Detection of the designer benzodiazepine metizolam in urine and preliminary data on its metabolism.

Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available via the Internet without control. Metizolam was ordered via the Internet and a 2 mg blue tablet was orally administered to a 54-year-old man. Urine samples were collected over 6 days in polypropylene tubes. After liquid/liquid extraction at pH 9.5, metizolam was analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) using a standard method devoted to benzodiazepines, and ions transitions, at m/z 328.9 > 275.0 and 328.9 > 300.0. Metizolam was detectable in hydrolyzed urine during the 46-h period, with concentrations always lower than 11 ng/mL. About 0.3% of the initial dose was excreted in urines as total unchanged metizolam during the first 24 h. The most relevant potential CYP- and UGT-dependent metabolites of metizolam were investigated in vitro using human liver microsome incubation and, subsequently, liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. Three mono-hydroxylated metabolites were produced including a hydroxylation compound at the 2-ethyl moiety of metizolam (M1) as quantitatively main metabolite, and a N-hydroxymetiazolam (M2). The structure of the third metabolite (M3) could not be elucidated because of a too low experimental production rate. Two authentic urine samples were analyzed using the same analytical method to search for metabolites of metizolam. M1, together with its glucuronide (M1-Glu), and M2 were observed in urine at the 8 h mark, whereas only M1 and M1-Glu were still detected in urine at 30 h post administration. Copyright © 2016 John Wiley & Sons, Ltd.

PKCε contributes to chronic ethanol-induced steatosis in mice but not inflammation and necrosis.

BACKGROUND: Protein kinase C epsilon (PKCε) has been shown to play a role in experimental steatosis by acute alcohol. The "two-hit" hypothesis implies that preventing steatosis should blunt more advanced liver damage (e.g., inflammation and necrosis). However, the role of PKCε in these pathologies is not yet known. The goal of this current work was to address this question in a model of chronic alcohol exposure using antisense oligonucleotides (ASO) against PKCε. METHODS: Accordingly, PKCε ASO- and saline-treated mice were fed high-fat control or ethanol (EtOH)-containing enteral diets for 4 weeks. RESULTS: Chronic EtOH exposure significantly elevated hepatic lipid pools as well as activated PKCε. The PKCε ASO partially blunted the increases in hepatic lipids caused by EtOH. Administration of PKCε ASO also completely prevented the increase in the expression of fatty acid synthase, and tumor necrosis factor α caused by EtOH. Despite these protective effects, the PKCε ASO was unable to prevent the increases in inflammation and necrosis caused by chronic EtOH. These latter results correlated with an inability of the PKCε ASO to blunt the up-regulation of plasminogen activator inhibitor-1 (PAI-1) and the accumulation of fibrin. Importantly, PAI-1 has been previously shown to more robustly mediate inflammation and necrosis (vs. steatosis) after chronic EtOH exposure. CONCLUSIONS: This study identifies a novel potential mechanism where EtOH, independent of steatosis, can contribute to liver damage. These results also suggest that PAI-1 and fibrin accumulation may be at the center of this PKCε-independent pathway.

Fatal diving accidents in western Norway 1983-2007.

Despite efforts to reduce their number, fatal diving accidents still occur. The circumstances and post-mortem findings in 40 fatal diving accidents in western Norway from 1983 through 2007 were investigated. Diving experience, medical history and toxicology reports were retrieved. The material consisted of recreational divers, professional saturation divers and professional divers without experience with saturation. In 33 cases the diving equipment was examined as part of the forensic investigation. In 27 cases defects in the diving equipment were found. For six divers such defects were responsible for the fatal accidents. Eighteen divers died on the surface or less than 10 m below surface. Five divers reached below 100 msw, and two of them died at this depth. The fatalities were not season-dependent. However, wave-height and strength of currents were influential factors in some cases. Twelve divers were diving alone. Twenty divers had one buddy, 9 of these divers were alone at the time of death. The cause of death was drowning in 31 out of 40 divers; one of them had a high blood-ethanol concentration, in two other divers ethanol was found in the urine, indicating previous ethanol consumption. Nine divers died from sudden decompression, pulmonary barotraumas, underwater trauma and natural causes. The study shows that most of the fatal diving accidents could be avoided if adequate diving safety procedures had been followed.

Disposition and metabolism of cumene in F344 rats and B6C3F1 mice.

