Recent attempted and actual weight change in relation to pregnancy loss: a prospective cohort study.

OBJECTIVE: To assess weight change and attempted weight loss during the 12-18 months before spontaneous conception in relation to the risk of pregnancy loss. DESIGN: Prospective cohort study. SETTING: United States, 2007-2011. METHODS: Women (n = 629) who were attempting pregnancy reported at baseline any weight loss attempts over the past 12 months, and their minimum and maximum weights during that time. Follow up lasted one to six menstrual cycles and throughout pregnancy. Using bodyweight measured at 4 weeks' gestation, participants were categorised as having weight loss ≥5%, weight gain ≥5%, both, or neither, over the previous 12-18 months. Log-binomial models adjusted for potential confounders. MAIN OUTCOME MEASURES: Risk ratio (RR) and 95% confidence interval (CI) of pregnancy loss. RESULTS: Attempted weight loss was reported by 44% of women and actual weight loss by 11%, but neither was consistently associated with pregnancy loss. The RR for recent weight gain ≥5% was 1.65 (CI 1.09, 2.49). CONCLUSIONS: Weight gain over the period spanning 12-18 months pre-conception to 4 weeks' gestation may increase the risk of pregnancy loss among fertile women with prior pregnancy losses. Attempted and actual weight loss were not associated with pregnancy loss; however, replication is needed from larger studies with data on particular weight-loss methods. TWEETABLE ABSTRACT: Recent weight gain before and around the time of conception may increase the risk of pregnancy loss.

Dogfish glucagon analogues counter hyperglycaemia and enhance both insulin secretion and action in diet-induced obese diabetic mice.

AIMS: To investigate the antidiabetic actions of three dogfish glucagon peptide analogues [known glucagon-like peptide-1 and glucagon receptor co-agonists] after chronic administration in diet-induced high-fat-diet-fed diabetic mice. MATERIALS AND METHODS: National Institutes of Health Swiss mice were pre-conditioned to a high-fat diet (45% fat) for 100 days, and control mice were fed a normal diet (10% fat). Normal diet control and high-fat-fed control mice received twice-daily intraperitoneal (i.p.) saline injections, while the high-fat-fed treatment groups (n = 8) received twice-daily injections of exendin-4(1-39), [S2a]dogfish glucagon, [S2a]dogfish glucagon exendin-4(31-39) or [S2a]dogfish glucagon-Lys(30) -γ-glutamyl-PAL (25 nmol/kg body weight) for 51 days. RESULTS: After dogfish glucagon analogue treatment, there was a rapid and sustained decrease in non-fasting blood glucose and an associated insulinotropic effect (analysis of variance, p < .05 to <.001) compared with saline-treated high-fat-fed controls. All peptide treatments significantly improved i.p. and oral glucose tolerance with concomitant increased insulin secretion compared with saline-treated high-fat-fed controls (p <.05 to <.001). After chronic treatment, no receptor desensitization was observed but insulin sensitivity was enhanced for all peptide-treated groups (p < .01 to <.001) except [S2a]dogfish glucagon. Both exendin-4 and [S2a]dogfish glucagon exendin-4(31-39) significantly reduced plasma triglyceride concentrations compared with those found in lean controls (p = .0105 and p = .0048, respectively). Pancreatic insulin content was not affected by peptide treatments but [S2a]dogfish glucagon and [S2a]dogfish glucagon exendin-4(31-39) decreased pancreatic glucagon by 28%-34% (p = .0221 and p = .0075, respectively). The percentage of ß-cell area within islets was increased by exendin-4 and peptide analogue treatment groups compared with high-fat-fed controls and the ß-cell area decreased (p < .05 to <.01). CONCLUSIONS: Overall, dogfish glucagon co-agonist analogues had several beneficial metabolic effects, showing therapeutic potential for type 2 diabetes.

Methyl isobutyl ketone-induced hepatocellular carcinogenesis in B6C3F1 mice: A constitutive androstane receptor (CAR)-mediated mode of action.

