O6-methylguanine DNA methyltransferase and glucose transporter 2 in foregut and hindgut gastrointestinal neuroendocrine neoplasms.

BACKGROUND: Streptozocin (STZ) is used for treating both pancreatic (PanNET) and gastrointestinal (GI-NET) neuroendocrine tumors but its therapeutic efficacy is relatively low in GI-NETs. Therefore, it has become pivotal to select GI-NET patients who could benefit from STZ treatment. STZ is transported via the glucose transporter 2 (GLUT2) into the cells and the loss of O6-methylguanine DNA methyltransferase (MGMT) also increases its therapeutic efficacy. Therefore, GLUT2 high and MGMT low status could be the surrogate markers of STZ. METHODS: In this study, we examined the MGMT and GLUT2 status in gastrointestinal neuroendocrine neoplasm (NEN). We studied 84 NEN cases: 33 foregut and 37 hindgut GI-NETs and 14 gastrointestinal neuroendocrine carcinomas (GI-NECs). RESULTS: In GI-NETs, MGMT scores of ≥2 and ≥ 3 were 77% (54/70) and 56% (39/70), respectively, and GLUT2 scores of ≥4 and ≥ 6 were 30% (21/70) and 4.3% (3/70), respectively. Methylation-specific polymerase chain reaction revealed that MGMT promoter methylation was detected only in 2/14 GI-NECs but none of the included GI-NETs. GLUT2 (GLUT2 score) and MGMT immunoreactivity (MGMT and H-scores) were both significantly correlated with Ki-67 labeling index (GLUT2 score: P = 0.0045, ρ = - 0.4570; MGMT score: P = 0.0064, ρ = - 0.4399; H-score: P = 0.0110, ρ = - 0.4135) and MGMT immunoreactivity were significantly correlated with GLUT2 immunoreactivity (MGMT score: P = 0.0198; H-score, P = 0.0004, ρ = 0.5483) in hindgut NETs, but not in foregut NETs. However, discrepancies from the above correlation between GLUT2 and MGMT immunoreactivity were detected in several GI-NET cases which could be potential candidates for STZ therapy. CONCLUSION: The evaluation of MGMT and GLUT2 status could provide an important information in planning STZ therapy in GI-NET patients.

The neurogenic effects of rosmarinic acid in a mouse model of type 2 diabetes mellitus

There is emerging evidence for a dysregulation of insulin signaling in the brains of patients with Alzheimer's disease (AD) with overlapping molecular features to Type 2 Diabetes Mellitus (T2DM). In addition, T2DM is a known risk factor of AD. The goal of this study was to investigate the neurogenic and neuroprotective potential of rosmarinic acid (RA) in a streptozotocin (STZ)-induced combined with high fat diet (HFD) mouse model of diabetes. Animals were divided into four experimental groups (control, diabetic, diabetic + RA, RA only). Behavioral analysis was performed to assess spatial learning and anxiety levels of animals, whereas quantitative real time PCR was carried out to assess the gene expression levels of neuronal markers of neurogenesis (Ki67, DCX and NeuN). A significant decrease in memory and spatial learning was observed in the diabetic mice, which was substantially improved by RA treatment. RA also increased the gene expression of NeuN, DCX and Ki67, which were dysregulated in the diabetic model. This study proposes RA as a potential therapeutic agent to mitigate neuronal dysfunction associated with T2DM by promoting adult hippocampal neurogenesis.

