Formation of an adduct by clenbuterol, a beta-adrenoceptor agonist drug, and serum albumin in human saliva at the acidic pH of the stomach: evidence for an aryl radical-based process.

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.

Dexamethasone-induced selective inhibition of the central mu opioid receptor: functional in vivo and in vitro evidence in rodents.

1. Endogenous corticosteroids and opioids are involved in many functions of the organism, including analgesia, cerebral excitability, stress and others. Therefore, we considered it important to gain information on the functional interaction between corticosteroids and specific opioid receptor subpopulations. 2. We have found that systemic administration (i.p.) of the potent synthetic corticosteroid, dexamethasone, reduced the antinociception induced by the highly selective mu agonist, DAMGO or by less selective mu agonists morphine and beta-endorphin administered i.c.v.. On the contrary dexamethasone exerted little or no influence on the antinociception induced by a delta 1 agonist, DPDPE and a delta 2 agonist deltorphin II. Dexamethasone potentiated the antinociception induced by the kappa agonist, U50,488. 3. In experiments performed in an in vitro model of cerebral excitability in the rat hippocampal slice, dexamethasone strongly prevented both the increase of the duration of the field potential recorded in CA1, and the appearance and number of additional population spikes induced by mu receptor agonists. 4. In both models pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the antagonism by dexamethasone of responses to the mu opioid agonists. 5. Our data indicate that in the rodent brain there is an important functional interaction between the corticosteroid and the opioid systems at least at the mu receptor level, while delta and kappa receptors are modulated in different ways.

Prescriptions for mesalazine and sulphasalazine: a prevalence estimate of patients treated for inflammatory bowel disease in Rome.

BACKGROUND: Sulphasalazine and 5-amino salicyclic acid drugs are specifically indicated for the treatment of inflammatory bowel disease. AIM: To use drug consumption by a given population as a marker to estimate the number of patients with inflammatory bowel disease. METHODS: Prescriptions for sulphasalazine and mesalazine were identified for the 133000 inhabitants of a local health unit in Rome. Other prescriptions received by the patients, who were users of sulphasalazine or mesalazine, were also studied. RESULTS: 99465 patients received at least one prescription for any drug in 1991. Three hundred and seventy-six patients were prescribed sulphasalazine and/or mesalazine, an average of 3.8 prescriptions per patient. These patients were exposed more frequently than the general population to other drugs often used in inflammatory bowel disease treatment, for example, corticosteroids, anti-diarrhoeal drugs and intestinal anti-infectives. We identified that 258 of 100000 inhabitants were prescribed either sulphasalazine or mesalazine; 127 of 100000 inhabitants received full-dose treatment for at least 30 days, and 42.8 of 100000 inhabitants received prescriptions of either drug, also associated with systemic corticosteroids. CONCLUSION: The consumption of drugs used specifically for inflammatory bowel disease may act as a marker for the prevalence of the condition in a community.

Long-term changes induced by developmental handling on pain threshold: effects of morphine and naloxone.

Mice pups were exposed daily to a stress-producing procedure (handling and saline injection) during the first 3 weeks of life. At 25 and 45 days of age, they were tested for differences in the tail-flick and hot-plate tests. The results indicate that chronic handling procedures during developmental stages can produce a long-lasting increase of the threshold for painful stimuli. This phenomenon is completely prevented by naloxone pretreatment and has enhancing effects on morphine analgesia, thus suggesting that postnatal handling can exert long-lasting interference on the sensitivity of some opioid receptor populations.

Effects of neonatal treatment with Tyr-MIF-1 and naloxone on the long-term body weight gain induced by repeated postnatal stressful stimuli.

Stressful stimuli repeatedly applied during the first postnatal weeks can induce body weight gain in the mouse during adulthood. This effect can be prevented by injecting naloxone concomitantly with stress. The peptides belonging to the Tyr-MIF-1 family have a great modulating activity on numerous stress-induced phenomena. The aim of the present work was to compare the effect of repeated neonatal injections of Tyr-MIF-1 or naloxone on the long-term body weight gain induced by a stressing procedure applied daily during the first three weeks of life. The results indicate that although naloxone blocked the development of the stress-induced effects, Tyr-MIF-1 potentiated them.

Chronic treatment with MIF-1 prevents the painful stimuli threshold elevation induced by neonatal handling in mice.

Chronic postnatal stressful handling results in a hyposensitivity to thermal nociceptive stimuli. This phenomenon is strongly affected by manipulations of the opioid system. In the present experiment, we report that chronic treatment with MIF-1 during the neonatal period prevents the behavioral alterations induced by handling while it is completely ineffective if injected acutely before antinociceptive testing by the tail flick test at 45 days of life.

MIF-1 can accelerate neuromotor, EEG and behavioral development in mice.

