Tumor necrosis factor-alpha and interferon-gamma inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement.

Nitric oxide has been implicated as one possible mediator of interleukin-1 beta (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN+TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF+IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF+IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.

Potentiation of temozolomide and topotecan growth inhibition and cytotoxicity by novel poly(adenosine diphosphoribose) polymerase inhibitors in a panel of human tumor cell lines.

Potent poly(ADP-ribose) polymerase (PARP) inhibitors have been developed that potentiate the cytotoxicity of ionizing radiation and anticancer drugs. The biological effects of two novel PARP inhibitors, NU1025 (8-hydroxy-2-methylquinazolin-4-[3H]one, Ki = 48 nM) and NU1085 [2-(4-hydroxyphenyl)benzamidazole-4-carboxamide, Ki = 6 nM], in combination with temozolomide (TM) or topotecan (TP) have been studied in 12 human tumor cell lines (lung, colon, ovary, and breast cancer). Cells were treated with increasing concentrations of TM or TP +/- NU1025 (50, 200 microM) or NU1085 (10 microM) for 72 h. The potentiation of growth inhibition by NU1025 and NU1085 varied between the cell lines from 1.5- to 4-fold for TM and 1- to 5-fold for TP and was unaffected by p53 status. Clonogenic assays undertaken in two of the cell lines confirmed that the potentiation of growth inhibition reflected the potentiation of cytotoxicity. NU1025 (50 microM) was about as effective as 10 microM NU1085 at potentiating growth inhibition and cytotoxicity, consistent with the relative potencies of the two molecules as PARP inhibitors. Potentiation of cytotoxicity was obtained at concentrations of NU1025 and NU1085 that were not toxic per se; however, NU1085 alone was 3-fold more cytotoxic (LC50 values ranged from 83 to 94 microM) than NU1025 alone (LC50 > 900 microM). These data demonstrate that PARP inhibitors are effective resistance-modifying agents in human tumor cell lines and have provided a comprehensive assessment protocol for the selection of optimum combinations of anticancer drugs, PARP inhibitors, and cell lines for in vivo studies.

Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells.

We have previously observed that a 6-day exposure of human pancreatic islets to a combination of cytokines (interleukin-1beta 50 U/ml + tumour necrosis factor-alpha 1000 U/ml + interferon-gamma 1000 U/ml) severely impairs beta-cell functions. In the present study, we examined whether this condition affects DNA integrity and viability of human islet cells. Cells were studied after 3, 6, and 9 days of cytokine treatment by both single cell gel electrophoresis (the "comet assay," a sensitive method for detection of DNA strand breaks) and by a cytotoxicity assay using the DNA binding dyes Hoechst 33342 and propidium iodide as indices for the number of viable, necrotic, and apoptotic cells. Cytokine treatment for 6 and 9 days resulted in a 50% increase in comet length (P < 0.01 vs. controls), indicating DNA strand breaks, as well as in a significant increase in the number of apoptotic cells (P < 0.02 vs. controls), but not in the number of necrotic cells. The arginine analogs N(G)-nitro-L-arginine and N(G)-monomethyl-L-arginine prevented nitric oxide formation by the cytokines but did not interfere with cytokine-induced DNA strand breaks and apoptosis. The present data suggest that prolonged (6-9 days) exposure of human pancreatic islets to a mixture of cytokines induces DNA strand breaks and cell death by apoptosis. These deleterious effects of cytokines appear to be independent of nitric oxide generation.

Endogenous nitric oxide induced by interleukin-1 beta in rat islets of Langerhans and HIT-T15 cells causes significant DNA damage as measured by the 'comet' assay.