In a National Toxicology Program (NTP) chronic inhalation study with methyl isobutyl ketone (MIBK), increases in hepatocellular adenomas and hepatocellular adenomas and carcinomas (combined) were observed in male and female B6C3F1 mice at 1800 ppm. A DNA reactive Mode-of-Action (MOA) for this liver tumor response is not supported by the evidence as MIBK and its major metabolites lack genotoxicity in both in vitro and in vivo studies. Constitutive androstane receptor (CAR) nuclear receptor-mediated activation has been hypothesized as the MOA for MIBK-induced mouse liver tumorigenesis. To further investigate the MOA for MIBK-induced murine liver tumors, male and female B6C3F1, C57BL/6, and CAR/PXR Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/day, 5 days/week for a total of 10 days. On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1-3 h following the final exposure. B6C3F1 and C57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, and no increase was observed in MIBK exposed CAR/PXR KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (CAR-associated) transcript and a slight increase in Cyp3a11(PXR-associated) transcript in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR-associated) or Cyp4a10 (PPAR-α-associated) transcripts. CAR/PXR KO mice exposed to 1800 ppm MIBK showed no evidence of activation of AhR, CAR, PXR or PPAR-α nuclear receptors via their associated transcripts. MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the CAR and PXR nuclear receptors and resultant hepatocellular proliferation leading to rodent liver tumors.

Genotoxicity screening via the γH2AX by flow assay.

The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Intra- and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef.

During the cooking of beef, the genotoxic heterocyclic aromatic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed. Little is known about the fate of these compounds in humans or the factors affecting it. We have developed assays based on capillary column gas chromatography-negative ion mass spectrometry capable of the simultaneous measurement of MeIQx, DiMeIQx, and PhIP in cooked meat and in human urine using stable isotope labeled analogues. Ten normal, healthy male volunteers were invited to consume a standard cooked meat meal (400-450 g lean beef, cooked as patties on a griddle hotplate) on four separate occasions over a period of 14 months. Following consumption of the test meals, urine was collected from 0 to 8 h, during which time all free amines were excreted and analyzed for MeIQx, DiMeIQx, and PhIP. Subjects ingested 240 +/- 9 (SEM) g cooked meat, which contained 2.2 +/- 0.2 ng MeIQx/g meat, 0.7 +/- 0.1 ng DiMeIQx/g meat, and 16.4 +/- 2.1 ng PhIP/g meat. The variability in relative systemic bioavailability was assessed from the percentage of ingested amine excreted unchanged in the urine. Subjects excreted 2.1 +/- 1.1% of MeIQx and 1.1 +/- 0.5% of PhIP ingested as unchanged amine in the urine. Levels of DiMeIQx in urine, if present, were below the sensitivity of our assay (20 pg/ml) and could not be detected in any of the samples analyzed. Irrespective of dose, urinary excretion of unchanged MeIQx or PhIP (expressed as a percentage of the ingested dose) remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low and the pooled interday (intrasubject) coefficients of variation for both compounds were only 19 and 3.4%, respectively. In contrast, inter-subject (intraday) variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Based on these studies, it should be possible to use the percentage excretion of MeIQx and PhIP to assess the relative bioavailability of these compounds in humans.

CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans.

The contribution of CYP1A2 to the metabolism of the dietary heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in vivo in humans, has been determined with furafylline, a highly selective inhibitor of this enzyme. The inhibitory potential of furafylline in vivo was first assessed by determining its effect on clearance of phenacetin to paracetamol by the model CYP1A2-dependent O-deethylation pathway. Furafylline inhibited this reaction by > 99% in all subjects, thus demonstrating its applicability to determining the contribution of CYP1A2 to a given reaction in vivo. A group of 6 healthy male volunteers received either placebo or 125 mg furafylline, in a double-blind balanced crossover design, 2 h prior to consuming a test meal of fried beef containing a known amount of amines. The excretion of PhIP and MeIQx in urine was determined during the subsequent 28 h, using gas chromatography-mass spectrometry. Following furafylline, the excretion of unchanged MeIQx increased 14.3-fold, while that of PhIP increased 4.1-fold (P < 0.01, paired t test). Elimination of both amines was first order and very rapid, with half-lives of < 5 h. The elimination rate constants did not change following furafylline, suggesting that total clearance is limited by hepatic blood flow. Because the elimination of the amines was first order, it was possible to calculate the contribution of CYP1A2 to the clearance of the amines. CYP1A2-catalyzed metabolism accounts for 91% of the elimination of ingested MeIQx and 70% of ingested PhIP, most likely via N-hydroxylation.

