted to determine if differential STZ metabolite accumulation is responsible for STZ sensitivity.

Expression and localization of the retinoblastoma gene during radiation-induced apoptosis in neonatal rat kidney.

An in vivo neonatal rat kidney model was used to study an association between expression and localization of the retinoblastoma tumor-suppressor gene (Rb), or its protein product (pRb), and localization of radiation-induced apoptosis. The rat kidney has two distinct zones of differentiation at birth-an outer nephrogenic zone, in which cells are undifferentiated and new nephrons are forming, and a differentiated zone internal to this zone that has essentially the adult kidney form. At 6 h after radiation (5 Gy), high levels of relatively synchronous apoptosis are induced in the nephrogenic zone, with little effect on the differentiated zone, and proliferation in the nephrogenic zone is almost totally inhibited by radiation treatment, again with little effect in the differentiated area. We have used our knowledge of this model to analyze control (sham-treated) and irradiated renal tissue for Rb mRNA transcript levels and localization (Northern blot and in situ hybridization (ISH)), pRb expression (Western blot and immunolocalization), apoptosis and mitosis (light and electron microscopy, and DNA gel electrophoresis for apoptosis), and cells in S-phase ([3H]thymidine uptake and autoradiography). Northern blots showed no detectable alteration in Rb transcript levels between control and irradiated tissues, whereas Western blots indicated increased expression of pRb in protein extracted from irradiated kidney compared with controls. ISH confirmed that Rb transcripts were not substantially altered in the nephrogenic and differentiated zones in control versus irradiated renal tissue. Immunolocalization of pRb demonstrated little effect in the differentiated zone, but in the nephrogenic zone pRb expression was increased, especially the S-shaped prenephrons, and was also found in many, but not all, apoptotic cells in this zone. The results link radiation-induced apoptosis and increased pRb expression in a zone of the neonatal kidney having a low level of cell differentiation.

Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse.

The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of beta-cell death responsible for the development of IDDM. Apoptotic cells were present within the islets of Langerhans in hematoxylin and eosin-stained sections of pancreases harvested from 3- to 18-week-old female NOD/Lt mice (a range of 11-50 apoptotic cells per 100 islets). Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Although some islets from age-matched control female NOD/scid mice contained apoptotic cells, virtually all of these cells were insulin negative as determined by immunohistochemistry. The small number of apoptotic insulin-positive cells identified in islets from NOD/scid mice (a range of 0-1 apoptotic cells per 100 islets) was not statistically significant, compared with the numbers recorded in NOD/Lt mice. All dying cells showed the morphological changes characteristic of cell death by apoptosis and stained positively with the TUNEL method for end-labeling DNA strand breaks. The maximum mean amount of beta-cell apoptosis occurring in NOD/Lt mice was at week 15 (50 apoptotic cells per 100 islets), which coincided with the earliest onset of diabetes as determined by blood glucose, urine glucose, and pancreatic immunoreactive insulin measurements. While there was no peak incidence of beta-cell apoptosis throughout the time period studied (weeks 3-18), the incidence of apoptosis decreased at week 18, by which time 50% of the animals had overt diabetes. The low levels of beta-cell apoptosis observed is indicative of a gradual deletion of the beta-cell population throughout the extensive preclinical period seen in this model and would be sufficient to account for the beta-cell loss resulting in IDDM. Apoptosis of beta-cells preceded the appearance of T-cells (CD3-positive by immunohistochemistry) in islets. Lymphocytic infiltration of islets (insulitis) was not detected until week 6. The results show that beta-cell apoptosis is responsible for the development of IDDM in the NOD/Lt mouse and that its onset precedes lymphocytic infiltration of the islets.

Combination of SCF, IL-6, IL-3, and GM-CSF increases the mitotic index in short term bone marrow cultures from acute promyelocytic leukemia (APL) patients.