We have used the comet assay (single cell gel electrophoresis) to measure nitric oxide-induced DNA damage in rat islets of Langerhans and insulin-containing HIT-T15 cells. Damage was induced following treatment with the nitric oxide donor SIN-1, which also releases superoxide, but was not reduced by exogenous superoxide dismutase, suggesting that nitric oxide itself, rather than superoxide or peroxynitrite may be the active species. The DNA damaging effect of nitric oxide was easily detectable at the earliest time point tested (15 min). Damage also resulted following induction of nitric oxide synthase by the cytokine interleukin-1 beta in both islets and HIT-T15 cells and was prevented by replacing the substrate, arginine, with nitromonomethyl arginine. Thus intracellular levels of nitric oxide generated by interleukin-1 beta-induced nitric oxide synthase were sufficient to cause DNA damage in islet cells and HIT-T15 cells.

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.

Comparison of inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans by streptozotocin and methyl and ethyl nitrosoureas and methanesulphonates. Lack of correlation with nitric oxide-releasing or O6-alkylating ability.

We have studied inhibition of glucose-stimulated insulin secretion in islets of Langerhans isolated from adult Sprague-Dawley rats and treated with different alkylating agents. Streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU) all released nitric oxide, as demonstrated by an increase in medium nitrite and cellular cyclic GMP. Methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), which do not possess a nitroso group, did not show evidence of nitric oxide release. All five compounds, however, decreased glucose-stimulated insulin release, suggesting that nitric oxide release was not necessary for the inhibition of secretion. Lack of involvement of nitric oxide was further suggested by the failure of oxyhaemoglobin to reverse STZ and MNU inhibition of insulin secretion. Since ENU was at least as effective as MNU in inhibiting insulin secretion, it appears that alkylation of DNA at the O6 position of guanine may not be involved in this process.

The effect of nitric oxide donors on insulin secretion, cyclic GMP and cyclic AMP in rat islets of Langerhans and the insulin-secreting cell lines HIT-T15 and RINm5F.

The aim of this study was to investigate whether short-term treatment with nitric oxide donors could mimic cytokine inhibition of insulin secretion. We tested the nitric oxide generating compounds 3-morpholinosydnonimine (SIN-1), S-nitroso-N-penicillamine (SNAP), S-nitrosoglutathione and hydroxylamine for their ability to inhibit insulin secretion, raise cyclic GMP and lower cyclic AMP levels in isolated rat islets of Langerhans and the insulin-secreting cell lines HIT-T15 and RINm5F. In islets, all nitric oxide donors inhibited glucose-induced insulin secretion and raised cyclic GMP levels. SIN-1 and S-nitrosoglutathione also reduced cyclic AMP, while SNAP and hydroxylamine had no effect. Insulin secretion in HIT-T15 cells was inhibited by SIN-1, SNAP and hydroxylamine and in RINm5F cells by hydroxylamine. Inhibition of HIT-T15 and RINm5F cell insulin secretion was not accompanied by an increase in cyclic GMP levels. The degree of inhibition of insulin secretion was unrelated to the extent of release of nitric oxide by the compounds as measured by nitrite and nitrate production. More effective inhibition by S-nitrosoglutathione and hydroxylamine versus SIN-1 and SNAP may be related to intracellular versus extracellular site of nitric oxide generation.

Nitric oxide donors decrease the function and survival of human pancreatic islets.

Nitric oxide (NO) has been proposed as a possible mediator of beta-cell damage in human IDDM. This hypothesis is based on in vitro studies with rodent pancreatic islets. In the present study we examined whether human beta-cells are affected by NO. In view of species differences in beta-cell sensitivity to damaging agents, rat islets were investigated in parallel. Isolated islets were exposed for 90 min to different concentrations of three chemically unrelated NO donors, SIN-1, GSNO or RBS. At the end of this incubation, human insulin release was mostly similar in control and NO-treated islets but, 48 h later, islet retrieval, islet DNA and insulin content, and glucose-induced insulin release were markedly lower in islets exposed to NO donors. Rat islets were already inhibited during the initial 90 min; 48 h later their loss in beta-cell function was similar to that in human islets. Nicotinamide or succinic acid monomethyl ester partially protected against SIN-1 induced islet cell loss, but not against the functional inhibition of human pancreatic islets. Exposure of human or rat islets to RBS was associated with significant DNA strand breakage, as judged by the comet assay (single cell gel electrophoresis) and by ultrastructural signs of cell damage. DNA damage was more severe in rat islet cells exposed to similar amounts of RBS. It is concluded that NO donors can damage human pancreatic islets, an effect paralleled by induction of nuclear DNA strand breaks.