Novel dual agonist peptide analogues derived from dogfish glucagon show promising in vitro insulin releasing actions and antihyperglycaemic activity in mice.

The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 ß-cells (p < 0.001) and reduced acute glycaemic responses following intraperitoneal glucose (25 nmol/kg) in healthy NIH Swiss mice (p < 0.05-p<0.001). The in vitro insulinotropic actions of [S2a]dogfish glucagon, [S2a]dogfish glucagon-exendin-4(31-39) and [S2a]dogfish glucagon-Lys(30)-γ-glutamyl-PAL, were blocked (p < 0.05-p<0.001) by the specific GLP-1 and glucagon receptor antagonists, exendin-4(9-39) and (desHis(1)Pro(4)Glu(9))glucagon amide but not by (Pro(3))GIP, indicating lack of GIP receptor involvement. These analogues dose-dependently stimulated cAMP production in GLP-1 and glucagon (p < 0.05-p<0.001) but not GIP-receptor transfected cells. They improved acute glycaemic and insulinotropic responses in high-fat fed diabetic mice and in wild-type C57BL/6J and GIPR-KO mice (p < 0.05-p<0.001), but not GLP-1R-KO mice, confirming action on GLP-1 but not GIP receptors. Overall, dogfish glucagon analogues have potential for diabetes therapy, exerting beneficial metabolic effects via GLP-1 and glucagon receptors.

Hospital-acquired Anemia in Critically Ill Dogs and Cats: A Multi-Institutional Study.

BACKGROUND: Hospital-acquired anemia is commonly described in people but limited information currently is available regarding its prevalence in animals. HYPOTHESIS/OBJECTIVES: Assess the prevalence of hospital-acquired anemia in hospitalized critically ill dogs and cats, and examine its relationship with phlebotomy practices, transfusion administration, and survival to discharge. ANIMALS: Eight hundred and fifty-one client-owned animals (688 dogs and 163 cats). METHODS: A multicenter, observational study was conducted in which packed cell volume (PCV) was recorded at the time of admission and on subsequent hospitalization days. Signalment, number of blood samples obtained, underlying disease, whether or not blood products were administered, duration of hospitalization, and survival to discharge were recorded. RESULTS: Admission anemia prevalence was 32%, with overall prevalence during the hospitalization period of 56%. The last recorded PCV was significantly lower than the admission PCV for both dogs (admission PCV, 42% [range, 6-67%]; last recorded PCV, 34% [range, 4-64%], P < .0001) and cats (admission PCV, 31% [range, 6-55%]; last recorded PCV, 26% [range, 10-46%], P < .0001). Patients that developed anemia had significantly more blood samples obtained (nonanemic, 5 blood samples [range, 2-54]; anemic, 7 blood samples [range, 2-49], P < .0001). Hospitalized cats were significantly more likely to develop anemia compared to dogs (P < .0001), but anemic dogs were significantly less likely to survive to discharge (P = .0001). Surgical patients were at higher risk of developing hospital-acquired anemia compared to medical patients (OR, 0.63; 95% CI, 0.4-0.9; P = .01). CONCLUSIONS AND CLINICAL RELEVANCE: Hospital-acquired anemia occurred frequently, especially in surgical patients. Additional studies focused on the direct effect of phlebotomy practices on the likelihood of anemia development in hospitalized animals are warranted.

Evidence for a single common Na+-dependent transport system for alanine, glutamine, leucine and phenylalanine in brush-border membrane vesicles from bovine kidney.

