The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison.

2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here, we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (TOP2B) covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide, which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination since both occur even under conditions of depleted ubiquitin. Like etoposide, since PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.

The general toxicology unknown. II. A case report: doxylamine and pyrilamine intoxication.

A general toxicology unknown case is presented to demonstrate our systematic approach. A 20-year-old male was found dead with multiple suicide notes. Overdose was suspected but substances were not known. Blood alcohol was negative. Urine was analyzed by enzyme-multiplied immunoassay technique and was negative for all drugs assayed. Urine was then extracted with ethyl acetate:hexane (1:1) at pH 10 and back-extracted into 1.0N sulfuric acid. The acidic layer was adjusted to pH 10, and re-extracted with ethyl acetate:hexane (1:1). The residue was analyzed by gas chromatography (GC) on a 3% OV-101 column. It was found to be negative for all commonly screened substances. However, several unknown peaks were observed. Electron impact mass spectra of these unknown peaks were obtained and searched for in our computer library of more than 25000 mass spectra. These unknown peaks were identified as doxylamine and pyrilamine by gas chromatography/mass spectrometry. The base peak and molecular ion for pyrilamine were at m/z 121 and 285, respectively. The base peak for doxylamine was at m/z 58. No molecular ion was observed for doxylamine. Both doxylamine and pyrilamine are antihistamines, but are promoted and used in the management of insomnia. Quantitation was performed on a GC using dexbrompheniramine as an internal standard. Blood concentrations for doxylamine and pyrilamine were 0.7 and 7.0 mg/L, respectively. Concentrations in other tissues were determined. Death was caused by combined doxylamine and pyrilamine intoxication; the manner of death was suicide.

A girl with headache, confusion and green urine.

The case of a 17-year-old girl with a history of headache, blurred vision, confusion, ataxia and syncope is presented. On admission, she had already recovered except for a slurring of speech. Her urine was found to be green. Screening for illegal drugs was negative, but gas chromatography with subsequent mass spectroscopy (GC-MS) revealed an extremely high concentration of flupirtine.

Nephrotoxicity of phenazopyridine.

A young man developed reversible acute renal failure after a large overdose of phenazopyridine. The renal failure was treated by peritoneal dialysis. Analysis of blood and urine samples failed to demonstrate the parent drug but a metabolite with similar ultraviolet absorption was detected. No parent drug or metabolites were detected in the peritoneal dialysate.

Methemoglobinemia from overdose of phenazopyridine hydrochloride.

A case of severe methemoglobinemia and hemolytic anemia following ingestion of 5.6 gm of phenazopyridine with suicidal intent by a 16-year-old woman was successfully treated with intravenous methylene blue. Methemoglobinemia was suspected in this patient because of her intense central cyanosis, chocolate-colored blood, and minimal dyspnea. The patient's peripheral blood smear exhibited "bite cells." To the best of our knowledge, this is only the third such case reported in the literature. The pathophysiology, diagnosis, and therapy are reviewed.

Poisoning with 4-aminopyridine: report of three cases.

Four-aminopyridine is an acutely toxic avicide considered by the manufacturer to be a bird "repellant" because only a small number of birds are acutely poisoned, become disoriented, and emit a distress cry frightening other members of the flock. Four-aminopyridine dramatically enhances transmission at the neuromuscular junction and other synapses, and has been employed clinically in the treatment of prolonged paralysis caused by antibiotics and muscle relaxants, and in the Eaton-Lambert syndrome. In this paper we report the results of an acute poisoning misadventure in three adult males. We review the animal toxicology, summarize the neurophysiological research using 4-AP as a potassium channel blocker, comment on clinical applications, and outline the management of overdose with this agent.

Treatment of methapyrilene toxicity with physostigmine.

Methypyrilene, 40 mg/kg, was administered to male mongrel dogs, resulting in both central and peripheral anticholinergic effects. Physostigmine in doses of 0.5 to 1.0 mg was effective in reversing the central effects completely and the peripheral effects to a limited degree.

Methemoglobinemia, muscle damage and renal failure complicating phenazopyridine overdose.

A 19-year-old woman with no history of renal or hematologic dysfunction developed methemoglobinemia, muscle damage, rhabdomyolysis and severe acute renal failure after an overdose of phenazopyridine hydrochloride (pyridium). The methemoglobinemia reversed after 24 h, but the patient remained oliguric, with myalgia and increased muscle enzymes. The patient received supportive treatment and recovered completely after 3 weeks. Since phenazopyridine is a commonly used drug, the physician should be alert to these possible severe complications.

Acute poisoning from over-the-counter sleep preparations.

All cases received by the Rocky Mountain Poison Center involving over-the-counter (OTC) sleep preparations were studied during an 18-month period to elucidate 1) the range of toxicity; 2) characteristic symptoms, and 3) the time of onset of symptoms. In 155 cases reviewed retrospectively, the three most commonly ingested agents were Sominex, Nytol and Sleepeze. Multiple ingestions were also involved. Symptomatology was equally divided among no symptoms, mild symptoms and possible life-threatening symptoms. The least amount taken to produce possible life-threatening symptoms was 16 Sominex, 18 Nytol and 15 Sleepeze, although the average amount producing the same symptoms was approximately twice that. These symptoms were seen within six hours in all but three of the 39 cases presenting with these symptoms. There were no deaths.

Paradoxical cerebral cortical hyperexcitability following flupirtine overdose.

We report the case of a patient with increased cerebral cortical excitability following intoxication with flupirtine, a centrally acting analgesic and antispastic drug. Typically, flupirtine exerts membrane stabilizing and hyperpolarizing effects through activation of neuronal G-protein regulated inwardly rectifying potassium channels (GIRK). Based on these properties, GIRK activators have been suggested as candidates for antiepileptic drug development. In contrast, our observation suggests that these substances may also display unexpected proconvulsant effects in vivo.