Publications by authors named "Gunnar Boysen"

72 Publications

Bioactivation of Isoxazole-Containing Bromodomain and Extra-Terminal Domain (BET) Inhibitors.

Metabolites 2021 Jun 15;11(6). Epub 2021 Jun 15.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

The 3,5-dimethylisoxazole motif has become a useful and popular acetyl-lysine mimic employed in isoxazole-containing bromodomain and extra-terminal (BET) inhibitors but may introduce the potential for bioactivations into toxic reactive metabolites. As a test, we coupled deep neural models for quinone formation, metabolite structures, and biomolecule reactivity to predict bioactivation pathways for 32 BET inhibitors and validate the bioactivation of select inhibitors experimentally. Based on model predictions, inhibitors were more likely to undergo bioactivation than reported non-bioactivated molecules containing isoxazoles. The model outputs varied with substituents indicating the ability to scale their impact on bioactivation. We selected OXFBD02, OXFBD04, and I-BET151 for more in-depth analysis. OXFBD's bioactivations were evenly split between traditional quinones and novel extended quinone-methides involving the isoxazole yet strongly favored the latter quinones. Subsequent experimental studies confirmed the formation of both types of quinones for OXFBD molecules, yet traditional quinones were the dominant reactive metabolites. Modeled I-BET151 bioactivations led to extended quinone-methides, which were not verified experimentally. The differences in observed and predicted bioactivations reflected the need to improve overall bioactivation scaling. Nevertheless, our coupled modeling approach predicted BET inhibitor bioactivations including novel extended quinone methides, and we experimentally verified those pathways highlighting potential concerns for toxicity in the development of these new drug leads.
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http://dx.doi.org/10.3390/metabo11060390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232216PMC
June 2021

Impacts of diphenylamine NSAID halogenation on bioactivation risks.

Toxicology 2021 06 6;458:152832. Epub 2021 Jun 6.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States. Electronic address:

Diphenylamine NSAIDs are highly prescribed therapeutics for chronic pain despite causing symptomatic hepatotoxicity through mitochondrial damage in five percent of patients taking them. Differences in toxicity are attributed to structural modifications to the diphenylamine scaffold rather than its inherent toxicity. We hypothesize that marketed diphenylamine NSAID substituents affect preference and efficiency of bioactivation pathways and clearance. We parsed the FDA DILIrank hepatotoxicity database and modeled marketed drug bioactivation into quinone-species metabolites to identify a family of seven clinically relevant diphenylamine NSAIDs. These drugs fell into two subgroups, i.e., acetic acid and propionic acid diphenylamines, varying in hepatotoxicity risks and modeled bioactivation propensities. We carried out steady-state kinetic studies to assess bioactivation pathways by trapping quinone-species metabolites with dansyl glutathione. Analysis of the glutathione adducts by mass spectrometry characterized structures while dansyl fluorescence provided quantitative yields for their formation. Resulting kinetics identified four possible bioactivation pathways among the drugs, but reaction preference and efficiency depended upon structural modifications to the diphenylamine scaffold. Strikingly, diphenylamine dihalogenation promotes formation of quinone metabolites through four distinct metabolic pathways with high efficiency, whereas those without aromatic halogen atoms were metabolized less efficiently through two or fewer metabolic pathways. Overall metabolism of the drugs was comparable with bioactivation accounting for 4-13% of clearance. Lastly, we calculated daily bioload exposure of quinone-species metabolites based on bioactivation efficiency, bioavailability, and maximal daily dose. The results revealed stratification into the two subgroups; propionic acid diphenylamines had an average four-fold greater daily bioload compared to acetic acid diphenylamines. However, the lack of sufficient study on the hepatotoxicity for all drugs prevents further correlative analyses. These findings provide critical insights on the impact of diphenylamine bioactivation as a precursor to hepatotoxicity and thus, provide a foundation for better risk assessment in drug discovery and development.
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http://dx.doi.org/10.1016/j.tox.2021.152832DOI Listing
June 2021

Effects of Genotype on the Detoxification of 1,3-Butadiene Derived Diepoxide and Formation of Promutagenic DNA-DNA Cross-Links in Human Hapmap Cell Lines.

Chem Res Toxicol 2021 01 31;34(1):119-131. Epub 2020 Dec 31.

University of Minnesota Masonic Cancer Center, Minneapolis, Minnesota 55455, United States.

Smoking is a leading cause of lung cancer, accounting for 81% of lung cancer cases. Tobacco smoke contains over 5000 compounds, of which more than 70 have been classified as human carcinogens. Of the many tobacco smoke constituents, 1,3-butadiene (BD) has a high cancer risk index due to its tumorigenic potency and its abundance in cigarette smoke. The carcinogenicity of BD has been attributed to the formation of several epoxide metabolites, of which 1,2,3,4-diepoxybutane (DEB) is the most toxic and mutagenic. DEB is formed by two oxidation reactions carried out by cytochrome P450 monooxygenases, mainly CYP2E1. Glutathione-S-transferase 1 (GSTT1) facilitates the conjugation of DEB to glutathione as the first step of its detoxification and subsequent elimination via the mercapturic acid pathway. Human biomonitoring studies have revealed a strong association between copy number and urinary concentrations of BD-mercapturic acids, suggesting that it plays an important role in the metabolism of BD. To determine the extent that genotype affects the susceptibility of individuals to the toxic and genotoxic properties of DEB, negative and positive HapMap lymphoblastoid cell lines were treated with DEB, and the extent of apoptosis and micronuclei (MN) formation was assessed. These toxicological end points were compared to the formation of DEB-GSH conjugates and 1,4--(guan-7-yl)-2,3-butanediol (-N7G-BD) DNA-DNA cross-links. negative cell lines were more sensitive to DEB-induced apoptosis as compared to positive cell lines. Consistent with the protective effect of GSH conjugation against DEB-derived apoptosis, positive cell lines formed significantly more DEB-GSH conjugate than negative cell lines. However, genotype did not affect formation of MN or -N7G-BD cross-links. These results indicate that genotype significantly influences BD metabolism and acute toxicity.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177101PMC
January 2021

Significance of Multiple Bioactivation Pathways for Meclofenamate as Revealed through Modeling and Reaction Kinetics.