Metabolic effects of B-glucans (Saccharomyces cerevisae) per os administration in rats with streptozotocin-induced diabetes / Efectos metabólicos de los β-glucanos (saccharomyces cerevisiae) a través de la administración oral en ratas con diabetes inducida por estreptozotocina

Introduction: beta-glucans (BG) derived from plant tissues are reported to show metabolic effects. In contrast, those fibers isolated from yeast seem to be more related to immune response modulation. Since diabetic individuals are more susceptible to exacerbation of inflammatory signs, the ingestion of fibers that could conjugate both metabolic and immune effects would be of great importance. Objective: we investigated the effect of BG - Saccharomyses cerevisae - ingestion on glycemic and lipoprotein profile of diabetic rats. Design: twenty-four adult Wistar rats were used, distributed into 4 groups in a design of entirely casualized delineation with a 2 x2 factorial model (with and without diabetes; with and without BG). Diabetes Mellitus was induced by an intraperitoneal injection of 80mg/kg of strepzotocin. Thus, animals with fasting glycemia of over 250mg/dl were considered diabetic. Forty-eight hours after induction, the rats received daily doses of 30 mg/kg of BG or saline solution by gavage during 28 days. Results and discussion: the Groups with DM presented a higher glycemic index and lower C peptide levels than the control groups, in addition to lower weight gain and higher ration consumption, water ingestion and urinary volume. Total cholesterol levels (CT), LDL-C + VLDL-C, plasma triacylglycerides (TAG) and alanine aminotransferase (ALT) were also higher in the diabetic animals (p<0.05). No histopathological hepatic alterations were observed in any of the groups. Furthermore, the diabetic animals present increase in villous:crypt ratio (V:C) in the duodenum, without interference of BG. No alterations in the carcass were observed between the groups. Conclusion: it was concluded that the use of BG significantly reduced the glycemic, TAG and ALT levels, showing its therapeutic potential (AU)

Introdución: los beta-glucanos (BG) derivados de tejidos vegetales se ha informado que muestran efectos metabólicos. Por el contrario, esas fibras aisladas de levadura parecen estar más relacionadas con la modulación de la respuesta inmune. Dado que los individuos con diabetes son más susceptibles a la exacerbación de los signos inflamatorios, la ingestión de fibras sí podría conjugar ambos efectos metabólicos e inmunológicos, lo cual sería de gran importancia. Objetivo: el objetivo de este estudio fue investigar los efectos de la ingestión de los BG —Saccharomyses cerevisiae— en el perfil glucémico y la lipoproteína de ratas diabéticas. Metodos: en el diseño de delineación, totalmente precario, fueron utilizadas 24 ratas Wistar macho adultas distribuidas en cuatro grupos, con un modelo factorial 2x2 (con y sin diabetes, con y sin BG). La diabetes mellitus fue inducida por la inyección intraperitoneal de un 80 mg/kg de estrepzotocina. Por lo tanto, los animales con glucemia en ayunas de más de 250 mg/dl fueron considerados diabéticos. Cuarenta y ocho horas después de la inducción, las ratas recibieron dosis diarias de 30 mg/kg de BG o solución salina mediante alimentación forzada durante 28 días. Resultados y discusión: los grupos con DM presentó el mayor índice glucémico y menores niveles de péptido C que los grupos de control, además de reducir el aumento de peso y un mayor consumo de la ración, la ingestión de agua y el volumen urinario. Los niveles de colesterol total (CT), LDL-C + VLDL-C, triacilglicéridos plasmáticos (TAG) y alanina aminotransferasa (ALT) también fueron más altos en los animales diabéticos (p<0,05), y había alteraciones en los niveles de HDL-C. La ingestión de BG redujo las concentraciones de glucosa en sangre (30%), TAG (32%) y ALT (41%) (p<0.05). No se observaron alteraciones hepáticas en ninguno de los grupos. Además, los animales diabéticos presentaron un aumento de la relación cripta:vellosidades (V:C) en el duodeno, sin interferencia de BG. No se observaron alteraciones en la carcasa entre los grupos. Conclusión: se concluyó que el uso de BG redujo significativamente la glucemia, los niveles de TAG Y ALT, mostrando su potencial terapéutico (AU)

Histopathological findings of the pancreas, liver, and carbohydrate metabolizing enzymes in STZ-induced diabetic rats improved by administration of myrtenal

