Entomotoxicology in burnt bodies: a case of maternal filicide-suicide by fire.

One of the most common methods of maternal filicide is by fire. In this case study, a 40-year-old female and her children were found completely burned in a burnt out car. All bodies showed a degree of destruction by fire consisting to a level 3 of the Crow-Glassman Scale (CGS) and early stage of insect activity. Toxicological analyses were performed on soft tissues and body fluids still available. The results were positive for diazepam and its metabolites only for children with blood concentrations consistent with therapeutic doses of benzodiazepines. Home video surveillance cameras confirmed sedation prior to death recording the mother while administering some drops of sedative drugs in a soft drink to the children just a couple of hours before setting fire to the car. Based on autopsy findings, all victims were still alive at the time of fire. The cause of death was determined as carbon monoxide poisoning and fatal thermal injuries by fire. This case study has a special focus on the entomotoxicology and the potential role of insects in death investigations of burnt bodies, supposed to be an inadequate substratum for insect colonization. It demonstrates that in burnt bodies, arthropod colonization can be quite immediate after fire is extinguished. Toxicological analyses performed on larvae actively feeding on the children's bodies were positive for diazepam and its metabolites in small amount compared with blood concentrations, whereas the larvae collected from the mother's body were totally negative. These data, according to the autopsy findings and the toxicological results from the victim's blood and tissues, supported the suspect of a non-lethal sedation prior to death, which is a common behaviour in maternal filicide.

Simultaneous determination of morphine, codeine and 6-acetyl morphine in human urine and blood samples using direct aqueous derivatisation: validation and application to real cases.

Opiates play a relevant role in forensic toxicology and their assay in urine or blood is usually performed for example in workplace drug-testing or toxicological investigation of drug impaired driving. The present work describes two new methods for detecting morphine, codeine and 6-monoacethyl morphine in human urine or blood using a single step derivatisation in aqueous phase. Propyl chloroformate is used as the dramatizing agent followed by liquid-liquid extraction and gas-chromatography-mass spectroscopy to detect the derivatives. The methods have been validated both for hydrolysed and unhydrolysed urine. For hydrolysed urine, the LOD and LOQ were 2.5ng/ml and 8.5ng/ml for codeine, and 5.2ng/ml and 15.1ng/ml for morphine, respectively. For unhydrolysed urine, the LOD and LOQ were 3.0ng/ml and 10.1ng/ml for codeine, 2.7ng/ml and 8.1ng/ml for morphine, 0.8ng/ml and 1.5ng/ml for 6-monoacetyl morphine, respectively. In blood, the LOD and LOQ were 0.44ng/ml and 1.46ng/ml for codeine, 0.29ng/ml and 0.98ng/ml for morphine, 0.15ng/ml and 0.51ng/ml for 6-monoacetyl morphine, respectively. The validated methods have been applied to 50 urine samples and 40 blood samples (both positive and negative) and they can be used in routine analyses.

Novel method for simultaneous aqueous in situ derivatization of THC and THC-COOH in human urine samples: validation and application to real samples.

The present work describes the validation of a novel aqueous in situ derivatization procedure with trimethyloxonium tetrafluoroborate (TMO) as methylating agent for the simultaneous, quantitative analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-Δ(9)-tetrahydrocannabinol carboxylic acid (THC-COOH) in human urine. The derivatizing agent is directly added to the urine sample and the methyl-derivatives are then recovered by liquid-liquid extraction procedure. Gas chromatography-mass spectrometry was used to detect the derivatives in selected ion monitoring mode. The limits of detection were 0.7 ng/mL for THC and 0.5 ng/mL for THC-COOH, whereas the limits of quantification were 1.9 and 0.9 ng/mL, respectively. The method has been applied to 60 real samples both positive and negative to immunochemical screening test resulting to be very useful and reliable in routine analysis of THC-COOH in human urine for toxicological and forensic purposes.

Previous exposure to (+/-) 3,4-methylenedioxymethamphetamine produces long-lasting alteration in limbic brain excitability measured by electroencephalogram spectrum analysis, brain metabolism and seizure susceptibility.

