Kenneth E. Ferslew

Blocking swelling‐activated chloride current inhibits mouse liver cell proliferation

1 A non‐transformed mouse liver cell line (AML12) was used to show that blocking swelling‐activated membrane Cl− current inhibits hepatocyte proliferation. 2 Two morphologically distinguishable cell populations exhibited distinctly different responses to hypotonic stress. Hypotonic stress (from 280 to 221 mosmol kg−1) to rounded, dividing cells activated an ATP‐dependent, outwardly rectifying, whole‐cell Cl− current, which took 10 min to reach maximum conductance. A similar anionic current was present spontaneously in 20 % of the dividing cells. Hypotonic stress to flattened, non‐dividing cells activated no additional current. 3 The Eisenman halide permeability sequence of swelling‐activated anionic current in the dividing cells was SCN− > I− > Br− > Cl− > gluconate. 4 Addition of either 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS), 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), tamoxifen or mibefradil inhibited swelling‐activated anionic current. Hyperosmolarity by added sucrose inhibited the spontaneous anionic current in dividing cells. 5 Added Cl− channel blockers NPPB (IC50= 40 μm), DIDS (IC50= 31 μm), tamoxifen (IC50= 1.3 μm) and mibefradil (IC50= 7 μm) inhibited proliferative growth of AML12 as determined by cell counts over 4 days or by protein accumulation over 2 days. Only the inhibitory effects of NPPB and mibefradil reversed with the drug washout. Hyperosmolarity by added sucrose (50 and 100 mm) also inhibited cell proliferation. 6 Of the hydrophobic inhibitors neither NPPB at 40 μm nor tamoxifen at 1.3 μm, added for 48 h, reduced cellular ATP; however, DIDS at 31 μm significantly reduced cellular ATP with an equivalent increase in cellular ADP. 7 We conclude that those membrane Cl− currents that can be activated by hypotonic stress are involved in mechanisms controlling liver cell growth, and that NPPB, tamoxifen and mibefradil at their IC50 for growth do not suppress the metabolism of mouse hepatocytes.

Morphine promotes Jurkat cell apoptosis through pro-apoptotic FADD/P53 and anti-apoptotic PI3K/Akt/NF-κB pathways

Opiates have been shown to inhibit cell growth and trigger apoptosis, but the underlying molecular mechanisms remain unclear. We have previously shown that morphine induces Fas expression and promotes Fas-mediated apoptosis. Here, we investigated the mechanisms by which morphine modulates apoptosis in human Jurkat cells. Morphine-induced apoptosis was inhibited by transfection with a dominant negative Fas-associated death domain (FADD) plasmid, revealing that morphine-induced apoptosis is dependent on FADD. Furthermore, suppression of endogenous p53 expression by RNA interference technology considerably attenuated the morphine-induced apoptosis. In addition, morphine-induced apoptosis seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K), as PI3K inhibition by the PI3K inhibitor LY294002 significantly enhanced morphine-induced apoptosis. Moreover, inhibition of Akt or nuclear factor-kappaB (NF-kappaB) expression by RNA interference technology also dramatically increased morphine-induced apoptosis. Our study thus demonstrates that morphine induces Jurkat cell apoptosis through FADD/p53, anti-apoptotic PI3K/Akt and NF-kappaB pathways.

Morphine promotes apoptosis via TLR2, and this is negatively regulated by β-arrestin 2

We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that beta-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and beta-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of beta-arrestin 2 by transfecting beta-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and beta-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.

Pharmacokinetics and bioavailability of the RRR and all racemic stereoisomers of alpha-tocopherol in humans after single oral administration

The plasma and red blood cell pharmacokinetics and bioavailability of the natural source (RRR, d) and all racemic (all rac, dl) stereoisomers of alpha‐tocopherol were studied in 12 men in a double‐blind randomized crossover study. Subjects were administered two 400‐mg soft‐gelatin capsules of either RRR or all rac alpha‐tocopherol. Plasma alpha‐tocopherol concentrations were determined by high‐performance liquid chromatography at various time intervals for up to 96 hours postadministration. Pharmacokinetic modeling of the data showed that alpha‐tocopherol was absorbed after a 2 to 4 hour lagtime and maximum plasma concentration occurred from 12 to 14 hours postadministration. There were no significant differences in the Ka, t1/2 α, β, or t1/2 β between RRR and all rac. Mean plasma alpha‐tocopherol concentrations were greater for RRR than all rac from 10 to 96 hours postadministration and significantly greater at 24 hours (P < .05). The red blood cell alpha‐tocopherol concentration from the RRR preparation was significantly greater than from the all rac preparation from 24 to 96 hours postadministration with Cmax for RRR (4.8 μg/mL) significantly greater than for all rac (4.0 μg/mL, P < .05). The RRR AUC0–96 for both plasma and red blood cells were significantly greater than the all rac AUC0–96 (P < .05) indicating a greater bioavailability of RRR versus all rac alpha‐tocopherol. This difference in overall bioavailability was apparently not due to a single pharmacokinetic component.