Newborn mice were injected SC daily with 1 mg/kg of MIF-1 or saline during the first 19 days of life. The progress of each pup was monitored for physical (body weight, eye and ear opening), neurobehavioral (reflexes) and neurophysiological (EEG) development until the weaning stage. In early adulthood (40 days of age) mice were tested on a maze learning task. Results indicate that MIF-1 can accelerate neurologic (days 3-9), somatic (days 10-14) and electroencephalographic (days 16-19) parameters, and that the effects of treatment last into the early adult stage with increased learning abilities in an appetitive task.

Effects of low doses of physostigmine on avoidance learning and EEG in two strains of mice.

The effects of the cholinomimetic drug, physostigmine (0, 0.01, 0.025, 0.05 and 0.1 mg/kg, i.p.), on shuttle-box avoidance learning and electroencephalographic (EEG) activity were investigated, in two separate studies, in mice belonging to the inbred C57BL/6 (C57) and DBA/2 (DBA) strains. The results of the behavioral investigation showed a consistent, significant enhancement of avoidance performance, on the whole of 5 daily training sessions, in C57 mice treated with the lowest dose (0.01 mg/kg) and in DBA mice treated with the highest doses (0.05 and 0.1 mg/kg) of the drug. Doses higher than 0.01 mg/kg, in C57 mice, and lower than 0.05 mg/kg, in DBA mice, had no significant effect. The avoidance improvements induced by physostigmine cannot be ascribed to general behavioral activation, since the doses that increased avoidance responses did not affect or even depressed spontaneous locomotor activity. The same doses of treatment which increased avoidance responding, also induced, in the same strains, consistent enhancement of 4-7 Hz (theta) EEG band power and decrease of 7-12 Hz (alpha) band power. Results suggest that the effects induced by physostigmine on the EEG and on the shuttle-box performance of mice are related to the same neurochemical systems, and are dependent upon the interaction of the dose with specific strain sensitivity.

Ultradian rhythms in the N1-P2 amplitude of the visual evoked potential in two inbred strains of mice: DBA/2J and C57BL/6.

Ultradian rhythmicity has been showed in many behavioural parameters in animals as well as in humans. We investigated the existence of a Basic Rest-Activity Cycle (BRAC) rhythm in amplitude fluctuations of mice's visual evoked potential (VEP) primary component. Two inbred strains of mice, C57BL/6 and DBA/2J, were used to verify the influence of genetics on biological rhythm as well. Results revealed the existence of an evident rhythm in the parameter under study and confirm previous reports of a 20 min. BRAC in avoidance behavior in DBA mice. This rhythm shows similar period within each strain, but significatively different periods between strains. Observed periods are near to species-specific reported BRAC cycle. Genetic hypothesys is suggested to explain differences between strains in expression of ultradian rhythm.

Reduced hippocampal CA1 Ca(2+)-induced long-term potentiation is associated with age-dependent impairment of spatial learning.

Expression of Ca(2+)-induced CA1 long-term potentiation (LTP) was analysed in hippocampal slices obtained from (1) 3-month-old and (2) 18-20-month-old Sprague-Dawley rats selected for their performances in the Morris water maze task. In all slices, a transient (10 min) increase of extracellular Ca2+ concentration (4 mM) caused a long-lasting enhancement of potentials evoked by electrical stimulation of radiatum fibers. However, a significant difference was found in the degree of potentiation among groups. In particular, increases of the CA1 response amplitudes were significantly lower in old rats impaired in spatial learning than in young at 30 (P < 0.05), 60, 90 and 120 min (P < 0.01) after restoring the normal Ca2+ concentration. On the contrary, no differences were observed between young animals and the old ones with good performances in spatial learning. The data suggest that amplitude of CA1 Ca(2+)-induced LTP in old rats is related to spatial learning abilities.

Effect of des-tyrosine-gamma-endorphin on neocortical spike-and-wave spindling in DBA/2J mice.

The effect of a beta-endorphin cleavage product devoid of opioid effects, des-tyrosine-gamma-endorphin (DT gamma E) on the neocortical spike-and-wave spindling episodes in the electrocorticogram (ECoG) of DBA/2J mice was studied. DT gamma E (0.01-1.0 mg/kg, i.p.) dose dependently reduced the spike-and-wave bursts duration. However, the low dose did not induce consistent modifications of the spike-and-wave bursts number while the dose of 0.1 and 1.0 mg/kg induced a progressive diminution. Furthermore, at all doses DT gamma E did not induce any alterations of the spike-and-wave bursts amplitude, frequency, and desynchronized activity when compared to the pre-drug period. These results indicate that this beta-endorphin fragment may affect brain excitability.

Orphanin FQ reduces morphine-induced dopamine release in the nucleus accumbens: a microdialysis study in rats.