This study aims to evaluate the efficacy of myrtenal, a natural monoterpene, for its antihyperglycemic effects and beta cell protective properties in streptozotocin (STZ)-induced diabetic rats. Oral administration of myrtenal at doses of 20, 40, and 80 mg/kg body weight to diabetic rats for 28 days resulted in a significant reduction (P < 0.05) in the levels of plasma glucose, glycosylated hemoglobin (HbA1c), and an increase in the levels of insulin and hemoglobin (Hb). Protection of body weight loss of diabetic rats by myrtenal was noted. The altered activities of the key metabolic enzymes involved in carbohydrate metabolism such as hexokinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, and hepatic enzymes AST, ALT, and ALP levels of diabetic rats were significantly improved by the administration of myrtenal in STZ-induced diabetic rats. Moreover, myrtenal treatment improved hepatic and muscle glycogen content in diabetic rats. Histopathological studies further revealed that the reduced islet cells were restored to near-normal conditions on treatment with myrtenal in STZ-induced diabetic rats. An alteration in liver architecture was also prevented by myrtenal treatment. Our results suggest that myrtenal possess antihyperglycemic and beta cell protective effects. Hence, myrtenal could be considered as a potent phytochemical for development as a new antidiabetic agent (AU)

Secoisolariciresinol diglucoside in high-fat diet and streptozotocin-induced diabetic nephropathy in rats: a possible renoprotective effect

Due to substantial morbidity and high complication rate of diabetes mellitus, which is considered as the third killer in the world, a search for the effective blockade of the progression of diabetic nephropathy (DN) remains a therapeutic challenge. Alternative antidiabetic drugs from natural plants are highly demanded nowadays. The aim of this study was to investigate the renoprotective effect of secoisolariciresinol diglucoside (SDG) on DN induced in rats. Diabetes was induced in male Sprague-Dawley rats by a high-fat diet (HFD) and an intraperitoneal 35 mg/kg streptozotocin (STZ) injection. Rats were divided into four groups: normal control rats, diabetic control rats, diabetic rats treated with SDG at 10 mg/kg/day for 4 weeks, and diabetic rats treated with SDG at 20 mg/kg/day for 4 weeks. At the end of the treatment, blood and renal tissue samples were collected for biochemical examination. The results revealed that SDG treatment significantly increased insulin level and decreased blood glucose, fructosamine, creatinine, and blood urea nitrogen levels in diabetic rats. Also, SDG significantly increased renal reduced glutathione, superoxide dismutase and decreased malondialdehyde and nitric oxide levels. In addition, SDG downregulated the renal nuclear factor kappa-B (NF-κB), tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) and upregulated renal survivin and B-cell lymphoma-2 (Bcl-2) expressions when compared with untreated diabetic control rats. This study demonstrated, for the first time, the renoprotective effects of SDG in HFD/STZ-induced DN in rats through correction of hyperglycemia; attenuation of oxidative/nitrosative stress markers; downregulation of renal expressions of inflammatory markers NF-κB, TNF-α, and iNOS; along with upregulation of renal expressions of antiapoptotic markers survivin and Bcl-2

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Modulation of neuropathic pain in experimental diabetes mellitus

The main objective of the current article is to investigate the diabetic polyneuropathy which represents a major preoccupation within the context of high incidence of diabetes mellitus (DM) and its complications. Moreover, neuropathy may develop despite intensive hyperglycaemic control. The effect of Zn and black grape seed polyphenols (BGSP) in streptozotocin diabetic rats was studied. Zn and BGSP were administered by gavage, daily, for 16 weeks to Wistar rats that have been rendered diabetic by a single i.v. injection of streptozotocin (55 mg/kg body weight). Dysalgesia was investigated under the conditions of nociceptive stimulation through the following tests: the thermoalgesic mechanism through the tail-flick test, the hot plate test and the plantar test, and the mechanoalgesic mechanism through the algesimetric test. Thermal hyperalgesia detected in the diabetic group is significantly reduced (p < 0.001) through the administration of polyphenols, or even better, of Zn. Diabetes-associated mechanical hyperalgesia decreased significantly (p < 0.001) probably through the inhibition of the NMDA receptors. Administration of Zn or BGSP to the diabetic group improves glycosylated haemoglobin (HbA1c) values but does not bring them to normal. The present data suggest a favourable effect of Zn and BGSP in inhibiting diabetic complications by several mechanisms

