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Biomedical subjects

A M Lynch

Publications and source records attributed to A M Lynch.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chronic haloperidol-induced changes in regional dopamine release and metabolism and neurotensin content in rats.

Chronic neuroleptic administration has previously been shown to alter in vivo measures of dopaminergic function and lead to regionally selective increases in neurotensin levels. In the current study, female rats were administered chronic haloperidol for 6 months via subcutaneous silastic implants. After 24 weeks of administration, microdialysis probes were inserted into the lateral caudate putamen and the medial prefrontal cortex. Basal samples were collected prior to infusion of a high K+ concentration (100 mM KCl). Extracellular concentrations of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were assessed using HPLC. Chronic haloperidol-treated rats showed increased basal dopamine metabolite levels in the caudate putamen and an altered response to the effects of high K+ on 3,4-dihydroxyphenylacetic acid; no significant differences were seen with other analytes in the caudate putamen. Although basal concentrations were not different between groups in the prefrontal cortex, haloperidol-treated rats showed a significant attenuation of response to the effects of high K+ infusion on dopamine metabolite concentrations. Radioimmunoassay measurement of tissue neurotensin content showed highly significant elevations of neurotensin concentrations in the caudate putamen and nucleus accumbens, but not in other brain regions analyzed. These results suggest a confluence of altered dopamine and neurotensin function in the caudate putamen which may be related to motor side effects of haloperidol, whereas changes in prefrontal dopamine function are not associated with altered neurotensin levels.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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)

Animals↗
PubMed1995

Longitudinal measurement of anticardiolipin antibodies during normal pregnancy: a prospective study.

We have previously shown that elevation of anticardiolipin antibodies (aCL) at the first prenatal visit is associated with increased fetal loss in normal pregnancy. The variation in aCL levels during normal pregnancy has not been established. To examine this question we measured IgG, IgM and IgA aCL levels five times during pregnancy at weeks 5-15, 16-25, 26-35, 36-37 and at delivery. Data were analyzed to determine: (a) the within and between subject variability of aCL during pregnancy; (2) the temporal trend of aCL; and (3) the relation of serial measures of aCL with maternal complications of pregnancy. We divided our cohort of 354 subjects into two groups. Group A included those subjects with consistently normal levels of aCL and group B those subjects with at least one elevated level of aCL. In group A the within subject variability was relatively low (28-34%). In group B we found wide fluctuations in aCL levels and a within subject variability of 88-91%. Subjects in group B had no increase in maternal complications of pregnancy. The present data suggest that aCL may fluctuate significantly during normal pregnancy and there is little clinical value in measuring aCL on a serial basis during pregnancy.

Adolescent↗
PubMed1995

Enzymatic studies of the activation of heterocyclic food mutagens in man.

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are amongst the most abundant of the heterocyclic aromatic amines formed during the cooking of beef. Both compounds are genotoxic and carcinogenic in rodents. These effects are manifested only after activation of the amines by P450. Human liver is very active at converting these amines to mutagenic products. Studies in vitro have established that, for both amines, mutagenicity with human liver microsomes is entirely via the N-hydroxylamine, essentially the only oxidation product of either amine. Both N-hydroxylation and mutagenicity of the amines can be almost completely inhibited by furafylline, a potent and highly selective inhibitor of CYP1A2 in man. These data, together with the work of others, show that the N-hydroxylation and hence the mutagenicity of both MeIQx and PhIP in man is catalyzed almost exclusively by CYP1A2. Liver from cynomolgus monkeys, unlike that from humans and marmosets, is very poor at activating MeIQx or PhIP to mutagenic products. Studies with anti-peptide antibodies of defined specificity revealed that this is due to the absence of CYP1A2, suggesting that this species should not be used to assess the possible risk posed to man by these amines. The marmoset would be a better species for this purpose.

Animals↗
PubMed1994

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.

Adult↗
PubMed1993

Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in food using a combined assay based on gas chromatography-negative ion mass spectrometry.