The effects induced by orphanin FQ (OFQ) on morphine-induced dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA) release in the nucleus accumbens were studied in rats by using microdialysis with electrochemical detection. Morphine administered intraperitoneally (i.p., 2, 5 and 10 mg/kg) dose-dependently increased DA and metabolites release in the nucleus accumbens. OFQ intracerebroventricularly (i.c.v.) administered at doses of 2, 5 and 10 nmol did not change DA and metabolites release in the nucleus accumbens. OFQ (10 nmol) administered i.c.v. 15 min before morphine (5 and 10 mg/kg, i.p.) significantly reduced morphine-induced DA and metabolites release in the nucleus accumbens. These effects suggest that OFQ may regulate the stimulant action linked to morphine-induced DA release in the nucleus accumbens.

Age and strain differences in rat place learning and hippocampal dentate gyrus frequency-potentiation.

Induction of post-tetanic potentiation (PTP) and long-term potentiation (LTP) was analyzed in hippocampal slices obtained from (i) young (6 months old) rats of different strains (Sprague-Dawley, SD; spontaneously hypertensive rats, SHR; and Wistar-Kyoto, WKY), and (ii) from aged (20-24 months old) SD and Fischer 344 (F 344) rats, each group showing a different performance in the Morris maze test. After the application of an electrical tetanus (1 s, 100 Hz, 50 microA) in the stratum moleculare, a significant difference was found in the percent of induction of the dentate PTP in hippocampal slices obtained from rats of different strains and ages. In particular, the induction of the dentate PTP was significantly (P < 0.01) higher in slices obtained from young SD or spontaneously SHR rats, having the better performance in the Morris maze than in slices obtained from old SD or F 344 rats or young WKY rats which had poorer performances in the Morris maze. On the contrary, no significant differences were found in the percent of induction of the LTP in the dentate area of hippocampal slices obtained from rats of different strains and ages. Moreover, after the application of an electrical tetanus (1 s, 100 Hz, 50 microA) in the stratum radiatum, no significant differences were found in the percent of induction of both PTP and LTP in the CA1 area of hippocampal slices obtained from rats of different strains and ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropin-related modulation of the human EEG and individual variability.

During a 6-h period in resting conditions, the blood concentrations at rest of cortisol, glucose and the adrenocorticotropic hormone (ACTH) varied spontaneously within physiological ranges in eight healthy male volunteers (24.5+/-1.7 years), without pulsatile changes, correlation among variables, or indications of stress response. The power of the 6.5-14.0 Hz physiological 'alpha' rhythm of the electroencephalogram (EEG) proved inverted-U correlated with the ACTH concentration (with maximum power at 12-14 pmol/l ACTH) but was independent from the extent of ACTH change or from cortisol/glucose concentrations. Two subgroups of subjects with low/high EEG power values could be separated depending on ACTH concentration, with estimated cut-off at 7-8 pmol/l. A direct ACTH modulation of brain electrophysiology or common factors (e.g. the corticotropin-releasing hormone) pacing both ACTH and EEG are suggested and may account for individual EEG differences.

Guinea-pigs treated with beta-endorphin fragments DTgammaE and DEgammaE exhibit reduced morphine inhibition of electrically-induced contractions.

The effect exerted by two gamma-endorphin derivatives (DTgammaE and DEgammaE) was investigated on morphine-induced inhibition on the electrically contractions of guinea pig ileum in vitro. Morphine (1x10(-8)-5x10(-8)-1x10(-7) M) dose dependently and significantly reduced the E.C. of guinea pig ileum, IC50=6.5x10(-8) M (Confidence limits: 3.7x10(-8)-9.1x10(-8)). DTgammaE and DEgammaEper se (1x10(-6)-5x10(-6)-1x10(-5) M) did not modify significantly the E.C. of guinea pig ileum. Furthermore, DTgammaE or DEgammaE injection 10-30-60 min before morphine, did not affect the inhibitory effect of morphine on the E.C. of guinea pig ileum. By contrast, ilea from guinea-pigs treated for 4 days with DTgammaE or DEgammaE (1 mg/Kg/i.p.) resulted less sensitive to the inhibitory effect of morphine, IC50=8.3x10(-7) M (Confidence limits: 1.4x10(-6)-3.5x10(-7)) for DTgammaE and IC50=7.7x10(-7) M (Confidence limits: 2.7x10(-6)-8.7x10(-7)) for DEgammaE. Our results indicate that chronic treatment of guinea pigs with DTgammaE or DEgammaE induces a significant reduction of the inhibitory effect of morphine on the E.C. of guinea-pig ileum thus confirming an important functional interaction between gamma-endorphin derivatives and opioid system.

Dexamethasone influence on morphine-induced analgesia in outbred Swiss and inbred DBA/2J and C57BL/6 mice.