Hepatoprotective effects of melatonin against pronecrotic cellular events in streptozotocin-induced diabetic rats

Oxidative stress-mediated damage to liver tissue underlies the pathological alterations in liver morphology and function that are observed in diabetes. We examined the effects of the antioxidant action of melatonin against necrosis-inducing DNA damage in hepatocytes of streptozotocin (STZ)-induced diabetic rats. Daily administration of melatonin (0.2 mg/kg) was initiated 3 days before diabetes induction and maintained for 4 weeks. Melatonin-treated diabetic rats exhibited improved markers of liver injury (P < 0.05), alkaline phosphatase, and alanine and aspartate aminotransferases. Melatonin prevented the diabetes-related morphological deterioration of hepatocytes, DNA damage (P < 0.05), and hepatocellular necrosis. The improvement was due to containment of the pronecrotic oxygen radical load, observed as inhibition (P < 0.05) of the diabetes-induced rise in lipid peroxidation and hydrogen peroxide increase in the liver. This was accompanied by improved necrotic markers of cellular damage: a significant reduction in cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) into necrotic 55- and 62-kDa fragments, and inhibition of nucleus-to-cytoplasm translocation and accumulation in the serum of the high-mobility group box 1 (HMGB1) protein. We conclude that melatonin is hepatoprotective in diabetes. It reduces extensive DNA damage and resulting necrotic processes. Melatonin application could thus present a viable therapeutic option in the management of diabetes-induced liver injury

Phlorizin pretreatment reduces acute renal toxicity in a mouse model for diabetic nephropathy.

Streptozotocin (STZ) is widely used as diabetogenic agent in animal models for diabetic nephropathy (DN). However, it is also directly cytotoxic to kidneys, making it difficult to distinguish between DN-related and STZ-induced nephropathy. Therefore, an improved protocol to generate mice for DN studies, with a quick and robust achievement of the diabetic state, without direct kidney toxicity is required. To investigate the mechanism leading to STZ-induced nephropathy, kidney damage was induced with a high dose of STZ. This resulted in delayed gastric emptying, at least partially caused by impaired desacyl ghrelin clearance. STZ uptake in the kidneys is to a large extent mediated by the sodium/glucose cotransporters (Sglts) because the Sglt inhibitor phlorizin could reduce STZ uptake in the kidneys. Consequently, the direct toxic effects in the kidney and the gastric dilatation were resolved without interfering with the ß-cell toxicity. Furthermore, pancreatic STZ uptake was increased, hereby decreasing the threshold for ß-cell toxicity, allowing for single low non-nephrotoxic STZ doses (70 mg/kg). In conclusion, this study provides novel insights into the mechanism of STZ toxicity in kidneys and suggests a more efficient regime to induce DN with little or no toxic side effects.

Antihyperglycemic and antioxidant effects of a flavanone, naringenin, in streptozotocin-nicotinamide-induced experimental diabetic rats

In the present study, the putative antihyperglycemic and antioxidant effects of a flavanone, naringenin, were evaluated in comparison with those of glyclazide, a standard drug for therapy of diabetes mellitus. Diabetes was induced experimentally in 12-h-fasted rats by intraperitoneal injections of first streptozotocin (50 mg/kg b.w.) and then of nicotinamide (110 mg/kg b.w.) after a 15-min interval. Untreated diabetic rats revealed the following in comparison with normal rats: significantly higher mean levels of blood glucose and glycosylated hemoglobin, significantly lower mean levels of serum insulin, significantly lower mean activities of pancreatic antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase), significantly lower mean levels of plasma non-enzymatic antioxidants (reduced glutathione, vitamin C , vitamin E), significantly elevated mean levels of pancreatic malondialdehyde (MDA) and significantly elevated mean activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Following oral administration of naringenin (50 mg/kg b.w./day) to diabetic rats for 21 days, the following observations were made in comparison with (..) (AU)

