Alterations in rat pancreatic B-cell function induced by prenatal exposure to cyproheptadine.

Treatment of pregnant rats with cyproheptadine during the last 8 days of gestation produced alterations in the function of the endocrine pancreas in the offspring. The abnormalities exhibited by 50-day-old progeny of drug-treated dams included glucose intolerance, a two-fold increase in levels of insulin in the pancreas, and an accentuated response to the insulin-lowering action of cyproheptadine in the endocrine pancreas. The alterations observed in these animals were limited to the insulin-containing cells, and no change was found in the pancreatic concentrations of glucagon and somatostatin. The results are the first to demonstrate that postnatal pancreatic B-cell function can be selectively altered by prenatal exposure to an exogenous chemical.

Inhibition of alloxan action in isolated pancreatic islets by superoxide dismutase, catalase, and a metal chelator.

The possible participation of superoxide anions, hydrogen peroxide, and hydroxyl radicals in the action of alloxan was investigated using isolated rat pancreatic islets. Exposure of islets for 5 min to alloxan (0.15 or 0.2 mg/ml) inhibited subsequent glucose-stimulated insulin release. The presence of superoxide dismutase (1000 U/ml), catalase (50 microgram/ml), or a metal chelator diethylenetriaminepentacetic acid (1 mM) markedly attenuated this effect of alloxan. Use of these agents afforded complete protection from the lower concentration of alloxan and partial protection from the higher concentration of the toxic compound. Inactivation of the enzymes or addition of excess iron to the chelating agent before its use with alloxan eliminated the protective action of these agents. The results are consistent with the proposal that hydroxyl radicals, generated via reactions that involve superoxide anions, hydrogen peroxide, and iron, mediate the deleterious effect of alloxan in pancreatic islets.

Inhibition of insulin release from rat pancreatic islets by drugs that are analogues of dopamine.

Various synthetic dopamine (DA) analogues have been shown to produce glucose intolerance and inhibit the compensatory increase in serum insulin during an oral glucose tolerance test (OGTT). To investigate the possibility that there is a direct action of dopamine analogues to inhibit glucose-stimulated insulin release from the endocrine pancreas, the following compounds were compared with the effects of epinephrine (EPI) on isolated rat pancreatic islets: apomorphine (APO), pergolide, lergotrile, TL-99 (2-dimethylamino-6,7-dihydroxytetralin), and RDS-127 (2-di-n-propyl-amino-4,7-dimethoxyindane). EPI, TL-99, and pergolide inhibited insulin release in a concentration-dependent fashion (10(-7)-10(-5) M), whereas lergotrile inhibited at 10(-5) M but not at 10(-6) M. RDS-127 and APO were ineffective at 10(-5) M, but produced a greater than 50% inhibition at 2 X 10(-4) M. The potencies of the DA analogues fell into two groups: compounds that are approximately as active as EPI (e.g., TL-99 and pergolide) or compounds that are relatively inactive (e.g., APO, lergotrile, and RDS-127). The inhibitory actions of EPI, TL-99, and pergolide were blocked by the alpha 2-adrenergic receptor antagonist yohimbine, whereas the DA receptor antagonist, sulpiride, had no effect, suggesting an action initiated at alpha 2-adrenergic receptors. Drugs from both groups produced marked glucose intolerance and inhibited the compensatory increase in insulin during an OGTT. Adrenodemedullation blocked the glucose intolerance and inhibition of insulin release caused by RDS-127, whereas these effects of TL-99 were not attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of rat insulin I and II messenger RNA synthesis: effects of fasting and cyproheptadine.