1. The influence of dexamethasone on morphine analgesia in three different strains of mice (Swiss, DBA/2J and C57BL/6) was studied by using the tail flick test. 2. I.c.v. as well as i.p. injections of dexamethasone did not modify nociceptive response in all strains. 3. I.c.v. injection of dexamethasone significantly reduced morphine analgesia in Swiss mice whereas no effects were observed in DBA/2J and C57BL/6 mice. 4. In addition, i.p. injection of dexamethasone significantly reduced morphine analgesia in all three strains. 5. These results suggest that the use of different genetic strains may provide an useful approach for studying dexamethasone-morphine analgesia interaction.

Dexamethasone pretreatment reduces the psychomotor stimulant effects induced by cocaine and amphetamine in mice.

1. The present study examined a comparison of the effect of DEX on psychomotor stimulant effects of cocaine and amphetamine in mice by using the locomotor activity test. 2. Cocaine (10 mg/kg/i.p.) and amphetamine (5 mg/kg/i.p.) increased markedly locomotor activity of mice whereas DEX per se (0.1-1.0-10 mg/kg/i.p.) did not modify the activity of control mice. 3. DEX pretreatment decreased the stimulating effects induced both by cocaine and amphetamine but no consistent dose-related effects were observed. 4. The results suggest that DEX may play an important role on the stimulating effects of cocaine and amphetamine and that it may be of some utility in the clinical management of psychostimulants abuse.

Taurine administration during lactation modifies hippocampal CA1 neurotransmission and behavioural programming in adult male mice.

Taurine plays a role in neuronal development. In this study, we examined whether postnatal taurine administration influences the long-term consequences induced by mild neonatal stressors (10 min maternal deprivation plus sham injection, applied daily to neonatal mice up to 21 days). At 30 days of age stressed mice showed higher pain threshold both in the tail-flick--which measures mostly the spinal mechanisms of pain--and in the hot-plate test--which reflects mainly the supraspinal mechanisms of pain. The latter effect was prevented completely by neonatal taurine administration, while the tail-flick test was not affected, thus suggesting that spinal pain is not sensitive to taurine treatment. At 140 days of age, mice which were stressed during the neonatal period showed consistent decrease in immobility time in forced swimming test, and taurine did not influence this parameter. At the same age, the fear/anxiety axis, measured with elevated plus maze test, did not show any consistent changes. Electrophysiological experiments in brain slices obtained from adult mice showed that input-output curves in hippocampal CA1 were increased by taurine administration in lactation. Hence, neonatal administration of taurine might permanently modify the functioning of hippocampus, a brain area which is known to be crucial for learning and memory.

Effect of naloxone on the long-term body weight gain induced by repeated postnatal stress in male mice.

Stress during the early postnatal life of laboratory animals can induce body weight gain in adulthood. Exposure of mice to repeated postnatal stress has been shown to increase body weight some weeks after the recording of a hyposensitivity to thermal nociceptive stimulation. Since the increase in nociceptive threshold after repeated environmental manipulations can be interpreted as a behavioral index of stress-induced endogenous opioid release, the present experiment was designed to study whether the stress-induced body weight gain could be prevented by a chronic treatment with the opioid antagonist naloxone. Sixteen litters of CD-1 male mice were used. Treatments took place from day 2 to day 19 of postnatal life. As well as body weight we measured spontaneous motor activity and food and water consumption. Body length and epididymal fat pad weight were also measured, at the end of the experiment. Stress (i.e. a daily saline injection schedule) increased the rate of body weight gain and the epididymal fat pad weight; a daily injection schedule of naloxone did not induce these effects. No significant differences were evident among groups regarding body length, motor activity, or food and water intake. The data suggest that repeated postnatal saline injections may induce long-term modifications in body and epididymal fat weights and that these effects may be mediated by endogenous opioids.

Visual, auditory, and somatosensorial evoked potentials in early and late treated adolescents with phenylketonuria.

Pattern reversal visual, auditory, and somatosensorial evoked potentials were recorded in two groups of phenylketonuric (PKU) adolescents after protracted exposition to high concentrations of phenylalanine following diet discontinuation. The first group consisted of 11 early treated (before age 3 months) PKU patients (ET-PKU); the second group consisted of 11 late detected (after age 8 months), symptomatic, PKU subjects (LT-PKU). Despite the relevant lag between the two groups in mental development and neurological status, no clear-cut difference in evoked potentials could be detected. Only the wave I latency of the brainstem auditory evoked potentials (BAEPs) was significantly shorter in ET- versus LT-PKU children. The P100 latency, I-V interpeak latency (IPL), and I-III IPL seem to discriminate the less severe form of PKU (ET-PKU type 3) from the most severe forms, ET-PKU type 1 plus 2 and LT-PKU. No correlations were found between clinical, biochemical, and neurophysiological parameters. The present data suggest that evoked potentials technique is of limited sensitivity in detecting central nervous system (CNS) alterations in PKU adolescents after diet discontinuation.