Seizures represent the most common neurological emergency in ecstasy abusers; however, no study addressed whether (+/-) 3,4-methylenedioxymethamphetamine ("ecstasy") per se might produce long-lasting alterations in brain excitability related to a pro-convulsant effect. C57 Black mice were treated with three regimens of (+/-) 3,4-methylenedioxymethamphetamine (5mg/kg x 2 for 1, 2 or three consecutive days). Following the last dose of (+/-) 3,4-methylenedioxymethamphetamine, during a time interval of 8 weeks, the following procedures were carried out: 1) cortical electroencephalographic recordings, including power-spectrum analysis; 2) administration of sub-threshold doses of kainate; 3) measurement of regional [(14)C]2-deoxyglucose uptake; 4) monoamine assay. We demonstrate that all mice pre-treated with (+/-) 3,4-methylenedioxymethamphetamine showed long-lasting encephalographic changes with frequencies peaking at 3-4.5 Hz at the power-spectrum analysis. This is concomitant with latent brain hyperexcitability within selected limbic brain regions, as shown by seizure facilitation and long-lasting latent metabolic hyperactivity which can be unraveled by phasic glutamate stimulation. This study sheds new light into the brain targets of (+/-) 3,4-methylenedioxymethamphetamine and discloses the occurrence of (+/-) 3,4-methylenedioxymethamphetamine-induced latent hyperexcitability within limbic areas, while it might provide a model to study in controlled experimental conditions limbic seizures and status epilepticus in C57 Black mice. Persistent changes produced by (+/-) 3,4-methylenedioxymethamphetamine in limbic brain excitability might be responsible for seizures and limbic-related disorders in chronic (+/-) 3,4-methylenedioxymethamphetamine abusers.

DNA damage and ubiquitinated neuronal inclusions in the substantia nigra and striatum of mice following MDMA (ecstasy).

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative, which is neurotoxic to both serotonin (5HT) and dopamine (DA) nerve terminals. Previous reports, carried out in rodents and non-human primates, demonstrated neurotoxicity to monoamine axon terminals, although no study has analyzed nigral and striatal cell bodies at the sub-cellular level. OBJECTIVE: In this study, we examined intrinsic nigral and striatal cells, and PC12 cell cultures to evaluate whether, in mice, MDMA might affect nigral and striatal cell bodies. METHODS: After administering MDMA, we analyzed effects induced in vivo and in vitro using high-performance liquid chromatography (HPLC) analysis, light- and electron microscopy with immunocytochemistry, and DNA comet assay. RESULTS: We found that MDMA (5 mg/kg x4, 2 h apart), besides a decrease of nigrostriatal DA innervation and 5HT loss, produces neuronal inclusions within nigral and intrinsic striatal neurons consisting of multi-layer ubiquitin-positive whorls extending to the nucleus of the cell. These fine morphological changes are associated with clustering of heat shock protein (HSP)-70 in the nucleus, very close to chromatin filaments. In the same experimental conditions, we could detect oxidation of DNA bases followed by DNA damage. The nature of inclusions was further investigated using PC12 cell cultures. CONCLUSIONS: The present findings lead to re-consideration of the neurotoxic consequences of MDMA administration. In fact, occurrence of ubiquitin-positive neuronal inclusions and DNA damage both in nigral and striatal cells sheds new light into the fine alterations induced by MDMA, also suggesting the involvement of nuclear and cytoplasmic components of the ubiquitin-proteasome pathway in MDMA toxicity.

Effects of liquid and freeze-dried grapefruit juice on the pharmacokinetics of praziquantel and its metabolite 4'-hydroxy praziquantel in beagle dogs.