Drug Metab Dispos 2021 02 25;49(2):133-141. Epub 2020 Nov 25.

Departments of Biochemistry and Molecular Biology (M.A.S, G.P.M.) and Environmental and Occupational Health (G.B.), University of Arkansas for Medical Sciences, Little Rock, Arizona (M.A.S.); Department of Pathology and Immunology, Washington University, St. Louis, Missouri (N.R.F., S.J.S.); Department of Chemistry, Hendrix College, Conway, Arizona (S.P.); and Independent Researcher (B.M.S.) and Department of Chemistry and Biochemistry (A.O.P., D.W.P.), Harding University, Searcy, Arkansas

Meclofenamate is a nonsteroidal anti-inflammatory drug used in the treatment of mild-to-moderate pain yet poses a rare risk of hepatotoxicity through an unknown mechanism. Nonsteroidal anti-inflammatory drug (NSAID) bioactivation is a common molecular initiating event for hepatotoxicity. Thus, we hypothesized a similar mechanism for meclofenamate and leveraged computational and experimental approaches to identify and characterize its bioactivation. Analyses employing our XenoNet model indicated possible pathways to meclofenamate bioactivation into 19 reactive metabolites subsequently trapped into glutathione adducts. We describe the first reported bioactivation kinetics for meclofenamate and relative importance of those pathways using human liver microsomes. The findings validated only four of the many bioactivation pathways predicted by modeling. For experimental studies, dansyl glutathione was a critical trap for reactive quinone metabolites and provided a way to characterize adduct structures by mass spectrometry and quantitate yields during reactions. Of the four quinone adducts, we were able to characterize structures for three of them. Based on kinetics, the most efficient bioactivation pathway led to the monohydroxy para-quinone-imine followed by the dechloro-ortho-quinone-imine. Two very inefficient pathways led to the dihydroxy ortho-quinone and a likely multiply adducted quinone. When taken together, bioactivation pathways for meclofenamate accounted for approximately 13% of total metabolism. In sum, XenoNet facilitated prediction of reactive metabolite structures, whereas quantitative experimental studies provided a tractable approach to validate actual bioactivation pathways for meclofenamate. Our results provide a foundation for assessing reactive metabolite load more accurately for future comparative studies with other NSAIDs and drugs in general. SIGNIFICANCE STATEMENT: Meclofenamate bioactivation may initiate hepatotoxicity, yet common risk assessment approaches are often cumbersome and inefficient and yield qualitative insights that do not scale relative bioactivation risks. We developed and applied innovative computational modeling and quantitative kinetics to identify and validate meclofenamate bioactivation pathways and relevance as a function of time and concentration. This strategy yielded novel insights on meclofenamate bioactivation and provides a tractable approach to more accurately and efficiently assess other drug bioactivations and correlate risks to toxicological outcomes.
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http://dx.doi.org/10.1124/dmd.120.000254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841419PMC
February 2021

Significance of Competing Metabolic Pathways for 5F-APINACA Based on Quantitative Kinetics.

Molecules 2020 Oct 20;25(20). Epub 2020 Oct 20.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

In 2020, nearly one-third of new drugs on the global market were synthetic cannabinoids including the drug of abuse -(1-adamantyl)-1-(5-pentyl)-1-indazole-3-carboxamide (5F-APINACA, 5F-AKB48). Knowledge of 5F-APINACA metabolism provides a critical mechanistic basis to interpret and predict abuser outcomes. Prior qualitative studies identified which metabolic processes occur but not the order and extent of them and often relied on problematic "semi-quantitative" mass spectroscopic (MS) approaches. We capitalized on 5F-APINACA absorbance for quantitation while leveraging MS to characterize metabolite structures for measuring 5F-APINACA steady-state kinetics. We demonstrated the reliability of absorbance and not MS for inferring metabolite levels. Human liver microsomal reactions yielded eight metabolites by MS but only five by absorbance. Subsequent kinetic studies on primary and secondary metabolites revealed highly efficient mono- and dihydroxylation of the adamantyl group and much less efficient oxidative defluorination at the -pentyl terminus. Based on regiospecificity and kinetics, we constructed pathways for competing and intersecting steps in 5F-APINACA metabolism. Overall efficiency for adamantyl oxidation was 17-fold higher than that for oxidative defluorination, showing significant bias in metabolic flux and subsequent metabolite profile compositions. Lastly, our analytical approach provides a powerful new strategy to more accurately assess metabolic kinetics for other understudied synthetic cannabinoids possessing the indazole chromophore.
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http://dx.doi.org/10.3390/molecules25204820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587938PMC
October 2020

Detection and Discrimination of DNA Adducts Differing in Size, Regiochemistry, and Functional Group by Nanopore Sequencing.

Chem Res Toxicol 2020 12 1;33(12):2944-2952. Epub 2020 Oct 1.

