Biomedical subjects

B Hellman

Publications and source records attributed to B Hellman.

At least 37 records · Page 2Linked to original sources

PubMed1998

In situ characterization of nonmitochondrial Ca2+ stores in individual pancreatic beta-cells.

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.

Animals↗

PubMed1997

Alkaline single-cell gel electrophoresis and human biomonitoring for genotoxicity: a pilot study on breast cancer patients undergoing chemotherapy including cyclophosphamide.

Alkaline single-cell gel electrophoresis (the 'comet assay') was used to evaluate DNA damage in lymphocytes from 17 breast cancer patients before and 1-21 h after chemotherapy including cyclophosphamide (600-1800 mg/m2). In order to control for the experimental variability over time, freshly isolated lymphocytes from female mice given physiological saline or cyclophosphamide (150 mg/kg b.wt.) were included as 'internal standards' in each individual electrophoresis run. There was an upward tendency of DNA damage in the mouse lymphocytes over the study period, but cyclophosphamide was constantly found to induce significant damage at all time points investigated (1-48 h). Although patients given up to 11 prior cycles of chemotherapy showed the same basal level of DNA damage as the patients coming to the clinic for their first treatment, the chemotherapy given at the time of the present blood sampling was associated with significant DNA damage in most samples. Considerable interindividual variations were observed both before and after the treatment. DNA single-strand breaks and alkali-labile sites in peripheral lymphocytes as evaluated by the comet assay seem to be useful molecular biomarkers for exposure to DNA damaging agents when monitoring ongoing exposures, but less impressive when monitoring accumulated exposures, at least in patients given high doses of cyclophosphamide and other antineoplastic agents.

Adult↗

PubMed1997

The role of N-acetylcysteine as a putative radioprotective agent on X-ray-induced DNA damage as evaluated by alkaline single-cell gel electrophoresis.

Samples of human whole blood from 8 different donors were incubated with physiological saline or N-acetyl-L-cysteine (NAC, 1 x 10(-3) M) before being irradiated in vitro with high-energy X-rays (0.7 or 2.0 Gy). Primary DNA damage was evaluated in isolated lymphocytes using alkaline single-cell gel electrophoresis. Whereas the lymphocytes from non-irradiated blood samples showed a similar 'background level' of damage, there was a difference in sensitivity towards the radiation-induced DNA damage, especially at 2.0 Gy. When the data were pooled there was a clear and dose-related increase (p < 0.001) in damage, both in the absence and presence of NAC. Using the two most sensitive 'comet parameters' for DNA damage, i.e., the tail inertia and tail moment, the radiation-induced damage was found to be significantly increased already at 0.7 Gy in the samples that had been irradiated without NAC. Overall, NAC was found to be without radioprotective effects. Instead, the incubation with NAC itself was found to be associated with a slightly increased level of DNA damage. If the present findings are relevant also in an in vivo situation using peripheral lymphocytes as a surrogate for non-malignant cells in the body, NAC seems to be of limited value as a radioprotective agent in the clinic, at least when it comes to the acute DNA-damaging effects of therapeutic doses of high-energy X-rays.

Acetylcysteine↗

PubMed1997

Cytoplasmic Ca2+ in glucagon-producing pancreatic alpha-cells exposed to carbachol and agents affecting Na+ fluxes.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured with dual wavelength fluorometry in glucagon-producing mouse pancreatic alpha-cells loaded with the indicator fura-2. Spontaneous rhythmic activity in terms of slow oscillations from a basal level was observed at 3 mM glucose. Like in the insulin-secreting beta-cells the generation of [Ca2+]i oscillations in the alpha-cells was affected by the activity of the Na/K pump. Blocking the pump with ouabain resulted in an initial rise of [Ca2+]i followed by gradual return to the basal level. The oscillations were transformed into sustained elevation of [Ca2+]i by 10 mM L-glycine, which is cotransported with Na+. A similar but less pronounced effect was obtained when Na+ was cotransported with 10 mM of the nonmetabolizable amino acid alpha-amino-isobutyric acid. L-glycine induced sustained increase of [Ca2+]i also when the oscillatory activity was suppressed by exposing the alpha-cells to 20 mM glucose in the presence of insulin. The observation that carbachol induces a [Ca2+]i response in isolated alpha-cells calls for reconsideration of current ideas that muscarinic stimulation of glucagon release is an indirect effect mediated by adjacent beta-cells.