Grapefruit juice changes the pharmacokinetic parameters of a variety of drugs metabolized primarily by cytochrome P450 3A. In a three-phase crossover study, six male beagle dogs were administered 100 ml of water (control), 100 ml of commercial liquid grapefruit juice, or 10 g of freeze-dried grapefruit juice (equivalent to 100 ml of liquid grapefruit juice) with 100 ml of water, followed after 2 h by single oral dose of praziquantel (30 mg kg(-1)). After treatment, the dogs were sampled at different times. Determination of praziquantel and its metabolite 4'-hydroxy praziquantel (identified by GC/MS) was performed by HPLC. Liquid and freeze-dried grapefruit juice preadministration increased the C(max) of praziquantel about three-fold and the AUC 2.5- and 2.3-fold, respectively. The T(max) (0.75 h) was unaffected by liquid or freeze-dried grapefruit juice, while T(1/2) was 2.3- and 1.7-fold higher compared with controls. The amount of 4'-hydroxy praziquantel was also affected by both liquid and freeze-dried grapefruit juice administration: the AUC and C(max) increased four- and three-fold, respectively and the T(max) was significantly enhanced. These findings demonstrate that both freeze-dried grapefruit juice and commercial liquid grapefruit juice significantly increase plasma concentrations and T(1/2) of praziquantel in dogs.

Pharmacokinetics and microsomal oxidation of praziquantel and its effects on the P450 system in three-month-old lambs infested by Fasciola hepatica.

Praziquantel (PZQ) is a broadly effective trematocide and cestocide, widely employed in veterinary and human medicine. In view of several differences in both its pharmacokinetic profile in different animal species and in the cytochrome P450-dependent system between ruminant and nonruminant species, the present study was undertaken to determine the pharmacokinetics of this drug, its effects on the P450 system and the involvement of cytochrome P450 in its metabolism in 3-month-old lambs infested by Fasciola hepatica. Following both oral and i.m. administration, PZQ disposition was best described by a linear one-compartment open model with a rapid absorption and elimination. Although the PZQ dose used by the i.m. route was only half of that used by the oral route, the mean PZQ plasma concentration was higher after i.m. than after oral treatment. Oral treatment with 30 mg/kg/day of PZQ did not modify the mono-oxygenase activities tested, whilst the administration of PZQ at a dose of 60 mg/kg/day for 2 days caused a significant decrease in the P450 3A-dependent erythromycin N-demethylase and 6beta testosterone hydroxylase activities. From the incubation of microsomes from lambs not treated with PZQ, a single metabolite (PZQ 11b-OH or PZQ 1-OH) was identified by GC/MS analysis. By selective inhibition of the 3A subfamily performed with triacetyloleandromycin, the production of this metabolite declined by about 90% suggesting a prominent role of P450 3A isoforms in this oxidation. These features indicate that agents or drugs which are able to modulate P450 3A-dependent catalysis may interfere with the metabolism, bioavailability and therapeutic effects of PZQ.

Comparison of HPLC and GC-MS methods for determination of embutramide (a component of Tanax or T-61) in biological specimens.

Tanax or T-61, a euthanasia solution commonly used in veterinary medicine, has been often involved in suicide attempts (humans) and malicious intoxications (animals). For forensic reasons, the identification of one or more of the three components (embutramide, mebenzonium iodide, and tetracaine hydrochloride) of Tanax is needed to confirm the hypothesis of intoxication. This study was performed with new high-performance liquid chromatographic and gas chromatographic-mass spectrometric methods to identify embutramide in biological matrices (blood, liver, kidney) from different animal species. The good sensitivity and specificity of both methods recommend their use in toxicological analysis in both human and veterinary medicine.

[Determination of opiates in urine: interpretation of comparison between the EIA and FPIA and confirmation of data with GC/MS]. / Determinazione degli oppiacei nelle urine: interpretazione del confronto tra EIA e FPIA e conferma dei dati con GC/MS.

BACKGROUND: The aim of this study was to evaluate some urine samples and to analyse them using two immunochemical screening methods. METHODS: A more reliable method has been used to obtain a critical analysis of the validity of the first analysis methods used. A total of 147 urine samples were examined using both the FPIA technique (Adx, Abbott) and the EIA technique (Random 120, Bracco); confirmation was obtained using GC/MS. RESULTS: When the threshold was altered from 200 to 300 ng/ml the EIA technique is more severely affected by changes in reference values compared to FPIA. In different values between two techniques, confirmation with GC/MS was possible only 5 cases. CONCLUSIONS: The study of data processing with immunochemical tests and after confirmation showed that the results with GC/MS are closer to Adx. The FPIA results are in agreement with the GC/MS technique both at 200 and 300 ng/ml with a percentage of 80.0%. The EIA technique has a different result, at 200 ng/ml it is in agreement for 40.0%, but at 300 ng/ml there is only 20.0%.

