Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits intracellular serine/threonine protein phosphatases causing disruption of actin microfilaments (MFs) and intermediate filaments (IFs) in hepatocytes. This study compared the effects of MCLR on the organization of MFs, IFs, and microtubules (MTs) in hepatocytes and nonhepatocyte cell lines and determined the sequence of toxin-induced changes in these cytoskeletal components. Rat renal epithelial cells and fibroblasts were incubated with MCLR at 100 or 200 microM for 6-18 hr. Rat hepatocytes in primary culture were exposed to the toxin at 1 or 10 microM for 2-64 min. Cells were fixed and incubated with primary antibodies against beta-tubulin, actin, and vimentin or cytokeratin IFs, followed by gold-labeled secondary antibodies with silver enhancement of the gold probe. The fraction of fibroblasts and hepatocytes with altered cytoskeletal morphology was evaluated as a function of MCLR dose and exposure time to assess the sequence of changes in cytoskeletal components. Changes in fibroblasts and some hepatocytes were characterized initially by disorganization of IFs, followed rapidly by disorganization of MTs, with the progressive collapse of both cytoskeletal components around cell nuclei. Many hepatocytes exhibited MT changes prior to effects on IF structure. Alterations in MFs occurred later and included initial aggregation of actin under the plasma membrane, followed by condensation into rosette-like structures and eventual complete collapse into a dense perinuclear bundle. The similarity of effects among different cell types suggests a common mechanism of action, but the independent kinetics of IF and MT disruption in hepatocytes suggests that there may be at least 2 sites of phosphorylation that lead to cytoskeletal alterations.

The presence of 6-monoacetylmorphine (6-MAM) is often used to distinguish between heroin (diacetylmorphine) and morphine exposures. 6-MAM, however, is rapidly metabolized to morphine and may not be present in detectable quantities in blood following heroin exposure. Recent studies have shown that 6-MAM may persist in cerebrospinal fluid (CSF) and this specimen may be preferable for establishing heroin exposure. This study reports postmortem distribution of 6-MAM, unconjugated morphine, and codeine in different tissues from 25 deceased individuals. In all cases, 6-MAM was detected in vitreous humor, and in CSF in 16 of the 25 cases (64%). When 6-MAM was detected in blood (13 of 25 cases), the level of 6-MAM in vitreous humor and CSF was higher than in blood, with a mean concentration ratio of 11.3 (range: 1.7-27) for vitreous humor and 6.6 (range: 2.6-17.3) for CSF. 6-MAM was not detected in liver in any of the cases examined. Free morphine levels were highest in liver, followed by blood, CSF, and vitreous humor. The concentration ratios (mean +/- standard deviation) for free morphine in vitreous humor, CSF, and liver to that in blood were 0.36 +/- 0.18, 0.64 +/- 0.27, and 2.99 +/- 2.12, respectively. The liver/blood ratio was consistent with previously reported values for morphine in heart and femoral blood. Codeine levels following heroin overdose were consistently low relative to the morphine concentration. For blood, liver, and CSF, the ratio of codeine to morphine was essentially the same (0.06), whereas the vitreous codeine/morphine concentration ratio was slightly higher (0.19). These results characterize the distribution of heroin metabolites in postmortem tissues. Vitreous humor appears to be a useful specimen for determining 6-MAM and establishing the morphine was derived from heroin.

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive, synthetic analog of the central nervous system stimulant cathinone. Its recent popularity as a recreational drug in the United States has led to numerous reports to poison control centers across the country. As with other synthetic cathinones, the recreational use of MDPV has resulted in death. MDPV is thought to exert its pharmacologic effects by inhibiting the reuptake of dopamine and norepinephrine. This report describes the case of an exposure of a 39-year-old male to MDPV, which resulted in his death. Postmortem concentrations of MDPV in various tissues were measured. The detection of MDPV in tissues and fluids was accomplished using gas chromatography-mass spectrometry analysis after solid-phase extraction. Blood analysis also demonstrated therapeutic levels of lamotrigine, fluoxetine, risperidone, benztropine, pseudoephedrine and ibuprofen. The detection of cathinones in hair was conducted using high-performance liquid chromatography-tandem mass spectrometry after solid-phase extraction. MDPV was uniformly distributed among multiple tissues (blood, brain, muscle, cerebrospinal fluid and lung) at concentrations of approximately 0.4 to 0.6 µg/mL. Tissue and fluids responsible for detoxification/excretion had higher concentrations of MDPV (kidney, liver and bile > 0.8 µg/mL). A blood concentration ≥ 0.4 µg/mL was judged sufficient to cause death. The cause of death was ruled MDPV intoxication resulting in cardiac arrhythmia.

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Skeletal remains of a domestic pig were assessed for relative distribution of amitriptyline, citalopram, and metabolites. Following acute exposure and outdoor decomposition for 2 years, drugs and metabolites were analyzed in 13 different bones. Bones were pulverized following a simple wash procedure, and drugs were extracted by passive incubation in methanol, followed by solid-phase extraction. Samples were analyzed by ultra-high performance liquid chromatography (UHPLC) and confirmed with gas chromatography-mass spectrometry. The Kruskall-Wallis test showed that bone type was a main effect with respect to drug level for all analytes, with levels varying from 33- to 166-fold. Ratios of levels of drug to that of the corresponding metabolite were less variable, varying roughly one- to eightfold. This suggests limitations in the interpretive value of drug measurements in bone and that relative levels of drug and metabolites should be further investigated in terms of forensic value.

Skeletal tissues from a domestic pig exposed to amitriptyline, diazepam, and pentobarbital were analyzed to determine the relative distribution of these drugs in bone. Following drug exposure and euthanasia, remains were allowed to decompose outdoors to complete skeletonization between summer 2007 and fall 2009. Remains were recovered and separated according to bone type. Twelve different bone types were pulverized and sampled in triplicate. Each bone sample underwent methanolic extraction (96 h, 50 °C), followed by solid-phase extraction and gas chromatography-mass spectrometry in the selected ion monitoring mode. Mass-normalized assay responses underwent ANOVA with post-hoc testing, revealing bone type as a main effect for all three drugs, but not for the diazepam metabolite (nordiazepam). The assay response varied with respect to bone type by factors of 27, 39, and 20 for pentobarbital, diazepam, and amitriptyline, respectively. The relative distribution between bone type was qualitatively similar for the three administered drugs analyzed for, with the largest response obtained from rib for all three drugs. This is the first study, to the authors' knowledge, of the distribution of different drugs in various decomposed skeletal tissues in a controlled experiment using an animal model of comparable physiology to humans. These data have implications for the interpretive value of forensic drug measurements in skeletal tissues.

Drug levels in decomposed individuals are difficult to interpret. Concentrations of 16 drugs were monitored in tissues (blood, brain, liver, kidney, muscle, and soil) from decomposing pigs for 1 week. Pigs were divided into groups (n = 5) with each group receiving four drugs. Drug cocktails were prepared from pharmaceutical formulations. Intracardiac pentobarbital sacrifice was 4 h after dosing, with tissue collection at 4, 24, 48, 96, and 168 h postdosing. Samples were frozen until assay. Detection and quantitation of drugs were through solid phase extraction followed by gas chromatograph/mass spectrometer analysis. Brain and kidneys were not available after 48 h; liver and muscle persisted for 1 week. Concentration of drugs increased during decomposition. During 1 week of decomposition, muscle showed average levels increasing but concentrations in liver were increased many fold, compared to muscle. Attempting to interpret drug levels in decomposed bodies may lead to incorrect conclusions about cause and manner of death.