Rino Froldi

Ethanol induces conditioned place preference in genetically selected alcohol-preferring rats.

Abstract The study of the biological mechanisms of ethanol reward has greatly suffered from problems to obtain ethanol-induced conditioned place preference (CPP) in rats. In the present study, CPP was obtained in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats, derived from Sardinian alcohol-preferring rats, following intragastric (IG) ethanol administration by means of a permanent IG catheter, but not after intraperitoneal (IP) injection or IG gavage. Rats with permanent IG catheter, received IG administration of 0.35, 0.7, 1.5 or 2.8 g/kg ethanol, as a 10% v/v solution. In ethanol-experienced rats 0.7 or 1.5, but not 0.35 or 2.8 g/kg ethanol significantly increased in comparison to controls the time spent in the ethanol-associated previously non-preferred compartment, which became preferred in the post-conditioning test. In ethanol-naive rats, only 0.7 g/kg ethanol significantly increased the time spent in the ethanol-associated compartment. On the other hand, no effect was observed in alcohol-experienced rats following IG gavage, or IP injection of 0.35, 0.7 or 1.5 g/kg ethanol. The present results provide evidence that ethanol possesses postingestive rewarding properties in msP rats, and that it can reliably induce CPP in them, provided that an appropriate method of administration is adopted.

Antidepressant-like effect of ethanol revealed in the forced swimming test in Sardinian alcohol-preferring rats

Abstract  Rationale: A large body of evidence indicates high comorbidity between depression and alcohol abuse. The self-medication hypothesis proposes that depressed subjects may abuse ethanol because it reduces the symptoms of depression. The present study evaluated whether ethanol may exert an antidepressant-like action in genetically selected alcohol-preferring rats, either Sardinian alcohol-preferring (sP) or Marchigian Sardinian alcohol-preferring (msP) rats, and for comparison in Sardinian alcohol-non-preferring (sNP) rats. Methods: The forced swimming test (FST) was used to evaluate the antidepressant-like action of ethanol; in this test the effect of ethanol ingestion on the immobility time was determined. Results: Ethanol-naive sP rats exhibited a longer period of immobility in comparison to sNP rats. Both in ethanol-naive sP and msP rats, voluntary ethanol drinking reduced the immobility time. A similar effect was obtained when repeated (five or nine) intragastric administrations of 0.7 g/kg ethanol were given during the 24 h prior to the test in msP and in sP, but not in sNP rats. Desipramine, like ethanol, sharply reduced immobility at doses of 5 or 20 mg/kg, given 3 times in the 24 h before the test in msP rats. The reduced immobility induced by ethanol in msP rats was apparently not the consequence of a general motor activation, because 9 IG administrations of ethanol, 0.7 g/kg, failed to alter locomotor activity in the open field test. Moreover, blood alcohol levels and rectal temperature of msP, sP and sNP after IG ethanol administration were not statistically different. Conclusions: The present results provide evidence for an antidepressant-like action of ethanol in sP and msP rats and suggest that this action may contribute to sustain their high ethanol drinking.

Mechanism of action for reduction of ethanol intake in rats by the tachykinin NK-3 receptor agonist aminosenktide.

Intracerebroventricular (i.c.v.) injection of tachykinin (TK) NK-3 receptor agonists inhibits alcohol intake in genetically selected alcohol-preferring rats. The present study investigated the mechanism of action by which the selective TK NK-3 receptor agonist aminosenktide (NH2-SENK) attenuates ethanol intake in Marchigian Sardinian alcohol-preferring (msP) rats. The effect of NH2-SENK was studied by i.c.v. injection in the conditioned taste aversion (CTA) and in the conditioned place preference (CPP) paradigms; moreover, the effect of NH2-SENK on blood alcohol levels (BAL) following intragastric ethanol administration was investigated. The i.c.v. dose of 125 ng/rat of NH2-SENK, that markedly reduces ethanol intake, did not modify BAL, nor did it increase the CTA induced by intraperitoneal injection of ethanol, 1 g/kg body weight. These findings suggest that the effect of NH2-SENK on alcohol consumption is not related to modification of the pharmacokinetics of ethanol, nor to increase of the aversive properties of ethanol. On the other hand, the same i.c.v. dose of NH2-SENK evoked a pronounced and statistically significant CPP. This finding indicates that the TK NK-3 receptor agonist NH2-SENK possesses rewarding properties in msP rats and suggests that its inhibitory effect on ethanol consumption may be due to substitution of the rewarding properties of ethanol, thus making its consumption redundant.