Cumene is a high-production volume chemical that has been shown to be a central nervous system depressant and has been implicated as a long-term exposure carcinogen in experimental animals. The absorption, distribution, metabolism, and excretion of [(14)C]cumene (isopropylbenzene) was studied in male rats and mice of both sexes after oral or intravenous administration. In both species and sexes, urine accounted for the majority of the excretion (typically ≥ 70%) by oral and intravenous administration. Enterohepatic circulation of cumene and/or its metabolites was indicated because 37% of the total dose was excreted in bile in bile duct-cannulated rats with little excreted in normal rats. The highest tissue (14)C levels in rats were observed in adipose tissue, liver, and kidney with no accumulation observed after repeat dosing up to 7 days. In contrast, mice contained the highest concentrations of (14)C at 24 h after dosing in the liver, kidney, and lung, with repeat dosing accumulation of (14)C observed in these tissues as well as in the blood, brain, heart, muscle, and spleen. The metabolites in the expired air, urine, bile, and microsomes were characterized with 16 metabolites identified. The volatile organics in the expired air comprised mainly cumene and up to 4% α-methylstyrene. The major urinary and biliary metabolite was 2-phenyl-2-propanol glucuronide, which corresponded with the main microsomal metabolite being 2-phenyl-2-propanol.

An avoidable death in police custody?

An obviously inebriated 36-year-old man was found in a helpless condition on a pavement. When he was brought to a police station, a doctor certified the individual to be fit to be held in custody. He was unresponsive when the officers tried to wake him the next morning but was allowed to sleep on until he was found dead in his cell at mid day. An autopsy revealed as cause of death an epidural hematoma and cerebral contusion with fracture of the calvarium. The expert report stressed the point that the monitoring of the state of consciousness had been objectively insufficient throughout the entire time period, both in terms of frequency as well as in terms of the monitoring methods. However, neither the police officers involved nor the doctor, was subjected to prosecution mainly because of the high burden of proof required by German criminal law. The article will discuss conclusions for the avoidance of such deaths (such as training of police officers, legal regulations on monitoring procedures, etc).

Effect of short-term ethanol administration on cadmium retention and bioelement metabolism in rats continuously exposed to cadmium.

The present study was performed to assess the effect of short-term ethanol administration on cadmium retention and accumulation as well as on bioelement metabolism (zinc, copper, calcium, and magnesium) in rats exposed to an aqueous solution of cadmium chloride for 8 weeks. Intoxication with cadmium led to accumulation of this toxic metal, particularly in the liver and kidney, which was linked to metallothionein synthesis as well as to a disturbance in the metabolism of zinc, copper, and calcium. These effects were dependent on the level of exposure. The administration of ethanol in the final phase of cadmium treatment increased cadmium retention and accumulation in the body with simultaneous elevation in liver and kidney metallothionein concentration. Ethanol alone or with cadmium caused or intensified the cadmium-induced changes in metabolism of zinc and copper. Calcium metabolism disturbed by cadmium was not influenced by ethanol. Neither agents had any effect on magnesium metabolism. We conclude that even short-term ethanol consumption in conditions of exposure to cadmium can increase this heavy metal body burden and lead to more serious disturbances in metabolism of important elements such as zinc and copper. Cadmium- and ethanol-induced changes in the homeostasis of these microelements are probably connected with the ability of both xenobiotics to cause metallothionein induction.

Mechanism of the alcohol cyclic pattern: role of the hypothalamic-pituitary-thyroid axis.

The cause of the cycle of urinary alcohol levels (UALs) in rats fed ethanol continually at a fixed rate is unknown. Rats were fed ethanol intragastrically at a constant dose for 2 mo, and daily body temperatures and UALs were recorded. Body temperature cycled inversely to UAL, suggesting that the rate of metabolism could be mechanistically involved in the rate of ethanol elimination during the cycle. To document this, whole body O(2) consumption rate was monitored daily during the cycle. The rate of O(2) consumption correlated positively with the change in body temperature and negatively with the change in UAL. Since the metabolic rate responds to changes in body temperature, thyroid hormone levels were measured during the UAL cycle. T(4) levels correlated positively with the O(2) consumption rate and negatively with the UALs. In a second experiment using propylthiouracil treatment, UALs did not cycle and a fall in body temperature failed to stimulate an increase in the rate of ethanol elimination. Consequently, rats died of overdose. Likewise, in a third experiment using rats with severed pituitary stalks, UALs failed to cycle and rats died of overdose. From these observations it was concluded that the UAL cycle depends on an intact hypothalamic-pituitary-thyroid axis response to the ethanol-induced drop in body temperature by increasing the rate of ethanol elimination.

Allopurinol prevents early alcohol-induced liver injury in rats.

