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Biomedical subjects

L Manzo

Publications and source records attributed to L Manzo.

At least 19 recordsLinked to original sources
PubMed2004

Development of a chromatographic bioreactor based on immobilized beta-glucuronidase on monolithic support for the determination of dextromethorphan and dextrorphan in human urine.

We here reported the development and application of an immobilized enzyme reactor (IMER) based on beta-glucuronidase to the on-line determination of urinary molar ratios of dextromethorphan (DOMe)/dextrorphan (DOH) for the assessment of the metabolic activity of CYP2D6, a genetically variable isoform of cytochrome P-450 (CYP). beta-Glucuronidase was immobilized on an HPLC monolithic aminopropyl silica support. Catalytic activity and stability of the chromatographic reactor were evaluated using 8-hydroxyquinoline glucuronide (8-HQG) as substrate. The IMER was coupled through a switching valve to a reversed-phase column (C8) for the simultaneous determination of dextromethorphan and its main metabolite dextrorphan. On purpose a selective reversed-phase ion pair HPLC method coupled with fluorescence detection was developed. Urine samples were first centrifuged to remove insoluble materials and then aliquots of the supernatants were injected into the coupled-column analyser. Linearity, precision and accuracy of the method were established. The method reliability was verified by comparing our data with previous data of a phenotyping study carried out by the Poison Control Centre of Pavia-Clinical Toxicology Division.

Chromatography, High Pressure Liquid↗
PubMed2002

Effect of sorbic acid administration on urinary trans,trans-muconic acid excretion in rats exposed to low levels of benzene.

Trans,trans-muconic acid (t,t-MA) is a biomarker of benzene exposure reflecting metabolic activation to trans,trans-muconaldehyde. t,t-MA background urinary levels are highly variable, thus limiting its use to exposure monitoring of levels over 1 ppm of benzene. Actually, sorbic acid (SA) is known to influence background excretion of t,t-MA in man, but only a few examples suggest that SA ingestion can enhance t,t-MA levels occurring together with benzene exposure. In this study, the effect of SA was investigated in benzene-exposed male Sprague-Dawley rats exposed to 1 ppm benzene for 6 h. Exposed animals had a 24-h urinary t,t-MA excretion higher than that observed in non-exposed animals (87+/-13 microg/kg vs 19+/-3 microg/kg body weight). The oral dose of 8 mg/kg body weight SA had no effect on urinary t,t-MA both in control and in benzene-exposed rats. Increases of t,t-MA levels in urine occurred at SA doses of 50-200 mg/kg body weight, and co-exposure to benzene and SA (50 and 100 mg/kg body weight) produced additive enhancement of t,t-MA excretion. These data demonstrate the dose-response relationship between SA administration and t,t-MA excretion. Our study showed that SA ingestion at doses equal to or greater than 50 mg/kg body weight significantly affects the t,t-MA urinary levels in rats exposed to 1 ppm of benzene for 6 h. These data support the conclusion that in man t,t-MA is not suitable for biomonitoring of low levels of benzene exposure.

Animals↗
PubMed2001

Direct analysis of urinary trans,trans-muconic acid by coupled column liquid chromatography and spectrophotometric ultraviolet detection: method applicability to human urine.

A coupled column liquid chromatographic (LC-LC) method for the direct analysis in human urine of the ring opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. The method was tested using urine samples collected from five refinery workers exposed to concentrations of airborne benzene (0.2-0.5 ppm), and from non-exposed volunteers. The analytical columns used were of 50 x 4.6 mm I.D. packed with 3 microm p.s. Microspher C18 material as the first column (C-1), and a 100 x 4.6 mm I.D. column packed with 3 microm p.s. Hypersil ODS material as the second one (C-2). The mobile phases applied consisted, respectively, of methanol-0.074% trifluoroacetic acid (TFA) in water (4:96, v/v) on C-1, and of methanol-0.074% TFA in water (10:90, v/v) on C-2. Under these conditions t,t-MA eluted 15 min after injection. The present method, coupling the LC-LC technique with UV detection at 264 nm, permits the quantitation of t,t-MA directly in urine at levels as low as 0.05 mg/l. The determination is performed with a sample throughput of 2 h(-1) requiring only pH adjustment and centrifugation of the sample. Calibration plots of standard additions of t,t-MA to pooled urine taken from five non-exposed subjects were linear (r>0.999) over a wide concentration range (0.05, 0.1, 0.5, 1.0, 2.0 mg/l). The precision of the method (RSD) was in the range of 0.5 to 3.8%, and the within-session repeatability on workers urine samples (levels 0.06, 0.1, 0.2, 1.0 mg/l) was in the range of 3 to 8%. The present method improves the applicability of routine t,t-MA analysis, where it is most desirable that a large number of biological samples can be processed automatically or with minimal human labour, at low cost, and with a convenient turn-around time.