Aminoisobutyric Acids↗

PubMed1997

Alkaline single cell gel electrophoresis of DNA fragments in biomonitoring for genotoxicity: an introductory study on healthy human volunteers.

Alkaline single cell gel electrophoresis (also known as the 'comet assay') is a rapid method for detecting DNA strand breaks in individual cells. Before the assay is used for biomonitoring in human populations the test conditions must be accurately characterised. Five healthy male volunteers donating capillary blood over a period of 20 weeks showed a fairly stable level of DNA damage in their lymphocytes. The values for tail moment and tail inertia, as evaluated by computerised image analysis of coded samples, were similar to those in lymphocytes from control mice but only 10% of those in lymphocytes from mice given cyclophosphamide (200 mg/kg b.wt.) 15-17 h before sacrifice. Inter- and intraindividual variations among the human subjects were related to both individual factors and laboratory conditions. When the comet assay is used for biomonitoring purposes it is suggested that each electrophoresis session should include not only the coded samples from the subjects, but also freshly isolated control cells and, in addition, cells that have been exposed to a well-characterised genotoxic insult.

Adult↗

PubMed1997

Do organic solvents induce changes in the dopaminergic system? Positron emission tomography studies of occupationally exposed subjects.

OBJECTIVES: The objective of this study was to test the hypothesis that long-term occupational exposure to organic solvents may effect the levels and turnover of dopamine in man. METHODS: A study was performed on 17 patients with neuropsychiatric symptoms due to occupational solvent exposure, and 11 healthy non-exposed male volunteers (controls). Positron emission tomography (PET) was used to assess striatal dopaminergic function, using L-[11C]DOPA, [11C]nomifensine and [11C]raclopride as tracers. RESULTS: The rate of dopamine synthesis was significantly increased among subjects with occupational exposure to organic solvents compared with non-exposed controls. After controlling for the difference in age between exposed and controls, the effect of solvent exposure became less apparent and was reduced from +32% (P = 0.009) to +25% (P = 0.07). There were no differences with regard to the binding of [11C]nomifensine. Patients with and without the diagnosis of toxic encephalopathy did not differ with regard to their putaminal uptake of L-[11C]DOPA, [11C]nomifensine and [11C]raclopride. CONCLUSION: The data support the hypothesis that long-term exposure to organic solvents may increase the rate of dopamine synthesis in the brain without affecting the number of presynaptic terminals or postsynaptic dopamine receptors.

Brain↗

PubMed1997

Oscillatory Ca2+ signaling in somatostatin-producing cells from the human pancreas.

Oscillatory Ca2+ signaling was studied in human somatostatin-releasing pancreatic delta cells identified by immunostaining. A ratiometric fura-2 technique was used for measuring cytoplasmic concentrations of Ca2+ and Sr2+ in delta cells exposed to the respective cation. Rhythmic activity in terms of slow (frequency, 0.1 to 0.4 per minute) oscillations from close to the basal level was seen in the presence of 3 to 20 mmol/L glucose during superfusion with medium containing 2.6 to 5 mmol/L Ca2+ or 5 mmol/L Sr2. These oscillations could be transformed into a sustained increase by decreasing extracellular Ca2+ or adding 1 mmol/L tolbutamide or 20 nmol/L glucagon. Addition of glucagon to a medium containing 20 mmol/L glucose resulted in the generation of short (< 30 seconds) transients, which disappeared upon exposure to 100 nmol/L of the intracellular Ca(2+)-adenosine triphosphatase (ATPase) inhibitor thapsigargin. When analyzing small aggregates of islet cells, it became evident that oscillatory activity in delta cells can be synchronous with that in adjacent non-delta cells. It is concluded that secretion of pancreatic somatostatin in man involves Ca2+ signaling similar to that regulating the pulsatile release of insulin.

Adult↗

PubMed1997

Induction of a glucose-dependent insulin secretory response by the nonmetabolizable amino acid alpha-aminoisobutyric acid.

