Terry Van Vleet | Abbvie Bioresearch Center (original) (raw)

Papers by Terry Van Vleet

Journal of Chromatography B, 2013

Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectr... more Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC-MS/MS analysis. Presented here are three LC-MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.

Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 2002

Poultry are some of the most sensitive species to the toxic effects of aflatoxin B(1) (AFB(1)), a... more Poultry are some of the most sensitive species to the toxic effects of aflatoxin B(1) (AFB(1)), and younger poultry are more sensitive to this mycotoxin. To elucidate the mechanisms for this age-related susceptibility, various enzyme activities relevant to AFB(1) were measured in liver microsomes prepared from male turkeys 9, 41 and 65 days of age. Hepatic microsomal o-dealkylation of methoxy- and pentoxyresorufin significantly increased, while that of ethoxyresorufin decreased with age. Microsomal AFB(1) activation to the reactive AFB(1)-8,9-epoxide (AFBO) was most efficient in the youngest birds, with apparent K(m) and V(max) values of 168 and 19, 110 and 6, and 116 microM and 10 nmol/mg/min for 9, 41 and 65-day-old birds, respectively. The activity of hepatic cytosolic glutathione S-transferases (GSTs) was deficient in the youngest age group, but were higher in the older groups. There was also an age-related increase in the expression of GST isoforms Yc, Yc(2), as well as AFB(1)-...

We have shown previously that the extreme sensitivity of turkeys to aflatoxin B 1 (AFB 1 ) is due... more We have shown previously that the extreme sensitivity of turkeys to aflatoxin B 1 (AFB 1 ) is due to a combination of efficient AFB 1 activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB 1 -relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB 1 to the putative toxic metabolite, the exo-AFB 1 -8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB 1 -relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB 1 detoxification by GST. #

Food and Chemical Toxicology

Toxicology and applied pharmacology, Jan 14, 2015

Mitochondrial toxicity can be difficult to detect as most cells can tolerate reduced activity as ... more Mitochondrial toxicity can be difficult to detect as most cells can tolerate reduced activity as long as minimal capacity for function is maintained. However, once minimal capacity is lost, apoptosis or necrosis occurs quickly. Identification of more sensitive, early markers of mitochondrial toxicity was the objective of this work. Rotenone, a mitochondrial complex I inhibitor, and 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor, were administered daily to male Sprague-Dawley rats at subcutaneous doses of 0.1 or 0.3mg/kg/day and intraperitoneal doses of 5 or 10mg/kg/day, respectively, for 1week. Samples of kidney, skeletal muscle (quadriceps femoris), and serum were collected for analysis of mitochondrial DNA (mtDNA) copy number and microRNA (miRNA) expression patterns. MtDNA was significantly decreased with administration of rotenone at 0.3mg/kg/day and 3-NP at 5 and 10mg/kg/day in the quadriceps femoris and with 3-NP at 10mg/kg/day in the kidney. Additionally, r...

Journal of Medical Genetics and Genomics Vol. 3 (4) pp. 74 - 76, April 2011 Available online http... more Journal of Medical Genetics and Genomics Vol. 3 (4) pp. 74 - 76, April 2011 Available online http://www.academicjournals.org/jmgg ISSN 2141-2278 ©2011 Academic Journals ... An alternative method for high throughput RNA ... Damir Simic*, Terry Van Vleet, Catherine Euler, ...

Gene expression, 2013

Notch signaling pathways are involved in the regulation of cell differentiation and are highly co... more Notch signaling pathways are involved in the regulation of cell differentiation and are highly conserved across species. Notch ligand binding leads to gamma-secretase-mediated proteolytic cleavage of the Notch receptor releasing the Notch intracellular domain, resulting in its subsequent translocation into the nucleus and gene expression regulation. To investigate the level of expression of Notch signaling pathway components in microanatomic regions following renal injury, kidneys from untreated, vehicle control, and puromycin aminonucleoside (PA, 150 mg/kg)-treated rats were evaluated. Frozen tissue sections from rats were microdissected using laser capture microdissection (LCM) to obtain glomeruli, cortical (proximal) tubules, and collecting ducts, and relative gene expression levels of Presenilin1, Notch1 and Hes1 were determined. In untreated rats, the Notch1 expression in glomeruli was higher than in the proximal tubules and similar to that in collecting ducts, whereas Presenil...