Animals↗
PubMed2001

Neurotoxicity and molecular effects of methylmercury.

The neurotoxicity of high levels of methylmercury (MeHg) and the high susceptibility of the developing brain are well established both in humans and experimental animals. Prenatally poisoned children display a range of effects varying from severe cerebral palsy to subtle developmental delays. Still unknown is the lowest dose that impairs neurodevelopment. The primary source of human exposure is the fish. The data obtained so far from epidemiological studies on fish-eating populations are not consistent. A reference dose of 0.1 microg MeHg/kg per day has been established by the U.S. Environmental Protection Agency based on a study on Iraqi children exposed to MeHg in utero. However, these exposures occurred at high level for a limited period of time, and consequently were not typical of lower chronic exposure levels associated with fish consumption. Major obstacles for estimation of a threshold dose for MeHg include the delayed appearance of the neurodevelopmental effects following prenatal exposure and limited knowledge of cellular and molecular processes underlying these neurological changes. In this respect, a strategy which aims at identifying sensitive molecular targets of MeHg at environmentally relevant levels may prove particularly useful to risk assessment. Here some examples of MeHg molecular effects occurring at low doses/concentrations are presented.

Animals↗
PubMed2001

Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene.

A coupled column liquid chromatographic (LC-LC) method for high-speed analysis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t-MA from urine samples was obtained using a 3 microm C18 column (50x4.6 mm I.D.) as the first column (C-1) and a 5 microm C18 semi-permeable surface (SPS) column (150x4.6 mm I.D.) as the second column (C-2). The mobile phases applied consisted, respectively, of methanol-0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol-0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol-0.05% TFA in water (25:75, v/v) was used for cleaning C-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC-LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50-4000 ng/ml) showed excellent linearity (r>0.999). The method was validated using urine samples collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%.

Animals↗
PubMed2001

Assessing effects of neurotoxic pollutants by biochemical markers.

Neurotoxins cause biochemical and molecular events which indicate early stage effects in exposed persons well before or well below the induction of overt disease. Monitoring these early events may represent a valid approach to developing markers of neurotoxicity in individuals exposed to environmental chemicals. In neurotoxicology, the use of biochemical markers is more problematic compared to other fields due to the complexity of central nervous system function, the multistage nature of neurotoxic events, and the inaccessibility of target tissue. Nevertheless, new biochemical assays have been developed in recent years to assess exposure, subclinical effects, and susceptibility to neurotoxic disorders. This paper reviews novel biomarkers of neurotoxicity and discusses perspectives and limitations of their use in occupational and environmental medicine.

Acetylcholinesterase↗
PubMed2001

Carbon monoxide cardiotoxicity.

Cardiac dysfunction including arrhythmias and myocardial ischemia have often been reported in carbon monoxide poisoning; scattered punctiform hemorrhages throughout the heart have been documented in autopsy samples. An appropriate diagnostic approach is crucial to assess carbon monoxide cardiac damage. This evaluation may be confounded by several factors, including the absence of overt symptoms and of specific ischemic changes in the electrocardiogram. In experimental studies, laboratory animals can develop cardiac changes similar to those seen in humans and therefore proved to be useful models to study the effects and the mechanisms of cardiac damage due to carbon monoxide. These investigations, as well as others performed in vitro, provide support for a direct action of carbon monoxide on the heart, in addition to systemic hypoxia produced by carboxyhemoglobin formation. This review focuses on the diagnostic aspects of carbon monoxide cardiotoxicity. Experimental results obtained in animals and in vitro models are also discussed.

Animals↗
PubMed2001

Cardiac damage in pediatric carbon monoxide poisoning.