Environmental and Occupational Health, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, Arkansas 72205, United States.

Chemically induced DNA adducts can lead to mutations and cancer. Unfortunately, because common analytical methods (e.g., liquid chromatography-mass spectrometry) require adducts to be digested or liberated from DNA before quantification, information about their positions within the DNA sequence is lost. Advances in nanopore sequencing technologies allow individual DNA molecules to be analyzed at single-nucleobase resolution, enabling us to study the dynamic of epigenetic modifications and exposure-induced DNA adducts in their native forms on the DNA strand. We applied and evaluated the commercially available Oxford Nanopore Technology (ONT) sequencing platform for site-specific detection of DNA adducts and for distinguishing individual alkylated DNA adducts. Using ONT and the publicly available ELIGOS software, we analyzed a library of 15 plasmids containing site-specifically inserted - or -alkyl-2'-deoxyguanosine lesions differing in sizes and regiochemistries. Positions of DNA adducts were correctly located, and individual DNA adducts were clearly distinguished from each other.
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http://dx.doi.org/10.1021/acs.chemrestox.0c00202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752846PMC
December 2020

Decoding the epitranscriptional landscape from native RNA sequences.

Nucleic Acids Res 2021 01;49(2):e7

Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.
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http://dx.doi.org/10.1093/nar/gkaa620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826254PMC
January 2021

Delivery of phosphatidylethanolamine blunts stress in hepatoma cells exposed to elevated palmitate by targeting the endoplasmic reticulum.

Cell Death Discov 2020 18;6. Epub 2020 Feb 18.

1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR USA.

Genetic obesity increases in liver phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio, inducing endoplasmic reticulum (ER) stress without concomitant increase of ER chaperones. Here, it is found that exposing mice to a palm oil-based high fat (HF) diet induced obesity, loss of liver PE, and loss of the ER chaperone Grp78/BiP in pericentral hepatocytes. In Hepa1-6 cells treated with elevated concentration of palmitate to model lipid stress, Grp78/BiP mRNA was increased, indicating onset of stress-induced Unfolded Protein Response (UPR), but Grp78/BiP protein abundance was nevertheless decreased. Exposure to elevated palmitate also induced in hepatoma cells decreased membrane glycosylation, nuclear translocation of pro-apoptotic C/EBP-homologous-protein-10 (CHOP), expansion of ER-derived quality control compartment (ERQC), loss of mitochondrial membrane potential (MMP), and decreased oxidative phosphorylation. When PE was delivered to Hepa1-6 cells exposed to elevated palmitate, effects by elevated palmitate to decrease Grp78/BiP protein abundance and suppress membrane glycosylation were blunted. Delivery of PE to Hepa1-6 cells treated with elevated palmitate also blunted expansion of ERQC, decreased nuclear translocation of CHOP and lowered abundance of reactive oxygen species (ROS). Instead, delivery of the chemical chaperone 4-phenyl-butyrate (PBA) to Hepa1-6 cells treated with elevated palmitate, while increasing abundance of Grp78/BiP protein and restoring membrane glycosylation, also increased ERQC, expression and nuclear translocation of CHOP, non-mitochondrial oxygen consumption, and generation of ROS. Data indicate that delivery of PE to hepatoma cells under lipid stress recovers cell function by targeting the secretory pathway and by blunting pro-apoptotic branches of the UPR.
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http://dx.doi.org/10.1038/s41420-020-0241-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028721PMC
February 2020

Use of electronic nicotine delivery systems by pregnant women II: Hair biomarkers for exposures to nicotine and tobacco-specific nitrosamines.

Tob Induc Dis 2019 6;17:50. Epub 2019 Jun 6.

Department of Environmental and Occupational Health, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, United States.

Introduction: Public awareness of electronic nicotine delivery systems (ENDS) has increased over time, and the perception that ENDS offer a safer alternative to cigarettes may lead some pregnant women to use them to reduce cigarette smoking during pregnancy. No previous studies have used metabolite levels in hair to measure nicotine exposure for ENDS users during pregnancy. We aimed to measure and compare levels of nicotine, cotinine, and tobacco-specific nitrosamines (TSNAs) in hair samples from pregnant women who were current ENDS users, current smokers, and current non-smokers. We also aimed to estimate the association between ENDS use/smoking and smallness for gestational age (SGA).

Methods: We used hair specimens from pregnant women who were dual users (ENDS and cigarettes), smokers, and non-smokers from a prospective cohort study to estimate exposure to nicotine, cotinine, and TSNAs. The exposure biomarkers and self-reports of smoking and ENDS use were used in log-binomial regression models to estimate risk ratios (RRs) for SGA among offspring.

Results: Nicotine concentrations for pregnant dual users were not significantly different from those for smokers (11.0 and 10.6 ng/mg hair, respectively; p=0.58). Similarly, levels of cotinine, and TSNAs for pregnant dual users were not lower than those for smokers. The RR for SGA was similar for dual users and smokers relative to nonsmokers, (RR=3.5, 95% CI: 0.8-14.8) and (RR=3.3, 95% CI: 0.9-11.6), respectively. Using self-reports confirmed by hair nicotine, the RR values for dual ENDS users and smokers were 8.3 (95% CI: 1.0-69.1) and 7.3 (95% CI:1.0-59.0), respectively.