Wfs1 mutation makes mice sensitive to insulin-like effect of acute valproic acid and resistant to streptozocin

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Valproic acid (VLP) is a widely used anticonvulsant and mood-stabilizing drug that relieves the endoplasmic reticulum (ER) stress response, a pathogenetic process related to diabetes. The aim of the present study was to evaluate whether acute valproic acid is able to interfere with glucose intolerance in two different diabetes models: The first model was a Wfs1 mutant mouse with an elevated ER stress response and the second model a streptozocin-induced diabetic mouse. VLP (300 mg/kg, i.p.) was administered to Wfs1 knockout (KO) mice and glucose tolerance test was performed 15 min later. VLP did not have an effect on the course of the glucose tolerance test in wild-type mice, while it did normalize the glucose intolerance in Wfs1 knockout mice. Acute valproic acid also lowered the blood glucose levels in streptozocin-treated mice and potentiated the effect of insulin in these mice. Thus, acute valproic acid is effective in lowering blood glucose levels possibly by potentiating insulin action in both Wfs1 KO mice and in streptozocin-induced type 1 diabetic mice (AU)

Beneficial effects of banana (Musa sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats

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Diabetes is a chronic health problem and major cause of death in most of the countries. Diet management plays an important role in controlling diabetes and its complications along with insulin and drugs. We have examined the effect of banana (Musa sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats. Our results indicated that banana flower and pseudostem have low glycemic index and have a high content of dietary fiber and antioxidants. Diabetic symptoms like hyperglycemia, polyuria, polyphagia, polydipsia, urine sugar, and body weight were ameliorated in banana flower- and pseudostem-treated rats. Increased glomerular filtration rate in the diabetic group (5.1 ± 0.22 ml/min) was decreased in banana flower-fed (2.5 ± 0.37 ml/min) and pseudostem-fed (3.0 ± 0.45 ml/min) groups and were significant at P < 0.001 and P < 0.01, respectively. Fructosamine and AGEs formed during diabetes were inhibited in treated groups when compared with the diabetic group. The diabetic group showed 11.5 ± 0.64 ìg of AGEs/mg protein in kidney, whereas, in banana flower- and pseudostem-fed groups, it was reduced to 9.21 ± 0.32 and 9.29 ± 0.24 ìg/mg protein, respectively, and were significant at P < 0.01. These findings suggest that banana flower and pseudostem have anti-diabetic and anti-AGEs properties and are beneficial as food supplements for diabetics (AU)

Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin.

AIMS/HYPOTHESIS: The role of selective uptake and alkylation in the diabetogenic action of streptozotocin was investigated in bioengineered RINm5F insulin-producing cells, with different expression levels of the glucose transporter GLUT2, by comparing the toxicity of streptozotocin with that of four chemically related alkylating compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N nitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). METHODS: GLUT2 expressing RINm5F cells were generated through stable transfection of the rat glucose transporter GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Cells expressing the glucose transporter GLUT2 were much more susceptible to streptozotocin toxicity than control cells due to the uptake of streptozotocin by this specific glucose transporter. In contrast, the GLUT2 expression had no effect upon the toxicity of MNU, ENU, MMS or EMS. Although the latter substances are, like streptozotocin, cytotoxic through their ability to cause DNA alkylation, they are not diabetogenic because they are not taken up through the glucose transporter GLUT2. CONCLUSION/INTERPRETATION: Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research.