Rats and mice retain a duplicated insulin (I) gene. Because the duplicated gene shares only incomplete homology with the ancestral insulin (II) gene it may be regulated differently. In the studies presented here we measured changes in abundance of these distinct insulin mRNAs and their precursors in response to fasting and fasting plus a single dose of cyproheptadine, two experimental manipulations that cause changes in the level of total insulin mRNA in rats. Both diminished rat insulin II mRNA to a greater extent than rat insulin I mRNA. Rat insulin II mRNA comprised 41% of the total insulin mRNA in 0 h controls and decreased to 33% of the total insulin mRNA after a 10-h fast. Insulin II mRNA decreased to 26% of the total insulin mRNA 10 h after treatment with cyproheptadine. To determine whether these manipulations had effects on insulin mRNA synthesis, precursors for each of the two mRNAs were quantified. Fasting for 24 h had only small effects on insulin I mRNA precursor, but diminished rat insulin II pre-mRNA to 32% of the 0 h control values. One and a half hours after fasting plus cyproheptadine administration, pre-mRNA for rat insulin II levels had decreased to 38%, while rat insulin I pre-mRNA remained at levels present in 0 h controls. Levels of rat insulin I and II pre-mRNAs were both maximally depressed at 10 h, but rat insulin II pre-mRNA decreased to 3%, while rat insulin I pre-mRNA diminished to only 49% of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired insulin release after exposure of pancreatic islets to autooxidizing dihydroxyfumarate.

Autooxidizing dihydroxyfumarate (DHF) is a known generator of superoxide anions and by hydroxyl free radicals. Isolated rat pancreatic islets were exposed for 5 min to 0.4 mg/ml DHF in an oxygenated buffer solution. As a result of this exposure, the islets exhibited a 60% reduction in glucose-stimulated insulin release. The presence of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, a metal chelator, during the exposure period protected the islets from the effects of autooxidizing DHF. This suggests that superoxide anions, hydrogen peroxide, and, ultimately, hydroxyl free radicals play a role in the insulin inhibitory effects caused by DHF exposure. Glucose (5 mg/ml), but not galactose, was also capable of protecting islets from DHF. The effects of DHF exposure on isolated islets appear to be similar to those previously reported for alloxan and lend support to the concept that hydroxyl free radicals generated during the oxidation of certain compounds can alter endocrine cell function.

Rapid alteration of pancreatic proinsulin and preproinsulin messenger ribonucleic acid in rats treated with cyproheptadine.

Cyproheptadine (CPH) appears to be unique among islet beta-cell toxicants by virtue of its ability to rapidly and reversibly inhibit insulin biosynthesis in rats. These studies examined the mechanism of CPH-induced insulin depletion by determining the time course for CPH-induced changes in pancreatic preproinsulin mRNA, proinsulin, and insulin levels. A single oral dose of CPH decreased proinsulin levels to 35% of the control value by 3 h. Proinsulin stayed depressed for up to 24 h. Preproinsulin mRNA declined more slowly, reaching 35% of the control value at 6 h, and increased more rapidly, returning to the control value by 24 h. Pancreatic insulin levels did not decrease significantly until 24 h. Exposure of isolated rat islets to CPH for 30 min in vitro selectively inhibited proinsulin synthesis by 62%, without affecting preproinsulin mRNA levels. The dissociation between changes in proinsulin and preproinsulin mRNA levels suggests that the decrease in preproinsulin mRNA in vivo is associated with but does not cause CPH-induced changes in insulin biosynthesis.

Acetaminophen elimination kinetics in neonates, children, and adults.

The elimination of acetaminophen (APAP) following an oral dose of 10 mg/kg in newborn infants, children, and adults was compared. Urinary excretion of unchanged APAP, APAP-sulfate, and APAP-glucuronide was complete within 30 hr at all ages. Higher percentages of the dose were excreted in the urine as APAP-sulfate in neonates (0-2 days old) and children (3-9 yr old) than in 12-yr old children and adults. A pharmacokinetic analysis indicated that the higher rate of APAP-sulfate formation in younger age groups apparently compensated for a deficiency in glucuronide formation. No dramatic age-related differences in the overall elimination rate constant for APAP were observed despite the quantitative changes in the metabolic pathways during early childhood.

Lack of effect of oral activated charcoal on imipramine clearance.