The effects of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) on insulin release were evaluated using beta cell-rich pancreatic islets from ob/ob mice. Both AIB and L-alanine promptly induced transient insulin release during column perifusion of islet cells. The secretory response was dependent on an elevated level of glucose and effectively suppressed by removal of Na+. The insulin release elicited by AIB fulfilled the criteria of a physiological event in being suppressed by clonidine or lowering of the temperature to 22 degrees C. AIB effectively promoted the increase in sodium (total as well as ionized cytoplasmic) obtained with ouabain blockage of the Na/K pump. When added to a medium containing 11 mM glucose, AIB altered cytoplasmic Ca2+ in terms of both an initial transitory rise and transformation of existing oscillations into a sustained elevation. It is concluded that amino acids can stimulate insulin release from mature beta cells by virtue of being cotransported with Na+.

Alanine↗

PubMed1997

No increased DNA damage in peripheral lymphocytes of sewage workers as evaluated by alkaline single cell gel electrophoresis.

OBJECTIVES: To study whether sewage workers are exposed to genotoxic substances. An increased risk of cancers among sewage workers has been noted. If this increased risk is due to an exposure to genotoxic agents, primarily DNA damage could be used as a biological marker of exposure. METHODS: In a cross sectional study, DNA damage in peripheral lymphocytes from 35 sewage workers and 30 controls was compared with alkaline single cell gel electrophoresis, a technique for detecting single strand breaks and alkali labile sites in DNA. The controls were selected from among municipal workers matched for age and smoking habit. Information about occupational exposures and possible confounders was collected by means of a questionnaire. RESULTS: No increase in DNA damage was found among the sewage workers when compared with the unexposed controls. CONCLUSIONS: The failure to detect increased damage to DNA in peripheral lymphocytes by alkaline single cell gel electrophoresis suggests that the sewage workers studied here were not exposed to genotoxic agents to a greater extent than other municipal workers. It may be, however, that the lymphocyte is not the appropriate target cell to study, or that sewage workers are exposed to carcinogens which do not damage the genetic material.

Animals↗

PubMed1997

Positron emission tomography studies of healthy volunteers--no effects on the dopamine terminals and synthesis after short-term exposure to toluene.

Despite extensive research, the mechanisms for the effects of organic solvents on the central nervous system are still unknown. One mechanism proposed is that solvents interfere with the synthesis of neurotransmitters. In the present study 11 male healthy volunteers were exposed during 15 min to 100 p.p.m. toluene at light physical exercise, and the dopamine decarboxylase activity and number of terminals in putamen were measured before and after exposure by positron emission tomography. Two different tracers were used [beta-11C]L-DOPA for decarboxylase activity during the in vivo synthesis of dopamine, and [11C]nomifensine to estimate the number of terminals. Although there was a slight increase in the rate of dopamine synthesis in the putamen after the exposure, this difference was not statistically significant (P = 0.4). No effect was observed with regard to the uptake of nomifensine. There was no significant relationship between the dose of toluene and rate of dopamine synthesis, and no significant correlation between the time from end of exposure to start of the PET-camera and DOPA. Our findings indicate that short term exposure to 100 p.p.m. of toluene does not affect the rate of dopamine synthesis or the number of presynaptic terminals.

Adult↗

PubMed1997

Oscillatory signaling and insulin release in human pancreatic beta-cells exposed to strontium.

Oscillatory signaling and insulin release were studied in isolated pancreatic islets and beta-cells obtained from human cadaveric organ donors. Taking advantage of Sr2+ as an analog for Ca2+, it was possible to demonstrate glucose-induced rhythmic activity in individual beta-cells identified by immunostaining. Glucose-induced slow oscillations of Sr2+ (frequency, 0.1-1.0/min) were sometimes seen at a sugar concentration as low as 3 mM. Addition of 20 nM glucagon resulted in a broadening of the oscillations or in their transformation into sustained elevation. Moreover, the presence of glucagon resulted in the appearance of short transients of Sr2+, which disappeared after exposure to the intracellular Ca2+-adenosine triphosphatase inhibitor thapsigargin. Digital image analyses indicated that slow oscillations can be synchronized among cells in small aggregates and intact islets. The rhythmic activity in the glucose-stimulated beta-cell had its counterpart in pulsatile insulin release when single islets were perifused with a Sr2+-containing medium. It is concluded that the human beta-cell has oscillatory signaling for insulin release similar to that observed in experimental animals.

