John F. Wyman

Alterations in Microtubules, Intermediate Filaments, and Microfilaments Induced by Microcystin-LR in Cultured Cells

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 μM for 6-18 hr. Rat hepatocytes in primary culture were exposed to the toxin at 1 or 10 μm for 2-64 min. Cells were fixed and incubated with primary antibodies against β-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.

Postmortem Tissue Distribution of MDPV Following Lethal Intoxication by "Bath Salts"*

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.

Blood pressure and hepatocellular effects of the cyclic heptapeptide toxin produced by the freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa strain PCC-7820

Laboratory rats and mice were used to investigate the hepatotoxicity caused by the cyclic heptapeptide (mol. wt 994) termed microcystin-LR. Microcystin-LR (also known as cyanoginosin-LR) is produced by the freshwater cyanobacterium (blue-green alga) M. aeruginosa strain PCC-7820. In time course histopathology studies with mice significant liver damage, with an absence of pulmonary emboli, were observed after 15 min. Pulmonary emboli did not appear until 1 hr. In rats, significant liver damage and the presence of occasional emboli were observed at 20 min. Pulmonary emboli did not contain fibrin nor appear life-threatening in any case and resembled the globular eosinophilic debris found in the liver sinusoids and central veins. Measurements of rat femoral arterial, jugular venous and hepatic portal venous blood pressures during the course of toxicity revealed a slowly declining arterial pressure and stable, normal venous pressures. Blood lactic acid levels rose in parallel with the fall in arterial pressure, a pattern typical of hemorrhagic shock. There was no indication of venous congestion that would accompany right heart failure. Isolated, perfused rat livers dosed with toxin showed rapid changes in the liver, including cessation of bile flow within 10 min and complete obliteration of normal lobular architecture within 60 min. No effect of the toxin was observed in isolated perfused rat heart. We conclude that in the mouse and rat, microcystin-LR is a potent, rapid-acting, direct hepatotoxin, with the immediate cause of death in acute toxicities being hemorrhagic shock secondary to massive hepatocellular necrosis and collapse of hepatic parenchyma.

Isolated rat liver perfusion studies with cyclic heptapeptide toxins of Microcystis and Oscillatoria (freshwater cyanobacteria)

Isolated perfused rat livers were used to study the dose-dependent effects of three cyclic heptapeptide toxins isolated from Norwegian freshwater bloom samples containing Microcystis aeruginosa, Oscillatoria agardhii var. and Oscillatoria agardhii var. isothrix. The high pressure liquid chromatography (HPLC) purified toxins had an i.p. LD50 in the rat and mouse of approximately 50, 500 and 1000 micrograms/kg, respectively. Hepatic insult of the toxins at concentrations of 0.5-4.0 times the rat i.p. lethal dose were assessed by monitoring bile flow, accumulation of total protein in the perfusate, release of intracellular enzymes and histopathologic examination of perfused liver tissue. One hundred micrograms of Microcystis toxin produced cessation of bile flow during a 1 hr perfusion period, while the two Oscillatoria toxins required 1000 and 2000 micrograms of toxin, consistent with their lower LD50 values. Hepatic cell membranes remained intact during the perfusion since release of enzymes and proteins into the perfusate was similar for toxin treated and control livers, and histopathologic examination of Trypan Blue infused livers revealed exclusion of the dye from the intracellular compartment of the parenchyma. Histopathologic findings for all three toxins showed hepatocellular disassociation that increased with toxin concentration. At the ultrastructural level, all three toxins caused dose-dependent vesiculation of rough endoplasmic reticulum, formation of concentric whorls composed of rough-ER, mitochondrial swelling, large cytoplasmic vacuoles and altered bile canaliculi. These changes were similar to those found for previous in vivo studies using Microcystis cyclic heptapeptides from Scotland and Australia. The Oscillatoria toxins required five to ten times more toxin to produce similar effects as the Microcystis toxin. At the higher concentrations, the Oscillatoria toxins also caused a proliferation of smooth-ER. The isolated perfused rat liver was found to be a good model for studying the hepatocellular effects of different cyclic peptide toxins from cyanobacteria.

High Temperature Decomposition of Military Specification L-23699 Synthetic Aircraft Lubricants:

Previous investigations reported the formation of a neurotoxicant, tri methylolpropane phosphate (TMP-P) during pyrolysis of synthetic, aircraft engine lubricating oils. Employing both sealed-tube and open-to-air methods of pyrolysis, we confirmed the formation of TMP-P during lubricant pyrolysis. Rodent bioassay and GC/MS analysis were performed for detection and quanti tation of neurotoxicant production. Maximal production of TMP-P for sealed- tube pyrolysis occured after 20 min at 500°C. A comparable amount of TMP-P was formed after 5 min at 650°C in open-to-air pyrolysis of lubricants. Our examination of TMP-P production was limited to L-23699 lubricants composed primarily of a trimethylolpropane (TMP) ester base stock. The formation of TMP-P was only dependent upon the presence of an organic phosphorous addi tive and TMP. Pyrolysed lubricants composed of a pentaerythritol base did not cause neurotoxicity in mice or rats following intraperitoneal (i.p.) injection. Other toxic bicyclophosphorous ester homologues were not detected in any pyrolyzed lubricants following GC/MS analysis. L-23699 lubricants which had been operationally employed in aircraft engines under rigorous test conditions, produced no neurotoxicity when intraperitoneally administered to rats.