The characteristics of the Na+-dependent transport of alanine, glutamine, leucine and phenylalanine were studied in bovine renal brush-border membrane vesicles. Inhibition of the transport of any one of these amino acids by any other was mutually competitive. The Ki value for the inhibition of alanine transport by leucine was similar to the Km for leucine transport; similar interrelationships existed for the other amino acids. Each amino acid was shown to exchange with each of the other amino acids across the membrane. From these and other results it is concluded that the Na+-dependent transport of these four amino acids is catalysed by a single common transport system.

Studies of the mechanism of activation of HIT-T15 cells by lactate.

L-Lactate, D-lactate, propionate and acetate (all 20 mM) caused a marked intracellular acidification in HIT-T15 cells loaded with 2'7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF), followed by recovery to more alkaline values. The effects of L- and D-lactate, but not propionate or acetate, were inhibited by 5 mM alpha-fluorocinnamate. Both L- and D-lactate caused a marked depolarisation and rise in cytosolic [Ca2+] in HIT cells as assessed by oxonol-V and quin2 fluorescence, respectively. Propionate had similar, though less marked, effects, whereas acetate exerted only a modest influence on membrane potential and cytosolic [Ca2+]. The rate of oxidation of L-lactate by HIT cells greatly exceeded that of D-lactate. alpha-Fluorocinnamate delayed, but did not prevent, the effects of L-lactate on HIT cell membrane potential or cytosolic [Ca2+]. L-lactate diminished the rate of efflux of 86Rb+ from preloaded HIT cells. Inhibition of calcium- and nucleotide-sensitive K+ channels with tetraethylammonium and tolbutamide also reduced the 86Rb+ efflux rate, and prevented any further reduction in response to L-lactate. However, such inhibition of K+ channels did not prevent a further depolarisation and rise in cytosolic [Ca2+] upon the subsequent addition of lactate. It is suggested that the activation of HIT-T15 cells by lactate is not the result of intracellular acidification or increased metabolic flux, and does not require diminished K+ permeability. An alternative mechanism is based upon the possible electrogenic flux of lactate across the plasma membrane.

Effects of nutrient and non-nutrient stimuli on cytosolic pH in cultured insulinoma (HIT-T15) cells.

Intracellular pH (pHi) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pHi, followed by a slower recovery to the resting pH value. Conversely the addition of N4Cl caused an increase in pHi followed by recovery. The addition of amiloride caused a fall in pHi; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pHi with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, alpha-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K+ or with Ba2+ was associated with a pronounced fall in pHi, followed by a gradual recovery. Insulin secretion from HIT cells was stimulated by glucose, glyceraldehyde, alpha-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na+-H+ exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pHi are discussed.

Massive haemorrhage associated with inadvertent incision of a suspected carotid artery pseudoaneurysm in a cat.

A 12-year-old, castrated male, domestic long-haired cat experienced massive haemorrhage associated with an incision of a swelling on the neck 2 weeks after right-sided ventral bulla osteotomy. Emergent control of haemorrhage was gained through unilateral carotid artery ligation. Cardiopulmonary resuscitation was provided in conjunction with massive blood transfusion. The cat made an unremarkable recovery. Carotid artery pseudoaneurysm due to surgical disruption of the carotid artery during ventral bulla osteotomy, specifically through the use of self-retaining retractors, was suspected. This case highlights the development of pseudoaneurysm as a potential complication of head and neck surgery, and additionally describes a case of massive transfusion in a cat.

Stable oxyntomodulin analogues exert positive effects on hippocampal neurogenesis and gene expression as well as improving glucose homeostasis in high fat fed mice.