Conclusions: We did not observe lower levels of nicotine, cotinine, and TSNAs for current dual users compared to smokers during pregnancy. The risk of SGA for offspring of pregnant dual users was similar to that for offspring of pregnant smokers. Future studies are needed to further estimate the magnitude of the association between ENDS use and smallness for gestational age.
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http://dx.doi.org/10.18332/tid/105387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662780PMC
June 2019

Use of Electronic Nicotine Delivery Systems (ENDS) by pregnant women I: Risk of small-for-gestational-age birth.

Tob Induc Dis 2019 21;17:44. Epub 2019 May 21.

Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, United States.

Introduction: The 2016 US Surgeon General's Report suggests that the use of electronic nicotine delivery systems (ENDS) is a fetal risk factor. However, no previous study has estimated their effect on adverse pregnancy outcomes. We assessed the prevalence of current ENDS use in pregnant women and explored the effect on birth weight and smallness-for-gestational-age (SGA), correcting for misclassification from nondisclosure of smoking status.

Methods: We conducted a cohort study with 248 pregnant women using questionnaire data and biomarkers (salivary cotinine, exhaled carbon monoxide, and hair nicotine). We evaluated the association between birth weight and the risk of SGA by applying multivariate linear and log-binomial regression to reproductive outcome data for 232 participants. Participants who did not disclose their smoking status were excluded from the referent group. Sensitivity analysis corrected for misclassification of smoking/ENDS use status.

Results: The prevalence of current ENDS use among pregnant women was 6.8% (95% CI: 4.4-10.2%); most of these (75%) were concurrent smokers. Using self-reports, the estimated risk ratio of SGA for ENDS users was nearly two times the risk in the unexposed (RR=1.9, 95% CI: 0.6-5.5), and over three times that for ENDS-only users versus the unexposed (RR=3.1, 95% CI: 0.8-11.7). Excluding from the referent group smokers who did not disclose their smoking status, the risk of SGA for ENDS-only use was 5 times the risk in the unexposed (RR=5.1, 95% CI: 1.1- 22.2), and almost four times for all types of ENDS users (RR=3.8, 95% CI: 1.3-11.2). SGA risk ratios for ENDS users, corrected for misclassification due to self-report, were 6.5-8.5 times that of the unexposed.

Conclusions: Our data suggest that ENDS use is associated with an increased risk of SGA.
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http://dx.doi.org/10.18332/tid/106089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662791PMC
May 2019

A simplified method for detection of N-terminal valine adducts in patients receiving treosulfan.

Rapid Commun Mass Spectrom 2019 Nov;33(21):1635-1642

Division of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel.

Rationale: Treosulfan is a substance that is being studied as part of the conditioning regimen given prior to allogeneic stem cell transplantation in patients with hematological malignancies. It is known to decompose into 1,2:3,4-diepoxybutane (DEB) under physiologic conditions. In this study, we investigate whether N-terminal valine adducts can be utilized to monitor differences in DEB formation of patients receiving treosulfan as part of the conditioning regimen for transplantation.

Methods: Blood samples were collected from a group of 14 transplant recipients and analyzed for N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) and 2,3,4-trihydroxybutylvaline (THB-Val) adducts as biomarkers for drug uptake and metabolism before treosulfan treatment and 6 days after treatment.

Results: A new direct injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated prior to clinical analysis. The assay precision was determined by 3 replicate analyses on 3 individual days using control globin spiked with known amounts of pyr-Val and THB-Val. The intra- and inter-day precision coefficients of variance (CVs) and accuracy were < 10% and 15%, respectively. In clinical specimens, the means ± SD of pyr-Val and THB-Val background were 0.29 ± 0.10 pmol/g HB and 5.17 ± 1.7 pmol/g HB, respectively.

Conclusions: These values are similar to those found previously. Treosulfan treatment leads to a significant increase in pyr-Val and THB-Val adducts in each patient (Student's t-test p <0.0001). The mean ± SD amounts of adduct formed were 245.3 ± 89.6 and 210 ± 78.5 pmol/g globin for pyr-Val and THB-Val, respectively. Importantly, these results show that this direct injection method can quantitate both background and treosulfan-induced pyr-Val and THB-Val N-terminal valine globin adducts in humans.
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http://dx.doi.org/10.1002/rcm.8509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817381PMC
November 2019

CYP2C19 and 3A4 Dominate Metabolic Clearance and Bioactivation of Terbinafine Based on Computational and Experimental Approaches.

Chem Res Toxicol 2019 06 10;32(6):1151-1164. Epub 2019 Apr 10.

Department of Biochemistry and Molecular Biology , University of Arkansas for Medical Sciences , Little Rock , Arkansas 72205 , United States.

Lamisil (terbinafine) is an effective, widely prescribed antifungal drug that causes rare idiosyncratic hepatotoxicity. The proposed toxic mechanism involves a reactive metabolite, 6,6-dimethyl-2-hepten-4-ynal (TBF-A), formed through three N-dealkylation pathways. We were the first to characterize them using in vitro studies with human liver microsomes and modeling approaches, yet knowledge of the individual enzymes catalyzing reactions remained unknown. Herein, we employed experimental and computational tools to assess terbinafine metabolism by specific cytochrome P450 isozymes. In vitro inhibitor phenotyping studies revealed six isozymes were involved in one or more N-dealkylation pathways. CYP2C19 and 3A4 contributed to all pathways, and so, we targeted them for steady-state analyses with recombinant isozymes. N-Dealkylation yielding TBF-A directly was catalyzed by CYP2C19 and 3A4 similarly. Nevertheless, CYP2C19 was more efficient than CYP3A4 at N-demethylation and other steps leading to TBF-A. Unlike microsomal reactions, N-denaphthylation was surprisingly efficient for CYP2C19 and 3A4, which was validated by controls. CYP2C19 was the most efficient among all reactions. Nonetheless, CYP3A4 was more selective at steps leading to TBF-A, making it more effective in terbinafine bioactivation based on metabolic split ratios for competing pathways. Model predictions did not extrapolate to quantitative kinetic constants, yet some results for CYP3A4 and CYP2C19 agreed qualitatively with preferred reaction steps and pathways. Clinical data on drug interactions support the CYP3A4 role in terbinafine metabolism, while CYP2C19 remains understudied. Taken together, knowledge of P450s responsible for terbinafine metabolism and TBF-A formation provides a foundation for investigating and mitigating the impact of P450 variations in toxic risks posed to patients.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692898PMC
June 2019

Injury to hypothalamic Sim1 neurons is a common feature of obesity by exposure to high-fat diet in male and female mice.