Journal of Histotechnology, 2015

ABSTRACT

Journal of Applied Toxicology, 2014

The objective of this study was to evaluate potential protective effects of vehicles containing d... more The objective of this study was to evaluate potential protective effects of vehicles containing d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which may impact nonclinical safety assessments of oxidative processes. This was achieved by evaluating plasma, liver and adrenal gland concentrations of d-α-tocopheryl succinate (TS) and d-α-tocopherol as well as oxidative status of plasma following oral dosing of TPGS-containing vehicles, intraperitoneal (IP) dosing of TS or ex vivo treatment of blood with H2 O2 . Male and female rats were dosed orally with formulations containing 5% or 40% TPGS (70 or 550 mg kg(-1) day(-1) TS, respectively) for 1 week. A control group was dosed orally with polyethylene glycol-400 (PEG-400; no vitamin E) and positive control animals received a single 100 mg kg(-1) day(-1) IP injection of TS. Whole blood from untreated animals was treated ex vivo with 5 or 50 mm H2 O2 , with or without TS (0.5, 5, 50 or 500 μm) or ascorbate (1 mm), for 1 h. Oral TPGS treatments did not affect d-α-tocopherol concentrations in plasma or adrenal glands and caused only transient increases in liver. Concentrations of TS in plasma, liver and adrenal glands were undetectable in control animals, but increased in all other groups. Oral administration of TPGS did not reduce plasma lipid peroxidation in vivo. Substantially greater TS concentrations used ex vivo (100× greater than in vivo) were also unable to reduce lipid peroxidation in H2 O2 -treated whole blood. These results provide evidence that administration of oral TPGS vehicles is unlikely to impact nonclinical safety assessments of pharmaceuticals. Copyright © 2014 John Wiley &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp; Sons, Ltd.

Current Protocols in Toxicology, 2001

In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are ... more In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed-color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell-impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed-color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual-labeled multinucleated cells have resulted from fusion.

Toxicology, 2013

The corticotrophin releasing factor (CRF) receptor I antagonist, BMS-764459 (evaluated as a poten... more The corticotrophin releasing factor (CRF) receptor I antagonist, BMS-764459 (evaluated as a potential treatment of affective disorders), was orally dosed to female Sprague-Dawley rats once daily for 2 weeks (vehicle control or 175mg/kg/day). To investigate the mechanism of BMS-764459-related liver weight increases, total liver RNA was isolated and evaluated for mRNA gene expression by microarray analysis (assessing the expression of approximately 24,000 genes) from snap-frozen tissue. Subsequently, mRNA and miRNA (microRNA) were also analyzed 5 years later from FFPE (Formalin Fixed Paraffin Embedded) samples via RT-PCR (about 800 miRNA evaluated). Genomic analyses showed that BMS-764459 induces AhR target genes with additional inductions of CYP2B, CYP3A, and Abcc3 consistent with the gene expression pattern of atypical CYP1A1 inducers. Analysis of miRNA expression identified a number of significantly affected miRNAs. To further evaluate their role in atypical CYP1A1 induction, an in silico evaluation of differentially expressed miRNA was performed and their putative mRNA 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-UTR (untranslated region) binding sequences were evaluated. MiR-680 and miR-29a were identified as potential regulators and biomarkers of atypical CYP1A1 induction by regulating Abcc3, CYP3A and CYP2B as well as a number of AhR targeted genes.

Toxicologic Pathology, 2006

Muraglitazar, a PPARα/γ agonist, dose-dependently increased urinary bladder tumors in male Harlan... more Muraglitazar, a PPARα/γ agonist, dose-dependently increased urinary bladder tumors in male Harlan Sprague-Dawley (HSD) rats administered 5, 30, or 50 mg/kg/day for up to 2 years. To determine the mode of tumor development, male HSD rats were treated daily for up to 21 months at doses of 0, 1, or 50 mg/kg while being fed either a normal or 1% NH 4 Cl-acidified diet. Muraglitazar-associated, time-dependent changes in urine composition, urothelial mitogenesis and apoptosis, and urothelial morphology were assessed. In control and treated rats fed a normal diet, urine pH was generally ≥ 6.5, which facilitates formation of calcium-and magnesium-containing solids, particularly in the presence of other prolithogenic changes in rat urine. Urinary citrate, an inhibitor of lithogenesis, and soluble calcium concentrations were dose dependently decreased in association with increased calcium phosphate precipitate, crystals and/or microcalculi; magnesium ammonium phosphate crystals and aggregates; and calcium oxalate-containing thin, rod-like crystals. Morphologically, sustained urothelial cytotoxicity and proliferation with a ventral bladder predilection were noted in treated rats by month 1 and urinary carcinomas with a similar distribution occurred by month 9. Urothelial apoptotic rates were unaffected by muraglitazar treatment or diet. In muraglitazar-treated rats fed an acidified diet, urine pH was invariably < 6.5, which inhibited formation of calcium-and magnesium-containing solids. Moreover, dietary acidification prevented the urothelial cytotoxic, proliferative, and tumorigenic responses. Collectively, these data support an indirect pharmacologic mode of urinary bladder tumor development involving alterations in urine composition that predispose to urolithiasis and associated decreases in urine-soluble calcium concentrations.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2002