The effect of oral activated charcoal on the pharmacokinetics of intravenous imipramine was studied in a randomized, crossover trial. Four normal men received intravenous imipramine (12.5 mg/70 kg) on two separate occasions, followed by either water or water plus high-surface-area activated charcoal (180 gm) in divided doses over 24 hours. Serum imipramine concentrations were measured from 0 to 24 hours after the imipramine infusion. There was no difference in the mean (+/- SE) t1/2 (9.0 +/- 0.8 vs. 10.9 +/- 1.6 hours), apparent volume of distribution (11.2 +/- 2.1 vs. 12.4 +/- 2.1 L/kg), or systemic clearance (992.2 +/- 138.3 vs. 930.3 +/- 101.9 ml/min/70 kg) of imipramine after dosing without and with oral activated charcoal, respectively (P greater than 0.05; paired t test). These results suggest that multiple oral doses of activated charcoal do not increase the clearance of imipramine in man.

Evidence that central dopamine receptors modulate sympathetic neuronal activity to the adrenal medulla to alter glucoregulatory mechanisms.

Previous reports suggest that analogs of dopamine (DA) can produce hyperglycemia in rats by interacting with DA receptors. Experiments reported here indicate the site of action and describe the metabolic sequalae associated with the hyperglycemic effect of apomorphine (APO), produced in conscious unrestrained rats. Apomorphine was more potent when administered by intracerebroventricular (i.c.v.) injection than when given subcutaneously (s.c.). Very small doses of the DA receptor antagonist pimozide, given intraventricularly, blocked the hyperglycemic effect of apomorphine administered subcutaneously. Sectioning of the spinal cord at thoracic vertebra T1-2 or sectioning the greater splanchnic nerve blocked apomorphine-induced hyperglycemia; whereas section of the superior colliculus or section at T5-6 had no effect. A dose of apomorphine or epinephrine (EPI) producing a similar degree of hyperglycemia elevated the concentration of EPI in serum to a similar degree, and the increase in EPI in serum preceded the increase in glucose in serum. Fasting animals for 2 or 18 hr had no significant effect on EPI- or apomorphine-induced hyperglycemia despite a reduction (91-93%) of the glycogen content of liver and skeletal muscle during the 18 hr fast. 5-Methoxyindole-2-carboxylic acid (MICA), an inhibitor of gluconeogenesis, blocked EPI- and apomorphine-induced hyperglycemia in rats fasted for 18 hr. However, 5-methoxyindole-2-carboxylic acid was ineffective in blocking hyperglycemia in animals fasted for 2 hr. Changes in insulin or glucagon in serum alone cannot account for the hyperglycemic action of apomorphine. These data demonstrate that apomorphine interacts with central DA receptors located in the hindbrain to activate sympathetic neuronal activity to the adrenal gland which subsequently releases epinephrine to alter homeostasis of glucose. Epinephrine may then, depending on the nutritional status, facilitate glycogenolytic or gluconeogenic processes to produce hyperglycemia.

Synthesis of an active hydroxylated glutethimide metabolite and some related analogs with sedative-hypnotic and anticonvulsant properties.

Two synthetic pathways are described for the preparation of 4-hydroxy-2-ethyl-2-phenylglutarimide (2), an active hydroxylated metabolite of glutethimide (1). Fourteen other glutethimide analogs were also synthesized and tested for biological activity. Most of the analogs exhibited sedative-hypnotic properties and compound 2 possessed the greatest activity compared to the parent drug. 4-Amino-2-ethyl-2-phenylglutarimide and 4-hydroxy-2-ethyl-2-phenylglutaconimide (13) exhibited the greatest potential as anticonvulsant agents. The structure-activity relationships of the series are discussed.

Cyproheptadine-induced alterations in clonal insulin-producing cell lines.