The weight-lowering and gluco-regulatory actions of oxyntomodulin (Oxm) have been well-documented however potential actions of this peptide in brain regions associated with learning and memory have not yet been evaluated. The present study examined the long-term actions of a stable acylated analogue of Oxm, (dS(2))Oxm(K-γ-glu-Pal), together with parent (dS(2))Oxm peptide, on hippocampal neurogenesis, gene expression and metabolic control in high fat (HF) mice. Groups of HF mice (n = 12) received twice-daily injections of Oxm analogues (both at 25 nmol/kg body weight) or saline vehicle (0.9% wt/vol) over 28 days. Hippocampal gene expression and histology were assessed together with evaluation of energy intake, body weight, non-fasting glucose and insulin, glucose tolerance, insulin sensitivity and lipids. Oxm analogues significantly reduced body weight, improved glucose tolerance, glucose-mediated insulin secretion, insulin sensitivity, islet architecture and lipid profile. Analysis of brain histology revealed significant reduction in hippocampal oxidative damage (8-oxoguanine), enhanced hippocampal neurogenesis (doublecortin) and improved hippocampal and cortical synaptogenesis (synaptophysin) following treatment. Furthermore, Oxm analogues up-regulated hippocampal mRNA expression of MASH1, Synaptophysin, SIRT1, GLUT4 and IRS1, and down-regulated expression of LDL-R and GSK3ß. These data demonstrate potential of stable Oxm analogues, and particularly (dS(2))Oxm(K-γ-glu-Pal) to improve metabolic function and enhance neurogenesis, synaptic plasticity, insulin signalling and exert protective effects against oxidative damage in hippocampus and cortex brain regions in HF mice.

Methyl isobutyl ketone exposure-related increases in specific measures of α2u-globulin (α2u) nephropathy in male rats along with in vitro evidence of reversible protein binding.

Chronic exposure to methyl isobutyl ketone (MIBK) resulted in an increase in the incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female Fischer 344 rats. Since a number of chemicals have been shown to cause male rat renal tumors through the α2u nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of α2u nephropathy including renal cell proliferation in male and female F344 rats following exposure to the same inhalation concentrations used in the National Toxicology Program (NTP) cancer bioassay (0, 450, 900, or 1800ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2u staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2u concentration. There was an exposure-related increase in all measures of α2u nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2u. The changes in HDA and α2u concentration were comparable to d-limonene, an acknowledged inducer of α2u nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2u, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to α2u and cause nephropathy. Together, the exposure-related increase in measures of α2u nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2u, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2u nephropathy-mediated mode of action (MoA).

Glucose increases cytosolic calcium concentration and inositol lipid metabolism in HIT-T15 cells.

We have investigated the effects of glucose on cytosolic free calcium concentration in the insulin-secreting cell line HIT-T15. Addition of glucose (10 mM) caused a 20-75% increase in cytosolic [Ca2+] within 5 minutes compared to controls in the absence of glucose. A maximal increase in cytosolic [Ca2+] was obtained with 5 mM glucose. The magnitude of the response was markedly dependent upon the concentration of extracellular Ca2+, and the rise in cytosolic [Ca2+] was inhibited by verapamil. Cytosolic [Ca2+] was greatly increased by depolarization of the cells with KCl (50 mM), whereas carbamylcholine had no apparent effect. Glucose and KCl were also effective in stimulating insulin release from HIT cells, although carbamylcholine was again ineffective. The secretory response to glucose was also found to be directly related to the concentration of extracellular [Ca2+]. Glucose and KCl, but not carbamylcholine, were found to slightly enhance the production of [3H]-inositol trisphosphate in HIT cells pre-labelled with myo-[3H]-inositol, indicating a modest stimulation of inositol lipid hydrolysis.

Lactate alters plasma membrane potential, increases the concentration of cytosolic Ca2+ and stimulates the secretion of insulin by the hamster beta-cell line HIT-T15.

We have studied the effect of lactate on a number of intracellular events which may be important in controlling the secretion of insulin by the hamster beta-cell line HIT-T15. Using the fluorescent dye Oxonol V, as well as intracellular recording techniques to measure changes in membrane potential, we found that lactate, glucose, K+ and tolbutamide caused depolarization of HIT cells, while valinomycin resulted in hyperpolarization. Consistent with these findings was the observation that 10 mM lactate caused an increase of 69.0 +/- 18.4% (S.E.M., n = 6) in the level of free cytosolic Ca2+ within HIT cells (assessed by fluorescence of quin 2). This was probably due to influx of Ca2+ through voltage-sensitive calcium channels, since it was dependent upon the concentration of extracellular Ca2+ and inhibited by verapamil. Lactate also caused cytosolic acidification in HIT cells and increased the secretion of insulin. These findings are consistent with the view that the electrogenic efflux of lactate could be a determinant in the activation of HIT cells by lactate and possibly by glucose.