BACKGROUND: Cardiovascular disorders including myocardial ischemia and heart failure have been described in both laboratory animals and humans following carbon monoxide poisoning. Carbon monoxide cardiotoxicity may be clinically occult and often remains undiagnosed because of the lack of overt symptoms and specific ischemic changes in the electrocardiogram. Routine myocardial necrosis markers have low diagnostic efficiency, particularly in patients with concomitant skeletal muscle necrosis or multiple organ failure complicating carbon monoxide poisoning. Carbon monoxide-induced cardiotoxicity has been investigated rarely in children. CASE REPORT: This paper describes carbon monoxide poisoning in a 12-year-old child who suffered from occult cardiac damage despite mild symptoms and low carboxy hemoglobin concentrations. Myocardial and mitral valve dysfunctions were observed, suggesting an ischemia-like syndrome. Cardiac damage was completely reversible within 1 month. CONCLUSION: This case report supports that a prolonged carbon monoxide exposure can cause cardiac damage in children even in the absence of specific symptoms, cerebral failure and high carboxyhemoglobin concentrations.

Carbon Monoxide Poisoning↗
PubMed2000

Antioxidants J811 and 17beta-estradiol protect cerebellar granule cells from methylmercury-induced apoptotic cell death.

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 microM to 1.5 microM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high-molecular-weight DNA fragments. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha-fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17beta-estradiol (10 microM) and the Delta(8,9)-dehydro derivative of 17alpha-estradiol J811 (10 microM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17beta-estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury.

Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death. Within 1 hr exposure, MeHg at 5-10 microM caused impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5-1 microM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 microM) exposure, long before the occurrence of nuclear condensation (6-9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg-induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z-VAD-fluoromethylketone and z-DEVD-chloromethylketone, the L-type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK-801. On the other hand, insulin-like growth factor-I partially rescued CGCs from MeHg-triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity.

Animals↗
PubMed2000

Mild ciguatera poisoning: Case reports with neurophysiological evaluations.

Ciguatera poisoning causes mainly gastrointestinal and neurological effects of variable severity. However, symptoms of peripheral neuropathy with paresthesias and paradoxical disturbance of thermal sensation are the hallmark. Electrophysiological studies are often normal, except in severe cases. We report four people who developed mild ciguatera poisoning after barracuda ingestion. Electrophysiological studies documented normocalcemic latent tetany. These findings are consistent with ciguatoxin's mechanism of toxicity, which involves inactivation of voltage-gated Na(+) channels and eventually increases nerve membrane excitability.

Adult↗
PubMed2000

Low-level exposure to methylmercury modifies muscarinic cholinergic receptor binding characteristics in rat brain and lymphocytes: physiologic implications and new opportunities in biologic monitoring.

Methylmercury (MeHg) affects several parameters of cholinergic function. These alterations are thought to play a role in MeHg neurotoxicity. In vitro experiments have indicated that MeHg acts as a strong competitive inhibitor of radioligand binding to muscarinic cholinergic receptors (mAChRs) in rat brain. Furthermore, rat brain mAChRs share several pharmacologic characteristics of similar receptors present on lymphocytes. Using the muscarinic antagonist [(3)H]quinuclidinyl benzilate (QNB) to label receptors, we investigated the in vivo interactions of MeHg with rat brain mAChRs. We also investigated whether MeHg-induced central mAChR changes are reflected by similar alterations in splenic lymphocytes. Exposure to low doses of MeHg--0.5 or 2 mg/kg/day in drinking water--for 16 days significantly increased (20-44% of control) mAChRs density (B(max)) in the hippocampus and cerebellum without affecting receptor affinity (K(d)). The effect of MeHg did not occur immediately; it was not apparent until 2 weeks after the termination of treatment. No significant changes in [(3)H]QNB binding were observed in the cerebral cortex. In splenic lymphocytes, mAChR density was remarkably increased (95-198% of control) by day 14 of MeHg exposure and remained enhanced 14 days after the cessation of treatment. These results suggest up-regulation of mAChRs in selected brain regions (hippocampus and cerebellum) after prolonged low-level ingestion of MeHg in rats. These cerebral effects are delayed in onset and are preceded by a marked increase in density of mAChRs on lymphocytes. In chronic MeHg exposure, peripheral lymphocytes may represent a sensitive target for the interaction of MeHg with mAChRs and, therefore, may be predictive indicators of later adaptive response involving cerebral mAChRs. Additionally, the effect of MeHg on lymphocyte mAChRs in vivo indicates that this receptor system should be investigated further as a possible target for MeHg immunotoxicity.

Administration, Oral↗
PubMed2000

Styrene-induced changes in amacrine retinal cells: an experimental study in the rat.