Determination of primary active constituents in Cannabis preparations by high-resolution gas chromatography/flame ionization detection and high-performance liquid chromatography/UV detection

Abstract For a complete quantitative analysis of primary active constituents in Cannabis preparations Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN), we have compared two different chromatographic techniques, high-resolution gas chromatography (HRGC)/flame ionization detection (FID) and high-performance liquid chromatography (HPLC)/UV. The two different methods have been validated using crude drug (hashish) with methyloleate and tetraphenylethylene as internal standard for HRGC/FID and HPLC/UV, respectively.

Hypericum perforatum CO2 Extract and Opioid Receptor Antagonists Act Synergistically to Reduce Ethanol Intake in Alcohol‐Preferring Rats

BACKGROUND Hypericum perforatum extracts attenuate ethanol intake in alcohol-preferring rats. The opioid receptor antagonists, naloxone and naltrexone, reduce ethanol intake in rats and humans. The combination of different agents that reduce ethanol intake has been proposed as an approach to the pharmacotherapy of alcoholism. This study evaluated the effect on ethanol intake of the combined administration of a CO2 H. perforatum extract and naloxone or naltrexone in genetically selected Marchigian Sardinian alcohol-preferring rats. METHODS Ten percent (v/v) ethanol intake was offered 2 hr per day at the beginning of the dark phase of the reverse light-dark cycle. H. perforatum CO2 extract was given intragastrically, 1 hr before access to ethanol. Naloxone or naltrexone was given by intraperitoneal injection 10 min before the extract. RESULTS H. perforatum CO2 extract reduced ethanol intake at 31 or 125 mg/kg, but not 7 mg/kg. These doses neither modified food or water intake during access to ethanol, nor reduce 0.2% saccharin intake. Naloxone reduced ethanol and food intake at 3 or 5 mg/kg, but not 1 mg/kg. When naloxone 1 mg/kg was combined with the three doses of H. perforatum CO2 extract, the attenuation of ethanol intake was more pronounced than that observed after the administration of the extract alone. Alcohol intake was also significantly reduced by 7 mg/kg of H. perforatum CO2 extract combined with naloxone 1 mg/kg. The combined treatments never modified the rats locomotor activity nor the simultaneous intake of food, water or 0.2% saccharin. Naltrexone reduced ethanol intake at 1 and 3 mg/kg, but not at 0.5 mg/kg. When naltrexone 0.5 mg/kg was combined with H. perforatum CO2 extract 7 mg/kg, ethanol intake was markedly reduced. CONCLUSIONS These findings provide evidence that H. perforatum CO2 extract and opiate receptor antagonists act synergistically to induce a pronounced and selective reduction of voluntary ethanol consumption in alcohol-preferring rats.

Conditioned Taste Aversion Induced by Ethanol in Alcohol-Preferring Rats: Influence of the Method of Ethanol Administration

A recent study of our group has shown that ethanol evokes conditioned place preference (CPP) in Marchigian Sardinian alcohol-preferring (msP) rats following intragastric (IG) administration by means of an indwelling IG catheter, but not following administration by gavage or by intraperitoneal (IP) injection. The present study evaluated in ethanol-naive msP rats the influence of the method of administration (IG injection by indwelling catheter vs. IP injection) on ethanol-induced conditioned taste aversion (CTA). The dose of 0.35 g/kg of ethanol did not evoke aversion either by IG or by IP administration. Following IG injection, 0.7 g/kg of ethanol, the amount that msP rats voluntarily ingest in a short (2-5 min) drinking episode, did not evoke CTA, and 1.5 g/kg induced a modest CTA. On the other hand, IP injection of 0.7 g/kg of ethanol evoked CTA, and 1.5 g/kg induced a very pronounced CTA. These findings show that the aversive properties of ethanol in msP rats are influenced by the method of administration, and suggest that the IG injection by catheter may reveal more faithfully than the IP injection the motivational properties of amounts of ethanol that alcohol-preferring rats voluntarily ingest.