Denervation impairs cutaneous microvascular function and blister healing in the rat hindlimb.

Skin sensory nerve nocifensor functions were investigated non-invasively in rats by measuring neurogenic inflammation and blister healing-rate after unilateral hindlimb denervation. Axon reflexes were evoked by transdermal iontophoresis of acetylcholine (ACh) or noxious electrical stimulation (TNS). Sodium nitroprusside (SNP) evoked direct dilator responses. Resultant changes in skin microvascular blood flux were measured by laser Doppler flowmetry. Compared with their sham-operated control limbs, denervation reduced inflammatory responses (ACh or TNS) by more than 85% and SNP responses by 28% (p < 0.05). Healing of dry-ice blisters raised on the hindpaw 14d post-denervation was significantly slower to complete healing (42d) than controls (26d) and initial inflammation was attenuated, confirming that innervation is important for inflammation and blister-healing.

Rapid actions of insulin on sensory nerve function.

The acute action of insulin on neurogenic flare was investigated using iontophoresis. Twenty-five patients with insulin-dependent diabetes mellitus (IDDM) and 25 age- and gender-matched controls were studied. Axon reflex vasodilatation was evoked by transdermal iontophoresis of acetylcholine (ACh) before and after skin treatment by the iontophoresis of insulin and measured using laser Doppler velocimetry. Axon reflex responses were reduced in IDDM patients compared with controls (p< 0.001) but were restored after the iontophoresis of insulin. Insulin iontophoresis had no effect on the size of the axon reflex response in control subjects (p > 0.05). This study confirms the reduction of the ACh-induced flare in human patients with IDDM and has demonstrated relatively rapid effects of insulin on this cutaneous neurogenic response.

Insulin sensitivity and sensory nerve function in non-diabetic human subjects.

The direct effect of reduced insulin sensitivity (measured by insulin tolerance test and fasting plasma insulin) on sensory nerve function was examined in non-diabetic human subjects. Thermal sensation (measured by warm and cold perception thresholds) deteriorated with fasting hyperinsulinaemia in the presence of normoglycaemia and normal glucose tolerance. The results suggest a possible role for insulin in sensory nerve function, also that deficits in insulin action per se may adversely affect the function of small sensory nerves independent of glycaemic levels, and may thus be implicated in the aetiology of diabetic neuropathy.

Use of the comet assay to investigate possible interactions of nitric oxide and reactive oxygen species in the induction of DNA damage and inhibition of function in an insulin-secreting cell line.

We have previously used the comet assay to demonstrate that the nitric oxide donor 3-morpholinosydnonimine (SIN-1) produces DNA damage in rat islets of Langerhans and in the SV40-transformed insulin-secreting hamster cell line, HIT-T15. Damage is not prevented by the addition of superoxide dismutase (SOD). In the present study, we have compared SIN-1, which generates nitric oxide, superoxide anion and hydrogen peroxide, with two other nitric oxide donors, S-nitrosoglutathione (GSNO) and the tetra-iron-sulphur cluster nitrosyl, Roussin's black salt (RBS). We have used the comet assay as a highly sensitive method to measure DNA-damaging ability, and also measured inhibition of DNA synthesis and inhibition of insulin secretion. We have examined the effect of SOD and catalase on each of these endpoints in HIT-T15 cells following a 30-min exposure to the compounds (24 h for DNA synthesis). All compounds produced a significant dose-dependent increase in strand-breakage formation and all inhibited DNA synthesis and glucose-stimulated insulin secretion. RBS was the most potent. SOD did not reduce the responses observed with any of the compounds. Catalase largely prevented DNA strand breakage, inhibition of DNA synthesis and inhibition of insulin secretion by SIN-1, but had no effect on responses to GSNO or RBS. Addition of SOD together with catalase gave no greater protection against SIN-1 than catalase alone. The nitric oxide and superoxide anion produced by SIN-1 are though to combine to form highly reactive peroxynitrite. In addition, H2O2 may be formed in the presence of SIN-1 and may form hydroxyl radical in the presence of a transition metal, such as Fe2+. It appears that in insulin-secreting cells, the effects of SIN-1 are largely mediated by this latter mechanism. In contrast, GSNO and RBS appear to act by a different mechanism, not overtly involving reactive oxygen species. GSNO and H2O2 show no significant interaction in the induction of DNA strand breaks. Both nitric oxide and H2O2 are effective, directly or indirectly, as DNA strand-breaking agents, inhibitors of DNA synthesis and inhibitors of insulin secretion.