Adult↗

PubMed1996

Crosstalk between the cAMP and inositol trisphosphate-signalling pathways in pancreatic beta-cells.

Glucose was found to induce large amplitude oscillations of cytoplasmic Sr2+ and Ca2+ in individual pancreatic beta-cells exposed to the respective cation. Subsequent addition of 20 nM glucagon or other agents raising cAMP triggered pronounced transients superimposed upon the large amplitude oscillations. Hyperpolarization with diazoxide prevented both the large amplitude oscillations and the superimposed transients. After short exposure to carbachol or ATP there was a temporary, and after addition of the Ca2+-ATPase inhibitor thapsigargin a permanent, disappearance of the transients with persistence of the glucose-induced large amplitude oscillations. The Ca2+ channel blocker methoxyverapamil exhibited opposite specificity in preventing the large amplitude oscillations under conditions when the transients often remained. In the presence of methoxyverapamil the transients disappeared during diazoxide hyperpolarization and were restored by subsequent K+ depolarization, which also elevated the content of inositol 1,4,5-trisphosphate (IP3) by 45%. The glucagon-induced transients were obliterated by 12-O-tetradecanoylphorbol 13-acetate, insensitive to ryanodine and paradoxically inhibited by high concentrations of caffeine. The IP3-mediated intracellular ion mobilization induced by carbachol was amplified by glucagon. The results indicate that depolarization-dependent formation of IP3 causes intracellular Ca2+ mobilization in individual beta-cells when the IP3 receptors are sensitized by cAMP. This mechanism may be an important determinant for the electrophysiological burst activity in intact pancreatic islets due to the presence of endogenous glucagon.

Animals↗

PubMed1996

Sensitivity of human pancreatic islets to peroxynitrite-induced cell dysfunction and death.

Nitric oxide and peroxynitrite (generated by the reaction of nitric oxide with the superoxide anion) may both be mediators of beta-cell damage in early insulin-dependent diabetes mellitus. We observed that acute exposure of primary cultured human pancreatic islets to peroxynitrite results in a significant decrease in glucose oxidation and islet retrieval. DNA strand breaks in single human and rat islet cells are detectable after acute peroxynitrite exposure, followed by a decrease in islet cell survival after 1 h and 24 h. Cell death appeared to occur via a toxic cell death mechanism (necrosis) rather than apoptosis, as suggested by vital staining and ultrastructural evidence of early membrane and organelle degradation, mitochondrial swelling and loss of matrix. This study demonstrates for the first time that cultured human pancreatic islets are susceptible to the noxious effects of peroxynitrite.

Animals↗

PubMed1996

Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca2+.

Pancreatic islets are known to respond to a raise of the glucose concentration with Ca2+ -induced 2-3-min pulses of insulin release. The reports of cyclic variations of circulating insulin in the fasting state made it important to explore whether insulin release is also pulsatile in the absence of stimulated entry of Ca2+. Individual pancreatic islets were isolated from a local colony of ob/ob mice and perifused under conditions allowing dual wavelength recordings of the cytoplasmic Ca2+ concentration ([Ca2+]i) with fura-2 and measurements of insulin with ELISA technique. At 3 mM of glucose, [Ca2+]i remained at a stable low level, but insulin was released in pulses with a frequency of 0.41+/-0.02 min-1, determined by Fourier transformation of original and autocorrelated data. Pulses of basal insulin release were also seen when glucose was omitted and 1 microM clonidine or 400 microM diazoxide was added to a glucose-free medium. The results indicate that pulsatile insulin release can be generated in the absence of stimulated entry of Ca2+. A tentative explanation for this phenomenon is inherent fluctuations in the ATP production of the beta cells.