A gas chromatographic-mass spectrometric assay has been developed for the measurement of 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) in food. Stable isotope-labelled analogues of MeIQx and PhIP are used as internal standards and the synthesis of deuterated PhIP is described. The mass spectrometer is operated in the electron-capture negative ion chemical ionisation mode and the amines are chromatographed as their di-3,5-bistrifluoromethylbenzyl derivatives. All three compounds can be measured in a single chromatographic run and detection limits of 0.05, 0.1 and 0.2 ng/g for MeIQx, DiMeIQx and PhIP, respectively, in food are obtained. Various home-cooked and commercially prepared foodstuffs were analysed with this assay and several were found to contain measurable amounts of one or more of the three amines. These results are presented and discussed.

Animals↗
PubMed1993

The cytochalasin-B micronucleus/kinetochore assay in vitro: studies with 10 suspected aneugens.

An in vitro micronucleus assay in low passage Chinese hamster Luc2 cells capable of detecting numerical and structural chromosome changes was developed. Chromosome loss was inferred by indirect visualisation of human CREST antikinetochore antibodies bound to centromeres in chemically-induced micronuclei of cytochalasin-B arrested binucleated cells. The assay was used to evaluate 10 chemicals which had been selected for their known or suspected effects upon various components of the cell-division apparatus. These chemicals were colchicine (COL), vinblastine (VBL), thiabendazole (TBZ), chloral hydrate (CH), thimerosal (TM), diazepam (DZ), pyrimethamine (PYR), hydroquinone (HQ), cadmium chloride (CdCl2) and econazole nitrate (EZ). Mitomycin-C (MMC) was used as a positive control for the induction of micronuclei. 8 of the core chemicals induced micronuclei in Chinese hamster Luc2 cells. 4 of the chemicals (COL, VBL, TBZ, CH) increased levels of micronuclei which were positive for kinetochore antibody labelling and hence chromosome loss. 3 of the chemicals (DZ, PYR, HQ) and the positive control (MMC) increased the levels of Mn which were negative for kinetochore antibody labelling. The results with TM were equivocal and EN was negative. The results of these studies suggest that the cytochalasin-B Mn/k assay is a cost-effective, simple and rapid alternative to classical cytogenetic assays for the detection of chemically induced aneuploidy.

Aneuploidy↗
PubMed1993

Molecular dosimetry of the food-borne carcinogen MeIQx using adducts of serum albumin.

Incubation of mouse serum albumin with the food borne carcinogen [2-14C]-Amino-3,8,-dimethylimidazo[4,5-f]quinoxaline (14C-MeIQx) in the presence of mouse hepatic microsomes and an NADPH-regenerating system in vitro resulted in the formation of adducts of MeIQx with albumin, which increased proportionately with time for at least 120 min (approximately 1 pmol equivalents/mg of protein/min). We have previously shown in male Swiss Webster mice in vivo that 14C-MeIQx bound covalently to serum proteins and that the formation of adducts was dose dependent. 14C-MeIQx (100 mg/kg, i.p.) was administered to male (MF1) mice which were killed 24 h later. Serum albumin was purified by affinity chromatography and covalent binding of 14C-MeIQx was assessed. Total covalent binding of MeIQx to albumin was 14.0 +/- 5.2 pmol per mg albumin, which was 5-fold greater than to haemoglobin. Following mild acid hydrolysis, 1.25 pmol MeIQx per mg albumin was liberated as free amine, as determined by gas chromatography negative ion mass spectrometry (GC-MS). This represents 9% of total MeIQx adducted to albumin in vivo (cf 1.3% adducted to haemoglobin). These results suggested that adducts of MeIQx with serum albumin should provide a significantly more sensitive dosimeter than those with haemoglobin. We therefore investigated this approach with serum protein samples from three volunteers. Human serum albumin and non-serum albumin protein fractions were separated by affinity chromatography, before being subjected to GC-MS analysis for hydrolysable adducts of MeIQx. The levels of MeIQx in control samples, and from the release of the putative sulphinamide adducts in hydrolysed samples were below the limits of detection of the GC-MS assay (29 +/- 2.6 amol MeIQx/mg albumin). Despite an increase of 2 orders of magnitude in sensitivity, compared with haemoglobin, it is unlikely that the sulphinamide adduct of MeIQx with human serum albumin can be used as a dosimeter for human aminoimidazoazaarene exposure.

Adult↗
PubMed1992

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.