Annual Review of Pharmacology and Toxicology, 2004

Numerous lines of evidence demonstrate that calpains, a family of 14 Ca(2+)-activated neutral cys... more Numerous lines of evidence demonstrate that calpains, a family of 14 Ca(2+)-activated neutral cysteine proteases, are involved in oncotic cell death in a variety of models. At this time, the biochemistry of most calpains and the specific roles of different calpains in physiology and pathology remain to be determined. A number of calpain substrates have been identified in cellular systems, including cytoskeletal proteins, and recent studies suggest that calpains mediate the increase in plasma membrane permeability to ions and the progressive breakdown of the plasma membrane observed in oncosis through the proteolysis of cystokeletal and plasma membrane proteins. Further, a number of reports provide evidence that the mitochondrial dysfunction observed in oncosis may be mediated by a mitochondrial calpain of unknown identity. Finally, a number of diverse calpain inhibitors have been developed that show cytoprotective properties in cellular systems and in vivo following diverse insults. It is suggested that future research be directed toward elucidation of the role(s) of specific calpain isozymes in physiological and pathological conditions; identifying and linking specific calpain substrates with altered cellular functions; and developing cell-permeable, potent, isozyme-selective calpain inhibitors.

AJP: Cell Physiology, 2006

Toxicology and applied pharmacology, 2002

Turkeys are among the most sensitive species to the toxic effects of the mycotoxin aflatoxin B 1 ... more Turkeys are among the most sensitive species to the toxic effects of the mycotoxin aflatoxin B 1 (AFB 1 ). In mammals, dietary antioxidants, such as butylated hydroxytoluene (BHT), have been shown to lessen the toxic effects of AFB 1 by various mechanisms. To test whether BHT protects against aflatoxicosis in turkeys, we supplemented the feed of 10-day-old male white turkeys with low (1000 ppm) and high (4000 ppm) BHT for 20 days. AFB 1 (1 ppm) was then added to the diets and continued for another 10 days. Birds in the AFB 1 -only group had a lower weight gain, a condition that had returned to near control in groups fed diets containing AFB 1 ؉ BHT. Significant elevations in serum aspartate transaminase, alanine aminotransferase, and lactate dehydrogenase, which were evident in the AFB 1 group, were reversed in the AFB 1 ؉ BHT groups. Histopathology revealed hepatic submassive necrotic lesions and biliary hyperplasia, the severity of which was lessened in the AFB 1 ؉ BHT-treated birds. Hepatocellular hydropic degeneration was observed in the BHT-only group, but not in the AFB 1 ؉ BHT groups. This condition associated with BHT treatment was found in a separate study to be reversible and without any long-term adverse effects. These results indicate that BHT counteracts many of the deleterious effects caused by AFB 1 and that this antioxidant may prove to be a viable feed additive for the reduction of aflatoxicosis in turkeys.