Animals↗

PubMed1996

Suppression of Ca2+ oscillations in glucagon-producing alpha 2-cells by insulin/glucose and amino acids.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was continuously monitored in single glucagon-producing alpha 2-cells isolated from the mouse pancreas and later identified by immunostaining. Up to 60% of the alpha 2-cells exhibited spontaneous [Ca2+]i oscillations (frequency 0.1-0.3/min) in a medium containing 3 mM glucose. In originating from a basal level of 60-100 nM, reaching peak values of 300-400 nM and promptly disappearing after blocking voltage-dependent Ca2+ channels with methoxyverapamil, the oscillations resembled those in insulin-releasing beta-cells stimulated by glucose. The oscillatory activity was suppressed when combining elevation of glucose to 20 mM with the addition of 2-2000 ng/ml insulin. Whereas 10 mM of L-arginine or l-glycine transformed the oscillations into sustained elevation of [Ca2+]i, there was no response to 1 mM tolbutamide or 0.1-1 mM gamma-aminobutyric acid. The observations that alpha 2-cells differ from islet cells secreting insulin and somatostatin in responding to adrenaline with mobilisation of intracellular calcium can be used for their rapid identification. It is suggested that the oscillations reflect periodic entry of Ca2+ due to variations of the membrane potential.

Amino Acids↗

PubMed1996

Demonstration of benzo(a)pyrene-induced DNA damage in mice by alkaline single cell gel electrophoresis: evidence for strand breaks in liver but not in lymphocytes and bone marrow.

Alkaline single cell gel electrophoresis (also known as the 'comet assay') was used to measure DNA strand breaks and alkali-labile sites in peripheral lymphocytes, bone marrow and liver cells of C57BL/6 mice orally exposed to benzo(a)pyrene. Although this polycyclic aromatic hydrocarbon is a well-known genotoxic agent, little is known about to what extent it actually induces DNA strand breaks in peripheral lymphocytes and other tissues after in vivo exposure. Significant and dose-related damage was observed in liver cells after three days of exposure (lowest observed effect level being 3 x 100 mg benzo(a)pyrene/kg b.wt. No such damage could be observed in the lymphocytes and bone marrow cells even after administration of 3 x 150 mg benzo(a)pyrene/kg b.wt. The reference substance cyclophosphamide produced pronounced DNA damage in lymphocytes and bone marrow cells already in a single dose of 100 mg/kg b.wt. The present mouse study questions the usability of DNA strand breaks in peripheral lymphocytes as an indicator of benzo(a)pyrene-induced genotoxicity.

Animals↗

PubMed1996

Glucose stimulation of somatostatin-producing islet cells involves oscillatory Ca2+ signaling.

The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured with fura-2 in individual mouse pancreatic delta-cells identified by immunostaining for somatostatin. A majority of the delta-cells responded to 3 mM glucose with slow oscillations of [Ca2+]i (frequency, 0.1-0.4/min). In originating from a basal level of 60-100 nM and reaching peak values of 200-500 nM, the oscillations resembled those in insulin-secreting beta-cells stimulated by glucose. The rise in glucose to 20 mM resulted in a minor increase in the oscillatory frequency and sometimes in transformation of the oscillations into sustained elevation of [Ca2+]i. The addition of 3 microM L-epinephrine effectively counteracted the increase in [Ca2+]i in response to glucose. The delta-cells reacted with a sustained elevation of [Ca2+]i after raising extracellular K+ to 30.9 mM or adding 1 microM tolbutamide. Analyses using the patch-clamp technique revealed the presence of K+ channels with properties similar to the ATP-sensitive channels in pancreatic beta-cells. It is concluded that regulation of somatostatin release mimics that of insulin, with glucose induction of [Ca2+]i oscillations.

Animals↗

PubMed1995

Ca2+ oscillations in pancreatic islet cells secreting glucagon and somatostatin.

Immunohistochemically identified glucagon-releasing alpha 2-cells from mouse pancreatic islets exhibited large amplitude oscillations of the cytoplasmic Ca2+ concentration in 3 mM glucose. Other small islet cells with similar oscillations in the presence of 20 mM glucose were identified as somatostatin-releasing alpha 1-cells. The oscillations in both cell types resembled those induced by glucose in the surrounding larger beta-cells in starting from the basal level and disappearing after addition of the voltage-dependent Ca2+ channel blocker methoxyverapamil. The discovery that the alpha 1- and alpha 2-cells have intrinsic abilities to generate oscillatory Ca2+ signals indicates that pulsatile release of somatostatin and glucagon do not require functional coupling to the beta-cells.

Animals↗