Effects of pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on methamphetamine pharmacokinetics and striatal dopamine losses.

We recently demonstrated that pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) exacerbates experimental parkinsonism induced by methamphetamine. The mechanism responsible for this effect remains to be elucidated. In this study, we investigated whether the exacerbation of chronic dopamine loss in DSP-4-pretreated animals is due to an impairment in the recovery of dopamine levels once the neurotoxic insult is generated or to an increased efficacy of the effects induced by methamphetamine. We administered different doses of methamphetamine either to DSP-4-pretreated or to intact Swiss-Webster mice and evaluated the methamphetamine-induced striatal dopamine loss at early and prolonged intervals. As a further step, we evaluated the striatal pharmacokinetics of methamphetamine, together with its early biochemical effects. We found that previous damage to norepinephrine terminals produced by DSP-4 did not modify the recovery of striatal dopamine levels occurring during several weeks after methamphetamine. By contrast, pretreatment with DSP-4 exacerbated early biochemical effects of methamphetamine, which were already detectable 1 h after methamphetamine administration. In addition, in norepinephrine-depleted animals, the clearance of striatal methamphetamine is prolonged, although the striatal concentration peak observed at 1 h is unmodified. These findings, together with the lack of a methamphetamine enhancement when DSP-4 was injected 12 h after methamphetamine administration, suggest that in norepinephrine-depleted animals, a more pronounced acute neuronal sensitivity to methamphetamine occurs.

A modification of the soxhlet apparatus for drug extraction from biological fluids.

The authors suggest a method to isolate drugs and toxic substances from biological fluids which appears to be of easier and better applicability than other systems, because it allows the extraction by means of a hot solvent, reduces manual operations and the waste of solvent, and shortens the time of execution for the researchers. A mathematical formula is proposed in order to evaluate the recovery-time curves of any drug shortly and with better accuracy.

High-performance liquid chromatography for the quantitative determination of the urinary metabolites of toluene, xylene, and styrene.

A new high-pressure liquid chromatography (HPLC) method for simultaneous quantitative determination of the urinary metabolites of toluene, m-xylene, and styrene (hippuric acid, m-methylhippuric acid, phenylglyoxylic acid, mandelic acid) is described. The extraction procedure was performed on acidified urines, after addition of 4-hydroxybenzoic acid as internal standard, using a butylchloride/isopropanol mixture and drying 0.5 ml of the organic layer under nitrogen flow. The residue obtained was dissolved in 0.1 ml water/acetonitrile and 5 microliters were injected into an HPLC apparatus equipped with a 0.26 X 25 cm HC ODS SIL X column. Absorbance measures were performed at 225 nm throughout the investigation. All metabolites were clearly separated in a short time (12 min) and the amounts of other urinary compounds affecting the analysis were so small that the measurement of low concentrations of the urinary metabolites could be easily performed. Linear calibration curves were obtained from 0.1 to 3 mg/ml and a correlation coefficient greater than 0.99 was found between concentrations of the standards and areas of the peaks. Statistical analysis confirms that this method, which has a high reproducibility, is simple, reliable, and useful for the biologic monitoring of industrial exposure to aromatic compounds.

Distribution and fate of alpha-naphthl-isothiocyanate (ANIT) in the organs and body fluids of the rat.

alpha-Naphthyl-isothiocyanate (ANIT) is a well known toxic substance which induces characteristic hepatic lesions. Its distribution in some organs and body fluids was investigated by using spectrophotometry, gas-chromatography and thin-layer chromatography. The results indicate that ANIT concentrates in the liver, kidneys and blood but not in the brain, urine and bile. Another five ANIT-like substances have also been examined, but no trace of them has been found in these organs and body fluids. The authors suppose that ANIT is metabolized to an unknown metabolite which is responsible for the toxic action of ANIT. None of the ANIT-like substances examined by us can be indentified with this metabolite.