Application of Micellar Electrokinetic Capillary Chromatography to Forensic Analysis of Barbiturates in Biological Fluids

Micellar electrokinetic capillary chromatography (MECC) is a form of capillary zone electrophoresis. Addition of a surfactant produces micelles in an aqueous/organic buffer. Separation of drugs is obtained via differences in the electrophoretic mobilities of the analytes within the capillary, resulting from their electrophoretic velocity and the electroosmotic flow of the buffer in a given electric field. The migration order is determined by the differential partitioning of the drugs between the micelles and the aqueous/organic phase. Barbiturates were extracted from various biological fluids at pH 4.5 with TOXI-TUBES B. MECC analyses were performed using a Waters Quanta 4000 Capillary Electrophoretic System with a 745 Data Module with a 75 microns x 60 cm capillary and an aqueous/organic buffer of 85% 10 mM borate, 10 mM phosphate, 100 mM sodium dodecyl sulfate and 15% acetonitrile at a pH of 8.5 with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: phenobarbital, 7.78 min.; butalbital, 8.01 min.; butabarbital, 8.23 min.; mephobarbital (internal standard), 8.88 min.; amobarbital, 9.41 min.; pentobarbital, 10.03 min. and secobarbital, 10.79 min. Correlation coefficients (r) between peak areas and concentration ranges of 3 to 60 micrograms/mL were from 0.964 to 0.999. Coefficients of variation (CV) ranged from 2.6 to 8.6% between days and 2.3 to 9.8% within day. Application of this methodology to four forensic cases of butalbital intoxication detected concentrations of 0.7 to 12.7 micrograms/mL in blood; 0.8 to 1.9 micrograms/mL in vitreous humor and 1.5 to 7.6 micrograms/mL in urine. MECC is applicable to forensic analysis of barbiturates extracted from biological fluids.

Glycogen synthase kinase-3 and p38 MAPK are required for opioid-induced microglia apoptosis

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. We previously reported that opioids inhibit cell growth and trigger apoptosis in lymphocytes. However, the underlying mechanism by which microglia apoptosis in response to opioids is not yet known. In this study, we show that morphine induces microglia apoptosis and caspase-3 activation in an opioid-receptor dependent manner. Morphine decreased the levels of microglia phosphorylated Akt (p-Akt) and p-GSK-3β (glycogen synthase kinase-3 beta) in an opioid-receptor dependent manner. More interestingly, GSK-3β inhibitor SB216763 significantly increases morphine-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. Moreover, co-treatment of microglia with SB216763 and morphine led to a significant synergistic effect on the level of phospho-p38 mitogen-activated protein kinase (MAPK). In addition, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly inhibited morphine-induced apoptosis and caspase-3 activation. Taken together, our data clearly demonstrates that morphine-induced apoptosis in microglial cells, which is mediated via GSK-3β and p38 MAPK pathways.

Postmortem Determination of the Biological Distribution of Sufentanil and Midazolam after an Acute Intoxication

A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a mu-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-micron-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260 degrees C; column 200 degrees C (0.8 min) 10 degrees C/min to 270 degrees C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide.

Prolonged, intravenous paraquat infusion in the rat: I. Failure of coinfused putrescine to attenuate pulmonary paraquat uptake, paraquat-induced biochemical changes, or lung injury

Paraquat (PQ) was administered to rats for 7 days by iv infusion from osmotic minipump at dosage rates of 250 and 500 nmol PQ/hr. The efficacy of putrescine in attenuating pulmonary PQ accumulation in vivo and the resulting PQ-induced biochemical changes and lung injury were assessed in these animals by coinfusion of putrescine at rates of 2500 or 5000 nmol/hr. Dose-dependent, steady-state blood levels of both PQ and putrescine were achieved by 18 hr and maintained throughout the infusion period. Lung PQ content at 7 days was dose-dependent and up to 18-fold greater than corresponding blood levels. No evidence of toxicity was observed in low-dose PQ animals while weight loss and overt toxicity was observed in high-dose PQ rats between Days 4 and 5. Histopathological examination of high-dose PQ rat lungs revealed qualitative changes typical of PQ toxicity. Significant (p less than 0.05) increases in lung glutathione and activities of glucose-6-phosphate dehydrogenase and GSSG reductase resulted from both PQ doses, reflecting PQ-induced oxidant stress and increased demand on lung NADPH. A net decrease in lung NADPH (p less than 0.05) was directly measured in high-dose PQ rats and may have contributed to the PQ-induced lung injury. Although putrescine is an effective inhibitor of pulmonary PQ uptake in vitro, the blood putrescine levels achieved in this study did not appear to inhibit this process in vivo. This was evidenced by putrescines failure to decrease 7-day lung PQ content, PQ-induced biochemical changes, or lung injury.

Spectral differentiation and gas chromatographic/mass spectrometric analysis of the lacrimators 2-chloroacetophenone and o-chlorobenzylidene malononitrile.

2-Chloroacetophenone (CN) and o-chlorobenzylidene malononitrile (CS) are the most common chemical agents used as lacrimators in the United States. There is a lack of complete spectral data on these compounds in the literature. Spectral data (ultraviolet, fluorescence, proton nuclear magnetic resonance, and infrared) and a gas-liquid chromatographic/mass spectrometric method are presented that differentiate and identify CN and CS. These methods and data were used to identify a forensic science specimen from an accidental intoxication.

Dose–response effects of chronic lithium regimens on spatial memory in the black molly fish

Lithium is widely used in the management of bipolar disorder, yet memory impairment is a serious side effect. To assess the effects of lithium on spatial working and reference memories, we have employed a plus maze utilizing spontaneous alternation (SA) and place-learning paradigms in two experiments with the black molly fish. Four treatment groups were gavaged with 20 microl of a 10, 100, or 1000 mM lithium chloride (LiCl) solution or ddH(2)O vehicle every 12 h for 22 to 24 days. On Day 15, subjects began an 8-day SA task or a 10-day place-learning task. Results indicate that there is a significant difference in SA performance among the treatment groups for Days 1, 2, and 3. Results of the place-learning task indicate that the 1 M dose group needed significantly more trials to reach criterion and made significantly fewer correct first choices than the other dose groups. Capillary ion analysis determinations of plasma and brain lithium levels illustrate linear dose-response relationships to doses administered. Regression analyses indicate that there is a relationship between SA performance and plasma/brain lithium levels during the initial part of testing. Collectively, the results indicate that chronic lithium administration impairs spatial working and reference memories.