Cyproheptadine (CPH) inhibits glucose-stimulated insulin synthesis and secretion, and reversibly depletes pancreatic insulin content in the rat. To examine whether the inhibitory actions of CPH on insulin cell function are linked to the ability of glucose to stimulate insulin synthesis and secretion, studies were performed in two different insulin-producing cell lines. CPH effects were compared in HIT-T15 cells, which respond to glucose with increased insulin synthesis and secretion, and in glucose-unresponsive RINm5F cells. CPH produced similar alterations in both cells lines. After a 48-hr culture period in the presence of 0, 0.1, 1.0 or 10.0 microM CPH, cellular insulin stores and media insulin levels were decreased in a concentration-dependent manner. At 10.0 microM CPH, RIN and HIT cell insulin content declined to 34 and 33% of controls respectively. Cellular insulin returned to control levels 48 hr after removal of CPH. In experiments designed to test a direct inhibitory effect on stimulated insulin secretion, 1 and 10.0 microM concentrations of CPH were found to inhibit glucose-stimulated insulin release from HIT cells, and K+, alanine and glyceraldehyde-stimulated release from RIN cells. CPH was also shown to inhibit insulin biosynthesis in both cell lines at concentrations that did not alter the synthesis of total cellular proteins. All of these alterations in cellular function were shown to occur at CPH concentrations that did not affect cell growth or viability. The results show that the actions of CPH do not appear to be dependent upon the existence of operational glucose signalling mechanisms for insulin synthesis and secretion.

Alloxan toxicity in isolated rat hepatocytes and protection by sugars.

Suspensions of isolated rat hepatocytes incubated in the presence of the diabetogenic agent alloxan exhibit time- and concentration-dependent damage. At concentrations of 3.5 mM and above, alloxan caused an increase in lactate dehydrogenase (LDH), glutamate-pyruvate transaminase (GPT) and intracellular potassium (K+) leakage, all of which are indices of plasma membrane damage, and decreased the intracellular reduced glutathione content (GSH) of the cells. Preincubation (10 min) in D-glucose (50 or 100 mM, but not 10 mM) partially protected the hepatocytes from LDH, GPT and K+ leakage and the decrease in GSH produced by alloxan (7 mM) during a 60-min incubation period. Other sugars (D-galactose, 2-deoxy-D-glucose, D-fructose, D-mannoheptulose and D-mannitol) were also found to protect hepatocytes against damage caused by alloxan. D-Fructose was found to be the most potent protective sugar. These results indicate that alloxan is not selectively toxic to the pancreatic beta-cell and that sugars can protect against alloxan-induced cytotoxicity in hepatocytes.

Workshop on human health impacts of halogenated biphenyls and related compounds.

A workshop on the Human Health Impacts of Halogenated Biphenyls and Related Compounds was held to assess the state of current research on these chemicals and to make recommendations for future studies. Participants discussed results from laboratory animal experiments on PCBs, PBBs, dioxins, and dibenzofurans which demonstrate a common mode of toxicological action while also revealing large variations in toxicological potency both within and between these chemical families. These variations demonstrate the importance of congener-specific analyses in future studies of effects of exposure to these compounds. Results from epidemiological studies of environmentally exposed adult and pediatric populations from the U.S., Japan, and Taiwan and occupationally exposed cohorts from around the world were considered. It was concluded that available evidence did not demonstrate serious adverse effects such as cancer, in exposed adult cohorts but did provide indications of possible neurobehavioral effects in children exposed in utero. In addition, workshop participants described newly developed markers of exposure and techniques for assessing endocrinological, immunological, and neurological effects and suggested these be applied to epidemiological studies of the effects of polyhalogenated compounds. Other recommendations included identification of other cohorts and development of a large registry of exposed individuals; performance of detailed studies of reproductive function and outcomes in exposed populations; and follow up of neurobehavioral effects in offspring of exposed women.

Assessment of human exposure to chemicals from Superfund sites.

Assessing human exposure to chemicals from Superfund sites requires knowledge of basic physical, chemical, and biological processes occurring in the environment and specific information about the local environment and population in the vicinity of sites of interest. Although progress is being made in both areas, there is still a tremendous amount to be done. Participants at this meeting have identified several of the areas in need of greater understanding, and they are listed below. Movement of dissolved and volatile organics, especially NAPLs, in the subsurface environment. This includes study of the partitioning of compounds between NAPLs, air, water, and soil. Partitioning of volatilized chemicals between gaseous and aerosol components of the atmosphere. This includes understanding how these components influence both wet and dry deposition. Long-term movement from sediments into biota and how these affect chronic toxicity to sediment biota. Broad validation of PBPK models describing partitioning of compounds from sediment and water into fish. Reactions of chemicals sorbed to atmospheric particles. This includes application of laboratory models to real and varied atmospheric conditions. Interactions between biotic and abiotic transformations in soil and sediment. Applicability of physiological pharmacokinetic models developed in laboratory studies of experimental animals and clinical investigations of humans to environmental chemicals, concentrations, and routes of exposure in humans. Use of human and wildlife behavioral and biomonitoring information to estimate exposure. This includes better understanding of human variability and the applicability of information gathered from particular wildlife species. To successfully address these gaps in our knowledge, much more analytical data must be collected.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered biologic activities of commercial polychlorinated biphenyl mixtures after microbial reductive dechlorination.