Adult↗
PubMed1992

Distribution of substance P-like immunoreactivity in guinea pig central nervous system.

The distribution of substance P-like immunoreactivity (SP-LI) in the guinea pig brain has been studied by immunohistochemistry and the results compared with the distribution in similar regions in the rat brain. In both species, dense SP-LI staining was found in the median eminence, arcuate hypothalamic nucleus, substantia nigra, dorsal raphe and dorsal tegmental nuclei, nucleus of the solitary tract, substantia gelatinosa of the spinal trigeminal nucleus, and spinal cord. Less dense staining was found in the caudate putamen, globus pallidus, nucleus accumbens, habenula, hypothalamic areas, and central grey. SP-LI cell bodies were found in areas previously described for the rat brain including several hypothalamic areas, limbic areas, central grey, and dorsal raphe and solitary tract nuclei. The major difference between the two species was found in the cortex and hippocampus. The guinea pig cortex contained many more SP-LI cells and fibres, distributed in layers II-VI, than the rat cortex. The guinea pig hippocampus contained marked staining, particularly in the pyramidal cell layer of CA1-3 fields of Ammon's horn and in the granular layer of the dentate gyrus, and SP-LI cells in the hilus of the dentate gyrus, whereas rat hippocampus contained few cells and no regions of dense staining. It is concluded that because the guinea pig brain has an extensive distribution of SP-LI in the cortex and hippocampus it resembles the primate brain more closely than does the rat brain.

Animals↗
PubMed1991

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.

Acetates↗
PubMed1991

The measurement of MeIQx adducts with mouse haemoglobin in vitro and in vivo: implications for human dosimetry.

We have investigated covalent binding of radiolabelled [14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to mouse haemoglobin in vitro and in vivo. Furthermore, we report the development of a capillary column gas chromatography negative ion mass spectrometry (GC-MS) assay capable of detecting MeIQx liberated from haemoglobin after acid or base hydrolysis. Following microsomal activation, the amount of radiolabelled material associated with haemoglobin in vitro increased with incubation time to 0.67 +/- 0.15 nmol/mg haemoglobin at 2 h (initial concentration 0.47 mM [14C]MeIQx, mean +/- SD, n = 6). Hydrolysis of these samples with acid revealed that 47-60% of the radiolabelled material covalently bound to haemoglobin was acid labile. Of this, 7.2-9.8% was recovered as MeIQx as determined by GC-MS. This liberated fraction should reflect the amount of sulphinic acid amide present which is formed when N-hydroxy-MeIQx reacts with sulphydryl-containing amino acids present in haemoglobin. In vivo, no radiolabelled material bound to haemoglobin could be detected in animals treated with the lowest dose of MeIQx (0.2 mg/kg). At higher doses, there was a dose-dependent increase in the covalent binding of radiolabel to haemoglobin (2.0-200 mg/kg). However, the GC-MS assay for hydrolysable adducts of MeIQx yielded detectable quantities of MeIQx (32.2 +/- 17.5 fmol MeIQx/mg haemoglobin) only at the highest dose used. Application of the GC-MS assay to human haemoglobin samples showed that acid-labile adducts of MeIQx, if present, were below the limit of detection of the assay. These results show that levels of sulphinamide adducts of the dietary aromatic amine MeIQx, with haemoglobin, are very low and the implications for future human dosimetry of this carcinogen are discussed.

Animals↗
PubMed1990

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.

Animals↗
PubMed1990

Imipenem resistance in a case of AIDS with relapsing Pseudomonas meningitis.

We describe an AIDS patient who had a recurrence of Pseudomonas meningitis to illustrate three points. First, the use of sulfamethoxazole-trimethoprim in AIDS patients for prophylaxis of Pneumocystis carinii pneumonia may cause the various body sites to be colonized with resistant species such as P aeruginosa. Second, Pseudomonas meningitis can recur in a patient with AIDS after a month of appropriate therapy. Finally, imipenem is a poor choice for Pseudomonas meningitis, even when alternative therapies appear much less attractive. High doses of imipenem should not be used for fear of seizures, and lower doses only produce resistant organisms in the CSF.

Acquired Immunodeficiency Syndrome↗
PubMed1989

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.

Acid-Base Equilibrium↗