J Neurochem 2019 04 11;149(1):73-97. Epub 2019 Feb 11.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

The hypothalamus is essential for regulation of energy homeostasis and metabolism. Feeding hypercaloric, high-fat (HF) diet induces hypothalamic arcuate nucleus injury and alters metabolism more severely in male than in female mice. The site(s) and extent of hypothalamic injury in male and female mice are not completely understood. In the paraventricular nucleus (PVN) of the hypothalamus, single-minded family basic helix-loop helix transcription factor 1 (Sim1) neurons are essential to control energy homeostasis. We tested the hypothesis that exposure to HF diet induces injury to Sim1 neurons in the PVN of male and female mice. Mice expressing membrane-bound enhanced green fluorescent protein (mEGFP) in Sim1 neurons (Sim1-Cre:Rosa-mEGFP mice) were generated to visualize the effects of exposure to HF diet on these neurons. Male and female Sim1-Cre:Rosa-mEGFP mice exposed to HF diet had increased weight, hyperleptinemia, and developed hepatosteatosis. In male and female mice exposed to HF diet, expression of mEGFP was reduced by > 40% in Sim1 neurons of the PVN, an effect paralleled by cell apoptosis and neuronal loss, but not by microgliosis. In the arcuate nucleus of the Sim1-Cre:Rosa-mEGFP male mice, there was decreased alpha-melanocyte-stimulating hormone in proopiomelanocortin neurons projecting to the PVN, with increased cell apoptosis, neuronal loss, and microgliosis. These defects were undetectable in the arcuate nucleus of female mice exposed to the HF diet. Thus, injury to Sim1 neurons of the PVN is a shared feature of exposure to HF diet in mice of both sexes, while injury to proopiomelanocortin neurons in arcuate nucleus is specific to male mice. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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http://dx.doi.org/10.1111/jnc.14662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438752PMC
April 2019

Glutaminase inhibitor CB-839 increases radiation sensitivity of lung tumor cells and human lung tumor xenografts in mice.

Int J Radiat Biol 2019 04 15;95(4):436-442. Epub 2019 Jan 15.

b The Winthrop P. Rockefeller Cancer Institute , University of Arkansas for Medical Sciences , Little Rock , AR , USA.

Purpose: The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses.

Materials And Methods: To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lungtumor xenografts in mice.

Results: Inhibition of GLS markedly reduced cell viability, producing ED values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ∼30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induced injury. In subsequent mouse xenografts, short-term CB-839 treatments reduced serum GSH by >50% and increased response to radiotherapy of H460-derived tumor xenografts by 30%.

Conclusion: The results support the proposed mechanistic link between GLS activity and GSH synthesis and suggest that GLS inhibitors are effective radiosensitizers.
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http://dx.doi.org/10.1080/09553002.2018.1558299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6622448PMC
April 2019

Novel isomeric metabolite profiles correlate with warfarin metabolism phenotype during maintenance dosing in a pilot study of 29 patients.

Blood Coagul Fibrinolysis 2018 Nov;29(7):602-612

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.

: For this pilot study, we leveraged metabolite patterns for warfarin patients to more accurately assess clinically relevant differences in drug metabolism. We tested our hypothesis that plasma metabolite levels correlate with the influence of clinical factors on R-warfarin and S-warfarin metabolism (warfarin metabolic phenotype). We recruited 29 patients receiving a maintenance dose and testing within targeted therapeutic range. We determined their CYP2C9 and vitamin K epoxide reductase genotype and profiled 14 isomeric forms of warfarin and its metabolites. We employed three novel types of clearance ratios using analyte levels to perform multiple-linear regression analyses with clinical factors impacting drug metabolism and dose-responses. Competitive clearance ratios correlated with seven clinical factors including lifestyle choices (smoking), genetics (CYP2C9 and vitamin K epoxide reductase 1), and drug interactions (omeprazole) along with age, weight, and malignancy. Significant competitive clearance ratio correlations (P = 0.04 to < 0.001) explained 21-95% variability. Their performances surpassed that of oxidative and metabolic clearance ratios based on the number and significance of correlations. Competitive clearance ratios may accurately assess significance of factors on maintaining levels of pharmacologically active forms of the drug and metabolites related to dose-responses and thus provide a strategy to minimize adverse events and improve safety during anticoagulant therapy. This unique capacity could provide a strategy in a future, higher power study with a larger cohort of patients to more accurately assess the significance of clinical factors on active drug levels contributing to warfarin dose-responses.
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http://dx.doi.org/10.1097/MBC.0000000000000752DOI Listing
November 2018

Response to Interpretation of Mass Spectral Data for the Cisplatin 1,2 Intrastrand Guanine-Guanine Adduct.