Acute promyelocytic leukemia (APL) is characterized cytogenetically by the presence of a reciprocal translocation between chromosomes 15 and 17 [t(15;17)(q22-q24;q11-q21)] in the bone marrow cells in the majority of patients. Cytogenetic evaluation of bone marrow cultures from patients with APL is often technically difficult, due to frequent difficult marrow aspiration and the suboptimal quality of cytogenetic preparations. This has important implications for the cytogenetic detection of residual disease. This study examined the proliferative ability of the recombinant human growth factors-stem cell factor (SCF), interleukin-6 (IL-6), interleukin-3 (IL-3), and granulocyte macrophage-colony stimulating factor (GM-CSF)-to determine if they would provide a consistent improvement over the standard cytogenetic culturing techniques in terms of mitotic index (MI). In all cases, the MI of the growth factor stimulated cultures showed a considerably higher (3.5-198 fold) and statistically significant (p < 0.01) increase compared to the unstimulated cultures. We conclude that the use of recombinant human growth factors is potentially an effective way of increasing the MI in bone marrow cultures from APL patients for the purposes of diagnosis and residual disease detection.

Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model.

Although insulin-dependent diabetes mellitus (IDDM) results from irreversible loss of beta cells, the mode of cell death responsible for this loss has not previously been categorized. In this study, the multiple low-dose streptozotocin (stz) model (intraperitoneal injection of stz at a concentration of 40 mg/kg body weight per day for five consecutive days) was used to investigate beta-cell death during the development of IDDM in male C57B1/6 mice. Apoptotic cells were evident by light microscopy within the islets of Langerhans of treated animals from day 2 (the day of the second stz injection) until day 17. Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Two peaks in the incidence of beta-cell apoptosis occurred: the first at day 5, which corresponded to an increase in blood glucose concentration, and the second at day 11, when lymphocytic infiltration of the islets (insulitis) was maximal. Insulitis did not begin until day 9, by which time treated animals had developed overt diabetes as revealed by blood glucose and pancreatic immunoreactive insulin (IRI) measurements. Beta-cell apoptosis preceded the appearance of T-cells in the islets and continued throughout the period of insulitis. Thus, whether induced by stz or a subsequent immune response, apoptosis is the mode of cell death responsible for beta-cell loss in the multiple low-dose stz model of IDDM.

Apoptosis in normal epithelium, premalignant and malignant lesions of the oropharynx and oral cavity: a preliminary study.

To explore the involvement of apoptosis in the development of oral and oropharyngeal squamous cell carcinoma (SCC) in vivo, biopsies were taken from patients with macroscopically normal (n = 6), leukoplakic (n = 12) or malignant (n = 8) mucosa. Leukoplakic lesions were divided histologically into dysplasia (n = 5) or carcinoma in situ (CIS: n = 7). Material was prepared for light and electron microscopy. The apoptotic index (AI), vertical cell position of apoptoses (cp), mitotic index (MI) and AI:MI ratio were calculated for each patient. AI increased from 0.12% +/- 0.07 S.E.M. (normal) to 0.58 +/- 0.13 (CIS) but fell to 0.14 +/- 0.14 in SCC. Apoptoses were suprabasal in normals, but generalised in CIS. MI increased from normal (0.20 +/- 0.06) to SCC (0.32 +/- 0.09), and AI:MI was at its maximum in CIS (1.57; SCC: 0.44). The results suggest that a change in apoptosis accompanies the onset of invasion in a premalignant lesion of the human oral cavity and oropharynx.

Adding food for thought. Structuring nutritional support for elderly trauma patients.

Of all elderly patients admitted to the acute setting, orthopaedic patients have been shown to be most severely undernourished or malnourished. The delivery of nutrition as part of patient care is undervalued and underdeveloped. Creating strategies within which effective treatment can be achieved is vital to its efficient delivery. Effective management of nutrition for those at risk will speed recovery, reduce the incidence of secondary complications and mortality and reduce nursing workload in both hospitals and community settings.

Hydroxyurea induces apoptosis and regular DNA fragmentation in a Burkitt's lymphoma cell line.