Preliminary investigation about the effects of streptozotocin-induced chronic diabetes on the nerve cell number and size of myenteric ganglia in rat colon

Estudiamos el efecto de la diabetes mellitus crónica, inducida por estreptozotocina, en cuanto a los aspectos cuantitativos de las neuronas del plexo mientérico del colon proximal de ratas. Utilizamos cuatro grupos de animales: dos grupos de animales diabéticos (D2 y D8). En dos grupos D2 y D8, sacrificamos animales de dos y ocho meses, respectivamente, después de la inducción de diabetes; y los otros dos grupos C2 y C8, fueron mantenidos como control de los grupos anteriores. Retiramos el colon de las ratas y lo sometimos a cortes histológicos que fueron teñidos con Hematoxilina-Eosina. Se obtuvieron preparados de membrana teñidos con el método Giemsa y NADH-diaphorasa. Constatamos que la mayoría de las neuronas, tanto de animales diabéticos como no diabéticos, poseen núcleo excéntrico y que, por lo tanto, este hallazgo no es indicativo de proceso degenerativo. Verificamos que animales sacrificados dos meses después de la inducción de diabetes, no sufrieron reducción significativa en el número de neuronas por área, mientras que en animales sacrificados ocho meses después de la inducción de diabetes, presentaron reducción significativa en el número de neuronas por área, comparados con el grupo control

STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells.

The glucose analog streptozotocin (STZ) has long been used as a tool for creating experimental diabetes because of its relatively specific beta-cell cytotoxic effect, but the mechanism by which systemic injection of STZ causes beta-cell destruction is not well understood. In the current study, we have used insulinoma (RIN) and AtT-20ins cell lines engineered for overexpression of GLUT2 or GLUT1 to investigate the role of glucose transporter isoforms in mediating STZ cytotoxicity. The in vivo effects of STZ were evaluated by implantation of RIN cells expressing or lacking GLUT2 into athymic nude rats. The drug had a potent cytotoxic effect on RIN cells expressing GLUT2, but had no effect on cells lacking GLUT2 expression, as indicated by histological analysis and measurement of the blood glucose levels of treated animals. The preferential cytotoxic effect of STZ on GLUT2-expressing cell lines was confirmed by in vitro analysis of GLUT2-expressing and untransfected RIN cells, as well as GLUT2- and GLUT1-overexpressing AtT-20ins cells. Consistent with these data, only GLUT2-expressing RIN or AtT-20ins cells transported STZ efficiently. We conclude that expression of GLUT2 is required for efficient killing of neuroendocrine cells by STZ, and this effect is related to specific recognition of the drug as a transported substrate by GLUT2 but not GLUT1.

Degradation products of streptozotocin do not induce hyperglycemia in rats.

The present study was conducted to determine whether degradation products of streptozotocin formed under various conditions induce hyperglycemia in rats. Streptozotocin is completely degraded in pH 7.4 buffer in 4 hr and even more rapidly in plasma. Streptozotocin degradation products resulting from incubation in pH 7.4 phosphate buffer or in plasma were not diabetogenic in rats.

Pharmacokinetic study of intraperitoneal streptozotocin.

Streptozotocin, a nitrosourea, has limited antitumour activity. However, in concentrations and exposures higher than those achieved after intravenous dosing, streptozotocin has been reported to sensitize various cell lines in vitro to other nitrosoureas or alkylating agents. We hypothesized that the intraperitoneal administration of streptozotocin would achieve concentrations and exposures in the peritoneal cavity sufficient for this sensitization to take place. The pharmacokinetics of streptozotocin in the peritoneal cavity and in plasma were determined in patients who received 1 g of streptozotocin via the intraperitoneal route. Fifteen courses were administered to 12 patients. The mean total area under the concentration versus time curves (AUC) was 183 +/- 31 (SE) mM min in the peritoneal cavity (5 courses) and 5.3 +/- 1.1 (SE) mM min in plasma (6 courses). The mean peritoneal to plasma AUC ratio was 64 +/- 23 (5 courses). The mean peak streptozotocin concentrations in the peritoneal cavity (5 courses) and plasma (6 courses) were 1.9 +/- 0.4 mM and 0.03 +/- 0.01 mM, respectively. No significant toxicity was observed on any course. We conclude that intraperitoneal administration of streptozotocin is feasible, and that drug concentrations and exposures are in an appropriate range in the peritoneal cavity to cause sensitization to other nitrosoureas.