The reductive dechlorination of polychlorinated biphenyls (PCBs) by anaerobic bacteria has recently been established as an important environmental fate of these compounds. This process removes chlorines directly from the biphenyl ring with replacement by hydrogen, resulting in a product mixture in which the average number of chlorines per biphenyl is reduced. In this study, dechlorination of commercial PCB mixtures (Aroclors 1242 and 1254) by microorganisms eluted from PCB-contaminated sediments of the River Raisin (Michigan) and Silver Lake (Massachusetts) caused a depletion in the proportion of highly chlorinated PCB congeners and an accumulation of lesser-chlorinated congeners. Dechlorination occurred primarily at the meta and, to a much lesser extent, para positions of biphenyl. The concentrations of the coplanar congeners including 3,3',4,4',5-pentachlorobiphenyl, the most potent dioxinlike congener, were significantly lowered by reductive dechlorination. Microbial reductive dechlorination of commercial PCB mixtures caused a substantial reduction in biologic activities in several instances. It significantly lowered or eliminated the inhibitory effects of Aroclors on fertilization of mouse gametes in vitro. Similarly, the dechlorinated product mixtures had substantially lower ethoxyresorufin-O-deethylase induction potencies and showed less ability to induce activating protein 1 transcription factor activity as compared to the unaltered Aroclors. In other assays the same dechlorinated product mixtures demonstrated biologic activities similar to the nondechlorinated Aroclors, including the ability of PCB mixtures to stimulate insulin secretion and cause neutrophil activation. The data presented here establish that the biologic activities of commercial PCB mixtures are altered by microbial reductive dechlorination and that an assessment of their toxic potential requires an array of tests that include the different mechanisms associated with PCBs.

Symposium overview: toxicity of non-coplanar PCBs.

Research into the mechanism of toxicity of PCBs has focused on the Ah receptor. However, it is becoming increasingly clear that certain ortho-chlorine-substituted, non-coplanar PCB congeners having low affinity for the Ah receptor exhibit important biological activities. Actions of non-coplanar PCB congeners in a variety of biological systems have been discovered and the mechanisms for these effects are being elucidated. The objectives of this symposium are to examine the state of knowledge concerning the mechanisms of toxic action of non-coplanar PCBs and to identify similarities and differences using a variety of biological systems. Effects to be considered will include: neurotoxicity, estrogenicity, insulin release, neutrophil function, calcium regulation, and relevant signal transduction systems. Finally, the symposium addresses the need to consider non-coplanar congeners within the context of risk assessment. The use of Ah-receptor binding and its associated biological effects to assess the total toxicity of PCBs may no longer be defensible because of the actions produced by non-coplanar congeners. This symposium provides documentation for that conclusion and focuses attention on emerging mechanisms of PCB action that have received relatively little attention to date. The topics presented should be of interest to toxicologists interested in mechanisms of action, in PCB risk assessment, and in regulatory toxicology.