Subchronic administration of clozapine, but not haloperidol or metoclopramide, decreases dopamine D2 receptor messenger RNA levels in the nucleus accumbens and caudate-putamen in rats.

The effects of unique profile antipsychotic drugs on dopamine D2 receptors and D2 receptor messenger RNA were assessed following subchronic administration in rats. Male, Sprague-Dawley rats were administered oral haloperidol, clozapine, metoclopramide or no drug for three weeks via their drinking water. Tissue from the medial nucleus accumbens and dorsolateral caudate-putamen was dissected and analyzed by Northern blot analysis for levels of dopamine D2 receptor messenger RNA and binding assays conducted with [3H]spiperone for dopamine D2 receptors. Haloperidol and metoclopramide, but not clozapine, significantly increased [3H]spiperone in the caudate-putamen, but not the nucleus accumbens. Clozapine significantly decreased D2 messenger RNA levels in the caudate-putamen and the nucleus accumbens, while metoclopramide and haloperidol had no significant effect in either brain region. The finding of decreased D2 receptor messenger RNA levels produced by subchronic clozapine may account for the lack of striatal D2 receptor up-regulation, which was robustly observed after subchronic haloperidol and metoclopramide. Furthermore, since haloperidol and metoclopramide have a high liability for motor side effects, the current results relate favorably to the low motor side effect profile of clozapine.

Tolerance-like attenuation to contingent and noncontingent cocaine-induced elevation of extracellular dopamine in the ventral striatum following 7 days of withdrawal from chronic treatment.

Time-dependent changes in mesolimbic dopamine (DA) function are believed to play a role in behavioral sensitization and drug craving experienced during withdrawal from chronic cocaine administration. The present study utilized intravenous (IV) cocaine self-administration coupled with intracranial microdialysis in rats to investigate time dependent changes during withdrawal from chronic cocaine exposure. Following 2 weeks of IV cocaine self-administration, rats were allowed contingent access to cocaine at 1 and 7 days of withdrawal while extracellular levels of DA were measured from the ventral striatum. A second group of animals received yoked, noncontingent cocaine for 2 weeks and were then administered noncontingent cocaine on days 1 and 7 of withdrawal. In addition, a third group of animals received 2 weeks of yoked saline followed by noncontingent cocaine 1 day after withdrawal. There were no significant differences between groups for the overall cocaine dosage or temporal pattern of infusions on days 1 and 7 of withdrawal. Basal extracellular DA concentrations did not differ between any treatment groups at either withdrawal time. Extracellular DA levels were increased throughout the session on both days; however, the increases at day 7 were significantly less than day 1 for both contingent and noncontingent conditions. DA overflow on day 1 did not differ between animals receiving chronic yoked cocaine or saline. These results suggest that tolerance-like attenuation to the DA-elevating effects of cocaine is not apparent early in withdrawal, but does develop by later time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic haloperidol potentiates stimulated glutamate release in caudate putamen, but not prefrontal cortex.

Rats were given haloperidol continuously for 6 months via subcutaneous implants. Extracellular concentrations of basal and high K(+)-stimulated GABA and glutamate in the lateral caudate putamen and the medial prefrontal cortex were then assessed using microdialysis. While there were no significant differences in basal extracellular concentrations in either brain region, chronic haloperidol-treated rats showed significantly greater increases in glutamate following stimulation with high K+ in the caudate putamen, but not the prefrontal cortex. This effect was accompanied by an attenuation of K(+)-stimulated GABA overflow in the caudate putamen. These results suggest regionally selective alterations in amino acid transmitter function which may be related to chronic neuroleptic-induced motor side effects.