Inhibition of Streptococcus mutans by the antibiotic streptozotocin: mechanisms of uptake and the selection of carbohydrate-negative mutants.

The antibiotic streptozotocin [2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranoside], an analog of N-acetylglucosamine (NAG), has been shown to be useful for the selection of carbohydrate-negative and auxotrophic bacterial mutants. We have adapted this method for use with the oral pathogen Streptococcus mutans, a gram-positive, aerotolerant anaerobe that uses predominantly carbohydrates as carbon sources for growth. Streptozotocin selectively kills growing cells of S. mutans GS-5, and under appropriate conditions it can reduce the number of viable cells in actively growing cultures by a factor of 10(3) to 10(4). However, unlike in enteric bacteria, which take up this antibiotic by a single NAG-specific transport system, streptozotocin appears to be taken up in S. mutans by both a NAG-specific system and a relatively nonspecific system that is also involved in glucose, fructose, and mannose uptake. Combining streptozotocin selection and a screening procedure involving indicator plates containing triphenyl-tetrazolium chloride, we developed a general method for the isolation of carbohydrate-negative and auxotrophic mutants of S. mutans. A preliminary characterization of both pleiotropic and specific carbohydrate-negative mutants isolated by using this procedure is presented.

Intracellular activation of cytotoxic agents: kinetic models for methylnitrosoureas and N-methyl-N'-nitro-N-nitrosoguanidine in cell culture.

The cytotoxic activity of N-methyl-N-nitrosourea (MNU), streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in cell culture by using a P388 cell growth rate inhibition assay. These agents appear to have very different activities when inhibition is related to the agent concentration in the culture medium: ED50(C0) = 40 microM for MNNG to 875 microM for streptozotocin. The mechanism of action of these three agents involves conversion to the active methanediazonium ion and subsequent methylation of cellular macromolecules. As a consequence, the rates of conversion of the parent agent to the methylating species in the medium and within the cell are important parameters that also need to be considered to reach a more detailed understanding of the mechanism of action. In order to do this, a kinetic model has been developed to calculate the concentration of drug that is converted to active methylating species within the cell during the assay incubation period. The use of cell culture kinetic models was extended from simple compounds activated through solvolytic reactions (nitrosoureas) to an agent that undergoes selective intracellular activation (MNNG). By use of measured values for initial drug concentration, incubation time, and cell volume, as well as extracellular and intracellular chemical activation rate constants, the intracellular concentration, [P4], which represents the cumulative intracellular reaction products formed during the incubation period, was calculated and related to cytotoxicity. All three agents showed an ED50[P4] between 140 and 180 microM, and for MNNG, this ED50 was independent of extracellular sulfhydryl concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethylidene glucose-substituted new analogue of streptozotocin cannot induce diabetes: study on the basis of structure and activity relationship.

4,6-O-Ethylidene glucose (ethylidene glucose), a specific inhibitor at the outer surface of a glucose transporter in the cell membranes, substituted analogue of streptozotocin was newly synthesized. This compound did not induce diabetes in rats and also did not show cytotoxic effect on pancreatic beta cells of neonatal rats in a monolayer culture system. The reasons why such a molecule was designed and why it showed no biological effects are discussed on the basis of a structure-activity relationship. Our results afford positive evidence for the presence of a glucose transport system or a glucose transporter on pancreatic beta cells and its involvement in the action of streptozotocin on beta cells.