Blood Cyanide Determination in Two Cases of Fatal Intoxication: Comparison Between Headspace Gas Chromatography and a Spectrophotometric Method*

Abstract:  Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen‐phosphorus detector (HS‐GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS‐GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS‐GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5–10 and 0.05–5 μg/mL, respectively. Detection limits were 0.2 μg/mL for VIS and 0.05 μg/mL for HS‐GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.

Unexpected Variation of the Codeine/Morphine Ratio Following Fatal Heroin Overdose

Postmortem samples from 14 cases of suspected heroin overdose were subjected to a preliminary systematic toxicological analysis in order to highlight the presence of unknown exogenous compounds (e.g., drugs of abuse, alcohol) that may have played a role in the mechanism of death. This analysis unveiled histories of poly-drug use in seven of the cases under investigation. Moreover, the concentrations of morphine and codeine in the brain were also investigated, and the results were compared with the data obtained from the blood specimens. The concentration of morphine in blood ranged from 33 to 688 ng/mL, while the concentration of codeine ranged from 0 to 193 ng/mL. However, in the brain, the concentration of morphine was found to be between 85 and 396 ng/g, while the levels of codeine ranged from 11 to 160 ng/g. The codeine/morphine ratio in the blood ranged from 0.043 to 0.619; however, in the brain, the same ratio was found to be between 0.129 and 0.552. In most cases, a significantly higher codeine/morphine ratio was found in the brain, suggesting the accumulation of codeine in brain tissue due its high lipophilicity as compared with morphine.

Hair analysis in order to evaluate drug abuse in driver's license regranting procedures.

In Italy, driving under the influence of drugs determines the suspension of the offenders drivers license. To regain the license the person must be drug free during an observation period. People whose license has been revoked or suspended can obtain, or re-obtain their drivers license subject to the judgment of a medical commission. The exclusion of illicit drug use is determined by means of toxicological analysis, mainly on urine or hair matrices. We reported the results of several years of experience of the forensic toxicology laboratory of the University of Macerata in the use of hair analysis for the assessment of past exposure to drugs in people suspected of driving under the influence of drugs. From 2004 to 2013, 8612 hair samples, were analyzed for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ(9)-THC) using gas chromatography/mass spectrometry (GC/MS) method. We used a cutoff (SoHT or national guidelines) to determine the positive data, regardless of the hair sample concentrations. 1213 samples resulted positive, 71.7% were positive for cocaine and metabolites, 19.8% for morphine and metabolites, 8.5% for Δ(9)-THC. We also studied the timeframe of the abuse, as well as gender and age distribution of positive subjects. Moreover, we analyzed the possible deterrent effect of the hair analysis on driving under the influence of psychoactive substances.

A case of fatal intoxication from metformin

A case of fatal intoxication from metformin is presented. The decedent was an obese 58‐year‐old‐woman with type II diabetes, in whom severe lactic acidosis secondary to metformin accumulation was precipitated by acute renal failure. She had been on metformin 500 mg twice a day. Postmortem blood was deproteinated with acetonitrile, washed with dichloromethane, and the resulting supernatant injected into high‐performance liquid chromatography system. Separation was performed on a analytical 125 × 4 mm i.d. RP‐8 column. The wavelength was set at 235 nm. The mobile phase was acetonitrile (40%), sodium lauryl sulfate, and sodium dihydrogen phosphate adjusted to pH 5.1 (60%) at a flow rate of 1.0 mL/min. The concentration of metformin in postmortem blood was 77.3 μg/mL. The qualitative result was also confirmed by LC/APCI/MS/MS analysis.