Journal of toxicology and environmental health. Part A, Jan 28, 2002

The mycotoxin aflatoxin B 1 ( AFB 1 ) is a hepatocarcinogen in many animal models and probably a ... more The mycotoxin aflatoxin B 1 ( AFB 1 ) is a hepatocarcinogen in many animal models and probably a human carcinogen. Besides being a dietary carcinogen, AFB 1 has been detected in dusts generated in the processing and transportation of AFB 1 -contaminated products. Inhalation of grain dusts contaminated with AFB 1 may be a risk factor in human lung cancer. Aflatoxin B 1 requires cytochrome P-450 ( CYP) -mediated activation to form cytotoxic and DNA-reactive intermediates, and this activation in human liver is mediated by the CYP 1A2 and 3A4 isoforms. Which isoforms are important in AFB 1 activation in human lung is not well understood. To investigate whether these CYPs can activate AFB 1 at low, environmentally relevant concentrations in human lung cells, SV40 immortalized human bronchial epithelial cells ( BEAS-2B) that were transfected with cDNA for CYPs 3A4 ( B3A4) or 1A2 ( B-CMV1A2) were used. B-CMV1A2 cultured in 15 nM AFB 1 produced the AFB 1 -glutathione conjugate (AFB 1 -GSH) and aflatoxin M 1 ( AFM 1 ) , while B3A4 cells produced only aflatoxin Q 1 ( AFQ 1 ) at 0.15 µM AFB 1 . Nontransfected BEAS-2B cells produced no metabolites, even at 1.5 mM AFB 1 . Microsomes prepared from B-CMV1A2 and B3A4 cells activated AFB 1 to AFB 1 8,9-epoxide ( AFBO) , while those from BEAS-2B cells did not produce AFBO. Cytosol from all three cell types was ineffective at glutathione S-transferase ( GST) -mediated trapping of enzymatically generated AFB 1 8,9-epoxide. B-CMV1A2 cells were 100-fold more sensitive to AFB 1 compared to B3A4 cells, and were 6000fold more sensitive than control BEAS-2B cells. Western immunoblots confirmed that only B-CMV1A2 cells expressed CYP 1A2 protein, while CYP 3A4 was only in B3A4 cells. B-CMV1A2 cells were the most sensitive to AFB 1 , followed by B3A4 cells. CYP 3A4, which has been predicted to activate AFB 1 primarily at higher AFB 1 concentrations, was also responsible for significant AFB 1 toxicity at low concentrations. These data indicate that human lung cells expressing these CYP isoforms are capable of activating AFB 1 , even at environmentally relevant concentrations.

Cancer research, 2002

Some epidemiological evidence suggests a link between the inhalation of aflatoxin B 1 (AFB 1 )-co... more Some epidemiological evidence suggests a link between the inhalation of aflatoxin B 1 (AFB 1 )-contaminated grain dusts and increased lung cancer risk. However, the mechanisms of AFB 1 activation and action in human lung are not well understood. We compared AFB 1 action in SV40 immortalized human bronchial epithelial cells (BEAS-2B) with two transfected cell lines that stably express human cytochromes P450 (CYPs) 1A2 (B-CMV1A2) and 3A4 (B3A4), the principal CYPs thought to activate this mycotoxin in human liver. All three cell types retained catalytically active glutathione S-transferase, the key phase II enzyme that detoxifies metabolically activated AFB 1 . B-CMV1A2 and B3A4 cells expressed methoxyresorufin-O-demethylase (MROD) and nifedipine oxidase activities, respectively, and were 3000-and 70-fold more susceptible, respectively, to the cytotoxic effects of AFB 1 than the control cell line (BEAS-2B). When cultured with a range of low, environmentally relevant AFB 1 concentrations (0.02-1.

The kidney is the target of numerous xenobiotic toxicants, including environmental chemicals. Ana... more The kidney is the target of numerous xenobiotic toxicants, including environmental chemicals. Anatomical, physiological, and biochemical features of the kidney make it particularly sensitive to many environmental compounds. Factors contributing to the sensitivity of the kidney include: large blood flow, the presence of a variety of xenobiotic transporters and metabolizing enzymes, and concentration of solutes during urine production. In many cases, the conjugation of environmental chemicals to glutathione and/or cysteine targets these chemicals to the kidney where inhibition of renal function occurs through a variety of mechanisms. For example, heavy metals such as mercury and cadmium target the kidney after glutathione/cysteine conjugation. Trichloroethlene and bromobenzene are metabolized and conjugated to glutathione in the liver before renal uptake and toxicity. In contrast, renal injury produced by chloroform and aristolochic acids is dependent on renal cytochrome P450 metabolism to toxic metabolites. Other compounds, such as paraquat or diquat, damage the kidney via the production of reactive oxygen species. Finally, the low solubility of ethylene glycol metabolites causes crystal formation within the tubular lumen and nephrotoxicity. This chapter explores mechanisms of nephrotoxicity by environmental chemicals, using these example compounds. What remains to be accomplished and by far the most difficult process is the elucidation of the detailed mechanisms of tubular cell injury after toxicant uptake and metabolism. The large number of individuals experiencing a decline in renal function with age makes the search for these mechanisms very compelling.