Dopamine (DA) is synthesized in amacrine cells and released upon membrane depolarization in a calcium-dependent way. Thus, it is recognized to function as a major neurotransmitter or modulator in vertebrate retina. Owing to DA modulating activity on cone-horizontal cells transmission, depletion or dysfunction of amacrine cells could interfere with chromatic processing, accounting for the acquired dyschromatopsia described among styrene-exposed workers. The present study has been designed to test the hypothesis that amacrine cells represent a vulnerable target of styrene in subchronically exposed rats. Ten female Sprague-Dawley rats were exposed to 300 ppm styrene 6 h/day, 5 days/week, for 12 weeks; ten rats exposed to fresh air served as a control group. Whole mounted retinas were used for the morphometry of tyrosine hydroxylase (TH) immunoreactive cells (IR). DA content and TH activity were measured by HPLC and electrochemical detection and glutathione (GSH) was measured by HPLC tandem mass spectrometry (LC-MS/MS). In treated rats, morphometric analysis showed a loss of TH-IR amacrine cells (6.2/mm2 vs. 8.7/mm2 recorded in controls, p = 0.002), without any peripheral-central variation in cell loss. DA content was also lower in exposed, as compared to control animals (208.64 vs. 267.98 microg/g w.w., p = 0.004). The activity of TH in the whole retina was similar in styrene-exposed and control rats when expressed as a function of the wet weight, whereas it was much higher in styrene-exposed rats (+64%) when expressed as a function of the number of TH-IR amacrine cells (p < 0.001). Finally, retinal GSH was reduced by 30% in exposed as compared to control rats (p = 0.01). In summary, retinal TH-IR cells were sensitive to styrene exposure, which seems to cause both structural and functional changes, represented by cell loss and DA depletion, respectively. These findings confirm the vulnerability of dopaminergic systems to styrene toxicity, providing some insights on the possible mechanism of loss in chromatic discrimination recorded among workers occupationally-exposed to styrene.

Animals↗
PubMed1999

Effect of styrene on monoamine oxidase B activity in rat brain.

Previous studies have indicated that workers exposed to styrene present a decreased activity of platelet monoamine oxidase B (MAO B), suggesting that this biochemical assay may represent a biomarker for styrene-induced neurotoxicity. This study was undertaken to determine whether exposure to styrene would cause changes in MAO B activity in the target organ--the brain. Groups of rats were exposed to styrene by inhalation at concentrations of 300 ppm for 4 wk or 50 ppm for 13 wk. Both treatments caused significant decreases of MAO B activity in several brain areas, while MAO A activity was not affected. Decreases in MAO B activity were also found in brainstem of rats given styrene (400 mg/kg) or styrene oxide (100 mg/kg) by i.p. injection for 2 wk. Styrene, styrene oxide, and other styrene metabolites (mandelic acid, phenylglyoxylic acid, and styrene glycol) had no direct inhibitory effect on brain MAO B activity when tested in vitro. These results indicate that exposure to low concentrations of styrene alters MAO B activity in rat brain, suggesting that the observed changes in human platelets may reflect alterations in the nervous system.

Administration, Inhalation↗
PubMed1999

Chronic exposure to aluminium impairs the glutamate-nitric oxide-cyclic GMP pathway in the rat in vivo.

Aluminium is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease, dialysis syndrome and other neurological disorders. The molecular mechanism of aluminium-induced impairment of neurological functions remains unclear. We showed that aluminium impairs the glutamate-nitric oxide-cGMP pathway in cultured neurons. The aim of this work was to assess by in vivo brain microdialysis whether chronic administration of aluminium in the drinking water (2.5% aluminium sulfate) also impairs the glutamate-nitric oxide-cGMP pathway in the cerebellum of rats in vivo. Chronic exposure to aluminium reduced NMDA-induced increase of extracellular cGMP by ca 50%. The increase in extracellular cGMP induced by the nitric oxide generating agent S-nitroso-N-acetylpenicillamine was higher (240%) in rats treated with aluminium than in controls. Immunoblotting experiments showed that aluminium reduced the cerebellar content of calmodulin and nitric oxide synthase by 34 and 15%, respectively. Basal activity of soluble guanylate cyclase was decreased by 66% in aluminium-treated rats, while the activity after stimulation with S-nitroso-N-acetylpenicillamine was similar to controls. Basal cGMP in the cerebellar extracellular space was decreased by 50% in aluminium-treated rats. These results indicate that chronic exposure to aluminium reduces the basal activity of guanylate cyclase and impairs the glutamate-nitric oxide-cGMP pathway in the animal in vivo.

Aluminum↗