Authors:
Gunnar Boysen

Chem Res Toxicol 2018 11 16;31(11):1108. Epub 2018 Oct 16.

Environmental and Occupational Health , University of Arkansas for Medical Sciences , 4301 West Markham St. , Little Rock , Arkansas 72205 , United States.

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http://dx.doi.org/10.1021/acs.chemrestox.8b00260DOI Listing
November 2018

PARP1 Is Up-Regulated in Non-small Cell Lung Cancer Tissues in the Presence of the Cyanobacterial Toxin Microcystin.

Front Microbiol 2018 6;9:1757. Epub 2018 Aug 6.

Department of Epidemiology, University of Arkansas for Medical Sciences, Little Rock, AR, United States.

Non-small cell lung cancer (NSCLC) is the major form of lung cancer, with adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being its major subtypes. Smoking alone cannot completely explain the lung cancer etiology. We hypothesize that altered lung microbiome and chronic inflammatory insults in lung tissues contribute to carcinogenesis. Here we explore the microbiome composition of LUAD samples, compared to LUSC and normal samples. Extraction of microbiome DNA in formalin-fixed, paraffin-embedded (FFPE) lung tumor and normal adjacent tissues was meticulously performed. The 16S rRNA product from extracted microbiota was subjected to microbiome amplicon sequencing. To assess the contribution of the host genome, expression levels were analyzed then integrated with altered NSCLC subtype-specific microbe sequence data. Surprisingly phylum was consistently observed in LUAD samples. Across the NSCLC subtypes, differential abundance across four phyla (, and ) was identified based on the univariate analysis (-value < 6.4e-4 to 3.2e-2). metagenomic and pathway analyses show that presence of microcystin correlates with reduced CD36 and increased PARP1 levels. This was confirmed in microcystin challenged NSCLC (A427) cell lines and positive LUAD tissues. Controlling the influx of particles or microcystin and the inhibition of PARP1 can provide a potential targeted therapy and prevention of inflammation-associated lung carcinogenesis.
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http://dx.doi.org/10.3389/fmicb.2018.01757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6087756PMC
August 2018

Diagnosis of lung tumor types based on metabolomic profiles in lymph node aspirates.

Cancer Treat Res Commun 2018 19;14:1-6. Epub 2017 Aug 19.

Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, United States; The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, United States. Electronic address:

Background: Treatment of lung cancer is evolving from the use of cytotoxic drugs to drugs that interrupt pathways specific to a malignancy. The field of metabolomics has promise with respect to identification of tumor-specific processes and therapeutic targets, but to date has yielded inconsistent data in patients with lung cancer. Lymph nodes are often aspirated in the process of evaluating lung cancer, as malignant cells in lymph nodes are used for diagnosis and staging. We hypothesized that fluids from lymph node aspirates contains tumor-specific metabolites and are a suitable source for defining the metabolomic phenotype of lung cancers.

Patients And Materials: Metabolic profiles were generated from nodal aspirates of ten patients with adenocarcinoma, ten with squamous cell carcinoma, and ten with non-malignant conditions using time-of-flight mass spectrometry. In addition, concentrations of selected metabolites participating in the kynurenine and glutathione pathways were measured in a second set of aspirates using tandem mass spectrometry.

Results: A list of consensus features that separated these three groups was identified. Two of the consensus features were tentatively identified as kynurenine and as oxidized glutathione. It was shown that metabolite concentrations in these pathways are different for patients with and without malignancy.

Conclusion: Together the data suggest that metabolomic analysis of lymph node aspirates can identify tumor-specific differences in cancer metabolism and reveal novel therapeutic targets. This proof-of-concept study demonstrates the validity to complement and refine diagnosis of lung cancer based on metabolic signature in lymph node aspirates.

Micro Abstract: Treatment of lung cancer is evolving from the use of cytotoxic drugs to drugs that interrupt metabolic pathways specific to a malignancy. We report here in that the metabolic phenotype of lung cancer can be determined in lymph node aspirates harboring malignant tumor cells. Knowledge about metabolic activity of malignant tumor cells may aide to personalize therapy.
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http://dx.doi.org/10.1016/j.ctarc.2017.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173633PMC
June 2019

Lamisil (terbinafine) toxicity: Determining pathways to bioactivation through computational and experimental approaches.

Biochem Pharmacol 2018 10 2;156:10-21. Epub 2018 Aug 2.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States. Electronic address:

Lamisil (terbinafine) may cause idiosyncratic liver toxicity through a proposed toxicological mechanism involving the reactive metabolite 6,6-dimethyl-2-hepten-4-ynal (TBF-A). TBF-A toxicological relevance remains unclear due to a lack of identification of pathways leading to and competing with TBF-A formation. We resolved this knowledge gap by combining computational modeling and experimental kinetics of in vitro hepatic N-dealkylation of terbinafine. A deep learning model of N-dealkylation predicted a high probability for N-demethylation to yield desmethyl-terbinafine followed by N-dealkylation to TBF-A and marginal contributions from other possible pathways. We carried out steady-state kinetic experiments with pooled human liver microsomes that relied on development of labeling methods to expand metabolite characterization. Those efforts revealed high levels of TBF-A formation and first order decay during metabolic reactions; actual TBF-A levels would then reflect the balance between those processes as well as reflect the impact of stabilizing adduction with glutathione and other biological molecules. Modeling predictions and experimental studies agreed on the significance of N-demethylation and insignificance of N-denaphthylation in terbinafine metabolism, yet differed on importance of direct TBF-A formation. Under steady-state conditions, the direct pathway was the most important source of the reactive metabolite with a V/K of 4.0 pmol/min/mg protein/µM in contrast to model predictions. Nevertheless, previous studies show that therapeutic dosing leads to accumulation of desmethyl-terbinafine in plasma, which means that likely sources for TBF-A would draw from metabolism of both the major metabolite and parent drug based on our modeling and experimental studies. Through this combination of novel modeling and experimental approaches, we are the first to identify pathways leading to generation of TBF-A for assessing its role in idiosyncratic adverse drug interactions.
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http://dx.doi.org/10.1016/j.bcp.2018.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188815PMC
October 2018

The Glutathione Conundrum: Stoichiometric Disconnect between Its Formation and Oxidative Stress.