Free radical formation caused by chronic ethanol administration could activate transcription factors such as nuclear factor-kappaB (NF-kappaB), which regulates production of inflammatory cytokines. Xanthine oxidase is one potential source of reactive oxygen species. Therefore, the purpose of this study is to determine whether allopurinol, a xanthine oxidase inhibitor and scavenger of free radicals, would affect free radical formation, NF-kappaB activation, and early alcohol-induced liver injury in rats. Male Wistar rats were fed a high-fat diet with or without ethanol (10-16 g/kg/day) continuously for up to 4 weeks with the Tsukamoto-French enteral protocol. Either allopurinol or saline vehicle was administered daily. Allopurinol had no effect on body weight or the cyclic pattern of ethanol in urine. Mean urine ethanol concentrations were 271 +/- 38 and 252 +/- 33 mg/dl in ethanol- and ethanol + allopurinol-treated rats, respectively. In the control group, serum aspartate aminotransferase and alanine aminotransferase levels were approximately 40 I.U./l and 25 U/l, respectively. Administration of enteral ethanol for 4 weeks increased serum transaminases approximately 5-fold. Allopurinol blunted these increases significantly by approximately 50%. Ethanol treatment also caused severe fatty infiltration, mild inflammation, and necrosis. These pathological changes also were blunted significantly by allopurinol. Furthermore, enteral ethanol caused free radical adduct formation, values that were reduced by approximately 40% by allopurinol. NF-kappaB binding was minimal in the control group but was increased significantly nearly 2.5-fold by ethanol. This increase was blunted to similar values as control by allopurinol. These results indicate that allopurinol prevents early alcohol-induced liver injury, most likely by preventing oxidant-dependent activation of NF-kappaB.

Effects of alcohol and nicotine on developing olfactory bulb: loss of mitral cells and alterations in neurotransmitter levels.

Previous research from our laboratory has shown that [ethanol (EtOH)] exposure during the brain growth spurt is detrimental to olfactory bulb development. This study extends those findings by examining the effects of EtOH, nicotine (NIC), and the combination of these drugs (EtOH/NIC) on olfactory bulb mitral cell numbers, as well as on various major neurotransmitter levels in neonatal rats. An artificial rearing paradigm was used in the present studies. These artificially reared pups were given 4 g/kg/day of EtOH and/or 6 mg/kg/day of NIC on postnatal day (PD) 4 to PD 9, except in the case of the acute neurochemistry study, in which the pups received treatment on PD 9 only. An artificially reared gastrostomy control group (GC) and a suckle control group were included. The mean total numbers of mitral cells in the EtOH and NIC groups were significantly reduced from that of the GC, as well as the volume of the left main olfactory bulb. There was no difference among any of the groups in mitral cell density. As for neurochemistry data, there was no difference in neurotransmitter levels among any of the groups in the repeat exposure regimen. There were, however, changes after the acute exposure (exposure on PD 9 only). Both serotonin and GABA levels were significantly increased only after NIC exposure. However, norepinephrine levels were significantly decreased after acute exposure in all three drug treatment groups, compared with that of the control group. Except for the GC control group, dopamine levels were not detected consistently after acute exposure to EtOH, NIC, or EtOH/NIC. Collectively, these findings demonstrate that exposure to EtOH or NIC individually during the brain growth spurt results in developmental deficits in the olfactory bulb, suggesting that both EtOH and NIC are neuroteratogens. Furthermore, this study demonstrated the capability of NIC to antagonize (protect) EtOH-induced mitral cell loss in the developing olfactory bulb.

Glycine accelerates recovery from alcohol-induced liver injury.

Glycine prevents hepatic damage caused by hypoxia-reoxygenation, diminishes mortality due to endotoxin and minimizes alcoholic liver injury by decreasing blood ethanol. Our purpose was to investigate the effect of dietary glycine during recovery from early alcohol-induced injury, using a model that mimics the clinical presentation and histopathology with alcoholics. Male Wistar rats were exposed to ethanol continuously for 6 wk via intragastric feeding that resulted in typical histology of alcoholic liver injury, including steatosis, inflammation, necrosis and increased serum levels of aspartate aminotransferase and alanine aminotransferase. After cessation of ethanol, one group of rats received a control diet, the other a glycine-containing diet for 2 wk. During this period, all parameters studied tended to return to baseline values. However, serum aspartate aminotransferase and alanine aminotransferase recovered about 30% more rapidly in rats fed glycine. Further, the hepatic pathology score was also significantly lower in the glycine group than in controls (0.5 vs. 2.6). After 1 wk, steatosis was reduced significantly more in the glycine group (5. 6%) than in controls (8.9%). Glycine also diminished numbers of infiltrating leukocytes and necrotic cells significantly more than in controls. This beneficial effect of glycine may be partly explained by the fact that glycine increased influx of chloride into Kupffer cells leading to diminished tumor necrosis factor-alpha production. These results indicate that a glycine containing diet expedites the process of recovery from ethanol-induced liver injury and may lead to its clinical application in alcoholic hepatitis.

Effect of specimen storage and preservation on toxicological analyses of urine.

1. The stability of nineteen classes of drugs or their metabolites in urine specimens, for the purpose of toxicological screening has been studied for periods of up to thirty-six weeks. 2. The effect of specimen preservation by refrigation and by the addition of boric acid, chloroform, sodium fluoride, mercuric chloride, and buffers, has been assessed. 3. With the exception of flurazepam, glutethimide, and secobarbital, specimens may be retained at room temperature for periods in excess of six weeks without deterioration. 4. Preservation by the addition of sodium fluoride (5 g/l) followed by freezing, thawing, and filtration significantly prevents specimen deterioration, and may be used as the method of choice.