Hydroxyurea (HU) is an S-phase-specific cytotoxic drug used in the clinical treatment of haematological malignancies. HU treatment has been shown to lead to accumulation of short DNA fragments which show direct correlation with cytotoxicity. Specific regular DNA fragmentation is a biochemical feature of apoptosis (programmed cell death) in some systems. We investigated the effect of HU on a neoplastic (Burkitt's lymphoma) cell line (BM13674) in vitro to determine the role of apoptosis in HU action. HU produced growth inhibition and cell death by apoptosis in BM13674 cells. Low dose HU (66 and 131 mumol/l) gave a growth inhibition effect only with no apoptosis being induced. Higher doses (0.66-13 mmol/l) induced apoptosis in a dose-dependent manner. Regular DNA fragmentation was detected by agarose gel electrophoresis of DNA and this correlated in time with the onset of apoptosis detected by light and electron microscopy. The results do not exclude the possibility that HU directly induces DNA strand breaks, which then initiate apoptosis and accompanying regular fragmentation of DNA in the apoptotic cells.

Reduction in apoptosis relative to mitosis in histologically normal epithelium accompanies fibrocystic change and carcinoma of the premenopausal human breast.

The aims of this study in 227 premenopausal women were (a) to determine the mitotic index (MI), the thymidine labelling index (LI), and the apoptotic index (AI) within the epithelial cells of histologically 'normal' human breast biopsy material removed away from the site of either a fibroadenoma or a carcinoma; and (b) to relate differences in the kinetic indices of the 'normal' epithelium to the pathology in the same breast diagnosed as fibroadenoma alone (125 patients), fibroadenoma with accompanying mild fibrocystic change (79 patients), or carcinoma (23 patients). Ratios of the average indices (AI/MI, AI/LI, MI/LI) were also calculated to minimize uncertainties related to the total cell population counted, the denominator in the LI, MI, and AI. All indices and ratios of indices were corrected for age, averaged over the cycle, and expressed as log-transformed values for analysis. Significant (P less than 0.001) reductions in AI and in apoptosis relative to mitosis (reduced AI/MI) were found in 'normal' epithelium from breasts having fibrocystic change (AI = 0.17 +/- 0.02; AI/MI = 1.01 +/- 0.18) and carcinoma (AI = 0.19 +/- 0.04; AI/MI = 0.88 +/- 0.29), compared with breast with fibroadenoma alone (AI = 0.27 +/- 0.03; AI/MI = 1.29 +/- 0.39). In the absence of significant differences in MI and LI between the 'normal' tissue groups, this finding raises the possibility that reduced epithelial cell apoptosis might be causally associated with the development of fibrocystic change and with an increased risk of development of carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat.

In this study we examined the possibility that regular or circadian fluctuations occur in the frequency of spontaneous spermatogonial apoptosis. Apoptosis of A2, A3 and A4 type spermatogonia occurring spontaneously in the normal rat testis was studied by light and electron microscopy. Normal and apoptotic A3 spermatogonia were quantified in 36 animals killed at two-hourly intervals over a 24 h period. Three sequential phases of spermatogonial apoptosis were defined and quantified separately: (i) an early phase in which cells showed margination of nuclear chromatin, (ii) an intermediate phase in which phagocytosed apoptotic bodies were partly degraded and (iii) a late phase in which only debris of degraded apoptotic bodies was evident. Groups of spermatogonia linked by intercellular bridges underwent apoptosis synchronously. Normal and apoptotic A3 spermatogonia occurred at a mean frequency of 33.4 and 9.6 per 10 seminiferous tubule profiles respectively; there was a large variation in these frequencies between animals, but no peaks or circadian periodicity were detected. Progressive degradation of apoptotic bodies was evident, the average ratios of intermediate and late bodies to early bodies being 1.5 and 3.5, respectively. Absence of a circadian rhythm in these data does not exclude the possibility that initiation of apoptosis in susceptible spermatogonial clones is synchronous, and that affected clones undergo lag periods of differing duration before expressing morphological apoptosis.

Cell death induced in a murine mastocytoma by 42-47 degrees C heating in vitro: evidence that the form of death changes from apoptosis to necrosis above a critical heat load.