Authors:
Gunnar Boysen

Chem Res Toxicol 2017 05 26;30(5):1113-1116. Epub 2017 Apr 26.

Department of Environmental and Occupational Health and The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205, United States.

Glutathione (GSH) is the most abundant antioxidant and is believed to maintain redox potential in tissues, cells, and individual compartments. However, GSH concentrations in some tumor cells and tissues have been reported to be as high as 1-10 mM, a concentration that is up to 10,000-fold higher than that of reactive oxygen species. Critical quantitative evaluation of glutathione's proposed functions suggests that glutathione is an amino acid checkpoint. In this role, glutathione contributes to regulating cell proliferation and apoptosis, pending amino acid availability.
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http://dx.doi.org/10.1021/acs.chemrestox.7b00018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920211PMC
May 2017

1,3-Butadiene-induced mitochondrial dysfunction is correlated with mitochondrial CYP2E1 activity in Collaborative Cross mice.

Toxicology 2017 03 9;378:114-124. Epub 2017 Jan 9.

Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR, United States; The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, United States. Electronic address:

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight hydrophobic compounds, including 1,3-butadiene, which is converted by CYP2E1 to electrophilic epoxide metabolites that covalently modify cellular proteins and DNA. Previous CYP2E1 studies have mainly focused on the enzyme localized in the endoplasmic reticulum (erCYP2E1); however, active CYP2E1 has also been found in mitochondria (mtCYP2E1) and the distribution of CYP2E1 between organelles can influence an individual's response to exposure. Relatively few studies have focused on the contribution of mtCYP2E1 to activation of chemical toxicants. We hypothesized that CYP2E1 bioactivation of 1,3-butadiene within mitochondria adversely affects mitochondrial respiratory complexes I-IV. A population of Collaborative Cross mice was exposed to air (control) or 200ppm 1,3-butadiene. Subcellular fractions (mitochondria, DNA, and microsomes) were collected from frozen livers and CYP2E1 activity was measured in microsomes and mitochondria. Individual activities of mitochondrial respiratory complexes I-IV were measured using in vitro assays and purified mitochondrial fractions. In air- and 1,3-butadiene-exposed mouse samples, mtDNA copy numbers were assessed by RT-PCR, and mtDNA integrity was assessed through a PCR-based assay. No significant changes in mtDNA copy number or integrity were observed; however, there was a decrease in overall activity of mitochondrial respiratory complexes I, II, and IV after 1,3-butadiene exposure. Additionally, higher mtCYP2E1 (but not erCYP2E1) activity was correlated with decreased mitochondrial respiratory complex activity (in complexes I-IV) in the 1,3-butadiene-exposed (not control) animals. Together, these results represent the first in vivo link between mitochondrial CYP2E1 activity and mitochondrial toxicity.
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http://dx.doi.org/10.1016/j.tox.2017.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5319481PMC
March 2017

Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines.

Biochim Biophys Acta 2016 Apr 26;1860(4):836-43. Epub 2016 Jan 26.

Department of Environmental and Occupational Health, 4301 W. Markham Street Slot #820, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Electronic address:

Background: Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems.

Methods: The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry.

Results: A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [(13)C5]glutamine demonstrated that by 12h >50% of excreted glutathione was derived from glutamine. Culturing in glutamine-free medium or treatment with BPTES, a GLS-specific inhibitor, reduced cell proliferation and viability and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES-induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity.

Conclusions: We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well.

General Significance: Glutamine is essential for synthesis and excretion of glutathione to promote cell growth and viability.
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http://dx.doi.org/10.1016/j.bbagen.2016.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768472PMC
April 2016

In HepG2 cells, coexisting carnitine deficiency masks important indicators of marginal biotin deficiency.

J Nutr 2015 Jan 19;145(1):32-40. Epub 2014 Nov 19.

Department of Biochemistry and Molecular Biology and

Background: A large number of birth defects are related to nutrient deficiencies; concern that biotin deficiency is teratogenic in humans is reasonable. Surprisingly, studies indicate that increased urinary 3-hydroxyisovalerylcarnitine (3HIAc), a previously validated marker of biotin deficiency, is not a valid biomarker in pregnancy.

Objective: In this study we hypothesized that coexisting carnitine deficiency can prevent the increase in 3HIAc due to biotin deficiency.

Methods: We used a 2-factor nutrient depletion design to induce isolated and combined biotin and carnitine deficiency in HepG2 cells and then repleted cells with carnitine. To elucidate the metabolic pathogenesis, we quantitated intracellular and extracellular free carnitine, acylcarnitines, and acylcarnitine ratios using liquid chromatography-tandem mass spectrometry.