Examination of the immunosuppressive effect of delta9-tetrahydrocannabinol in streptozotocin-induced autoimmune diabetes.

delta9-Tetrahydrocannabinol (delta9-THC) is capable of modulating a variety of immune responses, but has not been evaluated in models of immune-based diabetes. The objectives of the present study were: (a) to investigate the effect of delta9-THC in an established model of multiple low dose streptozotocin (MLDSTZ)-induced autoimmune diabetes; and (b) to determine the contribution of the immune response in the MLDSTZ model. CD-1 mice were treated with 40 mg/kg STZ for 5 days in the presence or absence of delta9-THC treatment. delta9-THC administered orally in corn oil at 150 mg/kg for 11 days attenuated, in a transient manner, the MLDSTZ-induced elevation in serum glucose and loss of pancreatic insulin. MLDSTZ-induced insulitis and increases in IFN-gamma, TNFalpha and IL-12 mRNA expression were all reduced on Day 11 by co-administration of delta9-THC. In separate studies, six doses of delta9-THC, given after completion of STZ treatment, was found equally effective in attenuating mice from MLDSTZ-induced diabetes. Studies performed using B6C3F1 mice showed moderate hyperglycemia and a significant reduction in pancreatic insulin by MLDSTZ in the absence of insulitis. In addition, MLDSTZ produced a less pronounced hyperglycemia compared to CD-1 mice that was not attenuated by delta9-THC. These results suggest that MLDSTZ can initiate direct beta-cell damage, thereby augmenting the destruction of beta-cells by the immune system. Moreover, these results indicate that delta9-THC is capable of attenuating the severity of the autoimmune response in this experimental model of autoimmune diabetes.

Isomeric specificity of diphenylmethylpiperidine in the production of rat pancreatic islet cell toxicity.

The relationship between chemical structure and the pancreotoxic potential of positional isomers of diphenylmethylpiperidine was investigated in rats. The chemical synthesis of these compounds and their N-methylated analogs is reported. Oral administration of 4-diphenylmethylpiperidine and its N-methylated derivative (130 and 260 micronmoles/kg) to rats for 14 days resulted in hyperglycemia, reduced pancreatic insulin content and the formation of large vacuoles in the cytoplasm of pancreatic islet cells. No effect on beta cell morphology or insulin content was observed after administration of 2- and 3-diphenylmethylpiperidine and their N-methylated analogs.

Age-related susceptibility to the insulin-depleting action of 4-diphenylmethylpiperidine in young rats.

Studies were undertaken to investigate age-related changes in the ability of 4-diphenylmethylpiperidine (4-DPMP) to reduce levels of pancreatic insulin in young rats. Oral doses of 4-DPMP (5.0 or 10.0 mg/kg) were given once daily for two days to 9-, 15- and 21-day-old rats. Twenty-four hours after the last dose, pancreatic insulin content, non-fasting serum glucose, and the amount of unchanged 4-DPMP present in the whole body were estimated. 4-DPMP treatment produced a decline in pancreatic insulin and the extent of this action was greater in younger animals. The observed changes in pancreatic insulin were not reflected in altered serum glucose levels, showing this parameter is a relatively poor indicator of pancreatic insulin loss. Younger animals had a larger fraction of the total dose of 4-DPMP in the body at the end of the experimental period when compared to the fraction retained by older rats. The age-related susceptibility of young rats to the diabetogenic action of 4-DPMP may be related to the differences in the rate of elimination of the chemical at different ages.

Polychlorinated biphenyls release insulin from RINm5F cells.

Polychlorinated biphenyls (PCBs) possess a variety of biological effects, including alterations in growth, development and metabolism, that may be dependent on insulin. However, no reports on the action of PCBs on cells which produce and secrete insulin are available. The current study examined the ability of a commercial mixture of PCBs (Aroclor 1254) and three specific PCB congeners, to alter the release of insulin using the hormone producing cell line RINm5F. Exposure of cells to Aroclor 1254 (A-1254) produced a concentration-dependent increase in media insulin reaching a peak, when expressed as percent of control, at 30 min. In spite of continued exposure, media insulin relative to control declined and no treatment-related difference was observed at 48 hrs. Cellular levels of the hormone declined as much as 50% by that time. The insulin releasing action of A-1254 was mimicked by each of the non-coplanar congeners 2,2',4,4'-tetrachlorobiphenyl (TCB) and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) but the coplanar congener 3,3',4,4'-TCB showed no significant activity. These results indicate that PCBs are capable of producing a release of insulin from RINm5F cells, an effect that is unlikely to be associated with coplanar congeners that initiate their action by binding to the Ah-receptor.