The pathogenesis of heat-induced cell death is controversial. Categorizing the death occurring after various heat loads as either apoptosis or necrosis might help to elucidate this problem, since it has been shown that these two processes differ in their mode of initiation as well as in their morphological and biochemical features. Log-phase cultures of mastocytoma P-815 x 2.1 were heated at temperatures ranging from 42 to 47 degrees C for 30 min. After 42 degrees C heating a slight increase in apoptosis was observed morphologically. However, after heating at 43, 43.5 and 44 degrees C, there was marked enhancement of apoptosis, and electrophoresis of DNA showed characteristic internucleosomal cleavage. With heating at 45 degrees C both apoptosis and necrosis were enhanced, whereas at 46 and 47 degrees C only necrosis was produced. DNA extracted from the 46 and 47 degrees C cultures showed virtually no degradation, which contrasts with the random DNA breakdown observed in necrosis produced by other types of injury; lysosomal enzymes released during heat-induced necrosis may be inactivated at the higher temperatures. It is suggested that apoptosis following heating may be triggered either by a limited increase in cytosolic calcium levels resulting from mild membrane changes or by DNA damage. Necrosis, on the other hand, is likely to be a consequence of severe membrane disruption.

Cell death by apoptosis following X-irradiation of the foetal and neonatal rat kidney.

A light and electron microscopic study was undertaken to determine the type of cell death induced by X-irradiation in the developing kidney. Five-day-old Sprague-Dawley rats were exposed to a whole-body dose of either 2 or 5 Gy, and foetuses in the eighteenth day of development were exposed to a dose of 4 Gy. The kidneys were examined at 4, 8 and 24 h, and at 1 and 2 weeks post-irradiation. The dying cells from both control and treated kidneys showed the morphological features of apoptosis, a distinct form of cell death that has been identified in mammalian tissues under physiological as well as pathological conditions. Necrosis was not detected. Apoptosis was infrequent in control kidneys and insignificant in extent when compared with the proliferative activity of the cells of the superficial nephrons. There was a pronounced increase in apoptosis during the first day after irradiation. The findings are in agreement with recent ultrastructural studies which report the presence of apoptosis following irradiation of rapidly proliferating adult cell populations, and irradiation of other immature mammalian tissues. There is evidence that apoptosis involves active cellular self-destruction, and it has been suggested that activation of apoptosis might bring about selective elimination of cells with critical DNA damage in irradiated tissues, thus minimizing propagation of genetic abnormalities.

Sertoli cell death by apoptosis in the immature rat testis following x-irradiation.

The importance of the morphological study of cell death has recently been emphasized by the recognition that the ultrastructural features of dying cells allow categorization of the death as either apoptosis or necrosis. This classification enables inferences to be drawn about the mechanism and biological significance of the death occurring in a particular set of circumstances. In this study, Sertoli cell death induced in the immature testis of three and four day old rats by 5 Gy (500 rads) x-irradiation was described by light and transmission electron microscopy with the objective of categorizing the death as apoptosis or necrosis. The testes were examined 1, 2, 3, 4, 8, and 24 h after irradiation. Following irradiation, there was a wave of apoptosis of the Sertoli cells starting in three to four hours and reaching a peak between four and eight hours. At 24 hours, only 61% of the expected number of Sertoli cells remained. These findings are in accord with recent ultrastructural reports that ionizing radiation induces cell death by apoptosis in rapidly proliferating cell populations. New insights into the pathogenesis of radiation-induced cell death might thus be expected to stem from future elucidation of the general molecular events involved in triggering apoptosis.

X-ray-induced cell death by apoptosis in the immature rat cerebellum.

The cells of the external granular layer (EGL) of the developing cerebellum are known to be particularly sensitive to radiation. In the past, changes induced in this layer by irradiation have been referred to by non-specific terms such as "pyknotic cells" and the mode of cell death has been assumed to be necrosis. However, in published light micrographs of these dying cells, the appearance is suggestive of apoptosis, a distinctive mode of cell death which occurs spontaneously in normal adult and embryonic tissues and can also be triggered by certain pathological stimuli. This light and transmission electron microscopic study of control and irradiated (7 h post-irradiation) rat cerebellum from 18 day fetuses and 5 day-old neonates showed that the cell death was effected by apoptosis. The apoptosis was markedly enhanced by x-irradiation and quantification of the cell death in the EGL of 5 day-old rats exposed to 4, 8, 25, 100, and 400 cGy x-irradiation demonstrated that there was a positive dose response relationship. The extent of cell death by apoptosis which was 0.2% in control, ranged from 0.8% after 4 cGy to 62.3% after 400 cGy x-irradiation. The recognition that cell death by apoptosis can be a major component of x-irradiation damage has important implications for radiobiological studies.