The Temporal Fate of Drugs in Decomposing Porcine Tissue

Abstract:  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.

Sequential ultrastructural and biochemical changes induced by microcystin-LR in isolated perfused rat livers

The cyanobacterial hepatotoxin, microcystin-LR (MCLR), is a potent protein phosphatase inhibitor that disrupts actin microfilament, cytokeratin intermediate filament, and microtubule networks in hepatocytes. To determine ultrastructural and biochemical changes that develop concurrently with microcystin-induced cytoskeletal disorganization, isolated rat livers were perfused with MCLR at 0.1 to 5.0 micrograms/ml for 5 to 40 min. Lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase changed over time, but trends for toxin-treated and control livers did not differ. The earliest toxin-induced ultrastructural changes, observed in livers perfused at 0.1 microgram/ml for 15-20 min or at 0.3 microgram/ml for 5-10 min, were loss of hepatocyte microvilli in the space of Disse, widening of sinusoidal fenestrae, disruption of sinusoidal endothelium, dilation of bile canaliculi with loss of microvilli, and widening of hepatocyte intercellular spaces. Lesions progressed with increasing toxin concentrations and exposure times. In livers perfused with MCLR at 0.5 microgram/ml for 10-20 min, hepatocytes had plasma membrane blebs and concentric whorls of rough endoplasmic reticulum, and there was marked disassociation of hepatocytes resulting in disrupted hepatic cords. At toxin concentrations of 2.0 or 5.0 micrograms/ml for 10-20 min, there was mild dilation of mitochondrial cristae, cytoplasmic vacuolization or invagination of plasma membranes, redistribution of organelles, and sometimes nuclear degenerative change. Some hepatocytes exhibited clusters of plasma membrane blebs radiating from round cytoplasmic structures, which may be composed primarily of condensed microfilaments.

EVALUATION OF SHIPBOARD FORMATION OF A NEUROTOXICANT (TRIMETHYLOLPROPANE PHOSPHATE) FROM THERMAL DECOMPOSITION OF SYNTHETIC AIRCRAFT ENGINE LUBRICANT

MIL-L-23699 lubricants that are composed principally of trimethylolpropane triheptanoate (TMP) and tricresyl phosphate (TCP) have been shown to form a neurotoxicant, trimethylolpropane phosphate (TMPP), during pyrolysis and/or combustion. Mechanistically, TMPP is thought to irreversibly inhibit the GABA-mediated inhibitory response and thereby produce epileptiform clonic/tonic seizures with convulsions followed by death. Thermal decomposition of MIL-L-23699 lubricant produces TMPP under laboratory conditions, but this product has not been detected in the workplace following actual fires. This study has examined whether TMPP is produced during an actual shipboard fire by placing the synthetic lubricant in a fire environment aboard the ex-U.S.S. Shadwell, Mobile, Alabama. Both biological and chemical analyses were performed on the thermally decomposed lubricant to ensure detection of the neurotoxic material. Under the conditions of this study, the formation of TMPP during a shipboard fire was confirmed. The implications of this finding for safe management of post-fire cleanup are discussed.

Effect ofpropylene glycol 1,2-dinitrate on cerebral blood flow in rats: a potential biomarker for vascular headache?

Two measurable indices of toxicity that can be correlated with exposure to propylene glycol dinitrate (PGDN) were evaluated along with its metabolism. Propylene glycol dinitrate was administered by rapid i.v. injection to male Fischer-344 rats. These rats demonstrated a dose-response of blood pressure (BP) to doses of PGDN ranging from 0.1 to 30 mg/kg; the maximum fall in systolic BP occurred within 1 min of dosing. The i.v. administration of PGDN to separate groups of animals resulted in an increase in cerebral blood flow that was correlated with the dose, but a clear dose-response was not obtained.

The Determination of the Repeated Oral Toxicity of Halocarbon Oil, Series 27-S

Halocarbon 27-S (HC 27-S), a polymer of chlorotrifluoroethylene (CTFE), is used as a lubricating oil for pumps in hyperbaric chambers. Although monomeric CTFE has been shown to produce renal lesions in rats, the toxicity of CTFE polymers have not been investigated. To assess the toxicity of repeated exposure to HC 27–S, three groups (N = 6/group) of male and female Fischer-344 rats were dosed with 2.5 g HC 27-S/kg for 7 or 21 consecutive days. Groups were sacrificed at 7, 21, and 35 days (14 days after the 21-day dosing). Corresponding control groups (N = 6) were dosed with deionized water. Decreased water consumption and urine output were apparent in all test groups. Statistically significant increases in fluoride excretion were noted in 24-hr urine samples assessed periodically during the study. Neurotoxic signs were observed in female rats but not in male rats. Significant increases in liver and kidney weights were seen in all rats, regardless of number of dosing days. The increased fluoride burden in treated animals appeared sufficient to alter bone calcium/phosphate ratios in male rats but not female rats. Gross liver enlargement and hepatocellular cytomegaly indicated that the liver was probably the primary target organ following repeated administration of HC 27-S. (Supported by US Navy through Air Force Contract # F33615-085-C-0532.)