Intracellular targets for nitric oxide toxicity to pancreatic beta-cells.

The radical nitric oxide (NO) may be a mediator of pancreatic beta-cell damage in early insulin-dependent diabetes mellitus (IDDM). Under the stimulus of cytokines, invading macrophages and the beta-cell themselves may produce large amounts of NO, leading to beta-cell dysfunction and death. It still remains to be determined which are the intracellular targets for NO-induced damage. Available data from rat islets indicate that the radical inactivates the mitochondrial enzyme aconitase, impairing substrate oxidation and ATP production. Ionic channels and complexes I and II of the mitochondrial electron transport chain are two other possible targets for NO effects which may impair insulin secretion. NO also leads to nuclear DNA damage in both rat and human pancreatic beta-cells, as evaluated by the 'comet assay'. The effects of NO at the DNA level are complex, and involve formation of N-nitrosoamines, deamination of purines and pyrimidines, or damage induced by peroxynitrite. Besides inducing over DNA damage. NO may also inactivate DNA repair/replication enzymes. The outcome of NO-induced beta-cell DNA damage can be cell death by apoptosis or, in some cases, necrosis. Upon cell damage beta-cells trigger cell repair mechanisms. This seems also to be the case following NO exposure, and insulin-producing cells are able to regain their function following treatment with non-lethal concentrations of NO. A better understanding of the mechanisms involved in NO-induced beta-cell damage and repair may be instrumental in developing new strategies for IDDM prevention.

Primates.

This article is an introduction to pet nonhuman primates and includes a discussion of basic life history and physiologic parameters of the most common pet species, diet and husbandry guidelines, and resources for further information for both the pet owner and the veterinarian. Preventive health care is emphasized, including diagnostic tests that should be included in annual examinations, recommended serologic testing, tuberculin testing, and vaccinations. Zoonotic diseases and preventive measures for the owner and the clinical staff are outlined. Common disease problems and recommended therapeutics are presented.

Therapeutics of companion exotic marsupials.

Exotic marsupials are becoming popular companion pets, yet little pharmacologic information is available to the practitioner. Species include sugar gliders (Petarus breviceps), South American (Brazilian) short-tailed opossums (Monodelphis domestica), brushtail possum or "phalanger" (Trichosurus vulpecula), and wallabies: Bennett's (Macropus rufogriseus rufogriseus) and Tammar or Dama (Macropus eugenii). Guidelines for logical choices of medications based on allometric scaling, gastrointestinal tract anatomy and physiology, and liver metabolism are given. An example of scaling and a formulary based on anecdotal, published medications are provided.

Palmar cold threshold test and median nerve electrophysiology in carpal tunnel compression neuropathy.

The diagnosis of median nerve compression neuropathy at the carpal tunnel is usually confirmed by clinical electrophysiology. The classical findings of a significantly slowed median nerve conduction velocity for both sensory and motor fibres, with a prolonged distal motor latency and a reduced amplitude compared to age-related norms are unambiguous, but these criteria are often present only in part. In such cases another quantitative indicator of compression neuropathy would be extremely helpful. The present study aimed to test whether measurement of warm and cold sensory acuity in cases of putative median nerve carpal tunnel compression would aid diagnostic certainty. Warm sensation is mediated by unmyelinated C-afferents, while cold sensation is conveyed by thinly myelinated A delta afferents. Because compression usually blocks larger diameter fibres first, cold perception on the skin of the palm distal to the compression should be more impaired than is warm perception. Standard electrophysiological measurements (median and ulnar motor and sensory nerve conduction velocities) were made, then perceptual thresholds for both warm and cold stimuli were measured on the skin of the wrist above the carpal tunnel and on the palm of the affected hand in 59 subjects. There was a significantly reduced median motor nerve conduction velocity and prolonged distal motor latency compared to normals. Further, although both thermal thresholds at the wrist were normal, those on the palm were elevated, cold being significantly raised (P less than 0.02) compared both to warm and to age-matched controls. Correlation of the nerve conduction velocity findings and thermal sensory acuity did not yield significant covariance of the positive and negative findings. Overall the results suggest that detection of preferentially elevated cold perceptual threshold (ie reduced cold sensory acuity) on the skin of the palm may aid in the diagnosis of putative carpal tunnel compression in patients with minimal or ambiguous electrophysiological data and provide a functional index of recovery after decompression.

Insulin sensitivity and sensory nerve function.

The central and peripheral nervous systems do not require insulin for glucose uptake. However, insulin receptors have been detected in these regions. The aim of this study was to examine peripheral sensory nerve function and its dependence on insulin using healthy non-diabetic control subjects, obese individuals, and diabetic (insulin dependent and non-insulin dependent diabetes mellitus) subjects. The results revealed that the warm and cold perception thresholds, reflecting the functional states of unmyelinated C-fibres and A-delta fibres respectively, increased with reduced insulin sensitivity and with increased fasting insulin concentrations. From such data in non-diabetic subjects with measured insulin sensitivity, it appeared that sensory nerve function was disturbed in normoglycemic but insulin resistant states, suggesting that insulin has an action on nervous tissue in addition to its effects on glucose metabolism. The mechanisms of this action remain to be elucidated.

Interleukin-1 beta effects on cyclic GMP and cyclic AMP in cultured rat islets of Langerhans-arginine-dependence and relationship to insulin secretion.

When islets were cultured with interleukin-1 beta (1 or 100 pmol/l) for 12 h in arginine-containing medium, cyclic GMP levels were increased 1.6- and 4.5-fold respectively. The arginine analogue, N-omega-nitro-L-arginine methyl ester, which blocks nitric oxide formation and partially reverses inhibition of insulin secretion by 100 pmol/l interleukin-1 beta, largely, but not completely, blocked generation of cyclic GMP. Treatment of islets with 100 pmol/l interleukin-1 beta for 12 h significantly decreased islet cyclic AMP generation in the absence of isobutylmethylxanthine (from 13.1 +/- 0.7 to 9.3 +/- 0.8 fmol/micrograms islet protein), this fall was arginine-dependent and may have resulted from an effect on a cyclic AMP phosphodiesterase, since it was masked if isobutylmethylxanthine was present. Isobutylmethylxanthine (0.4 mmol/l) reduced the inhibitory potency of interleukin-1 beta in 15 h slightly but significantly from 80.5 to 59.0%. The morpholinosydnonimine SIN-1, which is a nitric oxide donor, inhibited insulin secretion, raised islet cyclic GMP and lowered cyclic AMP; its effects were similar to those of interleukin-1 beta. However, 6-anilinoquinoline-5,8-quinone, [LY83583 (1-10 mumol/l)], inhibited insulin secretion, and significantly decreased cyclic GMP while 8-bromocyclic GMP stimulated insulin secretion. Both low- and high-dose interleukin-1 beta treatment give a large arginine-dependent and a small, yet significant, arginine-independent increase in cyclic GMP. The inhibitory effect of SIN-1 or interleukin-1 beta on insulin secretion seems to depend to a small extent on decreased islet cyclic AMP, though sustained increases in nitric oxide or depleted islet GTP may directly affect the secretory process.