Results: Relative to biotin-sufficient, carnitine-sufficient cells, intracellular acetylcarnitine increased by 90%, propionylcarnitine more than doubled, and 3HIAc increased by >10-fold in biotin-deficient, carnitine-sufficient (BDCS) cells, consistent with a defensive mechanism in which biotin-deficient cells transesterify the acyl-coenzyme A (acyl-CoA) substrates of the biotin-dependent carboxylases to the related acylcarnitines. Likewise, in BDCS cells, the ratio of acetylcarnitine to malonylcarnitine and the ratio of propionylcarnitine to methylmalonylcarnitine both more than tripled, and the ratio of 3HIAc to 3-methylglutarylcarnitine (MGc) increased by >10-fold. In biotin-deficient, carnitine-deficient (BDCD) cells, the 3 substrate-derived acylcarnitines changed little, but the substrate:product ratios were masked to a lesser extent. Moreover, carnitine repletion unmasked biotin deficiency in BDCD cells as shown by increases in acetylcarnitine, propionylcarnitine, and 3HIAc (each increased by >50-fold). Likewise, ratios of acetylcarnitine:malonylcarnitine, propionylcarnitine:methylmalonylcarnitine, and 3HIAc:MGc all increased by >8-fold.

Conclusions: Our findings provide strong evidence that coexisting carnitine deficiency masks some indicators of biotin deficiency and support the potential importance of the ratios of acylcarnitines arising from the acyl-CoA substrates and products for biotin-dependent carboxylases in detecting the biotin deficiency that is masked by coexisting carnitine deficiency.
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http://dx.doi.org/10.3945/jn.114.201343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264022PMC
January 2015

Multiple UDP-glucuronosyltransferases in human liver microsomes glucuronidate both R- and S-7-hydroxywarfarin into two metabolites.

Arch Biochem Biophys 2014 Dec 19;564:244-53. Epub 2014 Oct 19.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA. Electronic address:

The widely used anticoagulant Coumadin (R/S-warfarin) undergoes oxidation by cytochromes P450 into hydroxywarfarins that subsequently become conjugated for excretion in urine. Hydroxywarfarins may modulate warfarin metabolism transcriptionally or through direct inhibition of cytochromes P450 and thus, UGT action toward hydroxywarfarin elimination may impact levels of the parent drugs and patient responses. Nevertheless, relatively little is known about conjugation by UDP-glucuronosyltransferases in warfarin metabolism. Herein, we identified probable conjugation sites, kinetic mechanisms and hepatic UGT isoforms involved in microsomal glucuronidation of R- and S-7-hydroxywarfarin. Both compounds underwent glucuronidation at C4 and C7 hydroxyl groups based on elution properties and spectral characteristics. Their formation demonstrated regio- and enantioselectivity by UGTs and resulted in either Michaelis-Menten or substrate inhibition kinetics. Glucuronidation at the C7 hydroxyl group occurred more readily than at the C4 group, and the reaction was overall more efficient for R-7-hydroxywarfarin due to higher affinity and rates of turnover. The use of these mechanisms and parameters to model in vivo clearance demonstrated that contributions of substrate inhibition would lead to underestimation of metabolic clearance than that predicted by Michaelis-Menten kinetics. Lastly, these processes were driven by multiple UGTs indicating redundancy in glucuronidation pathways and ultimately metabolic clearance of R- and S-7-hydroxywarfarin.
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http://dx.doi.org/10.1016/j.abb.2014.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258421PMC
December 2014

Inhibitory potency of 4-carbon alkanes and alkenes toward CYP2E1 activity.

Toxicology 2014 Apr 18;318:51-8. Epub 2014 Feb 18.

Department of Environmental and Occupational Health, College of Public Health, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 820. Little Rock, AR 72205, Unites States; The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States. Electronic address:

CYP2E1 has been implicated in the bioactivation of many small molecules into reactive metabolites which form adducts with proteins and DNA, and thus a better understanding of the molecular determinants of its selectivity are critical for accurate toxicological predictions. In this study, we determined the potency of inhibition of human CYP2E1 for various 4-carbon alkanes, alkenes and alcohols. In addition, known CYP2E1 substrates and inhibitors including 4-methylpyrazole, aniline, and dimethylnitrosamine were included to determine their relative potencies. Of the 1,3-butadiene-derived metabolites studied, 3,4-epoxy-1-butene was the strongest inhibitor with an IC50 of 110 μM compared to 1700 μM and 6600 μM for 1,2-butenediol and 1,2:3,4-diepoxybutane, respectively. Compared to known inhibitors, inhibitory potency of 3,4-epoxy-1-butene is between 4-methylpyrazole (IC50 = 1.8 μM) and dimethylnitrosamine (IC50 = 230 μM). All three butadiene metabolites inhibit CYP2E1 activity through a simple competitive mechanism. Among the 4-carbon compounds studied, the presence and location of polar groups seems to influence inhibitory potency. To further examine this notion, the investigation was extended to include structurally and chemically similar analogues, including propylene oxide and various butane alcohols. Those results demonstrated preferential recognition of CYP2E1 toward the type and location of polar and hydrophobic structural elements. Taken together, CYP2E1 metabolism may be modified in vivo by exposure to 4-carbon compounds, such as drugs, and nutritional constituents, a finding that highlights the complexity of exposure to mixtures.
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http://dx.doi.org/10.1016/j.tox.2014.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747242PMC
April 2014

Differences in butadiene adduct formation between rats and mice not due to selective inhibition of CYP2E1 by butadiene metabolites.

Toxicol Lett 2013 Nov 8;223(2):221-7. Epub 2013 Sep 8.

Department of Chemistry and Biochemistry, University of Arkansas at Fayetteville, Fayetteville, AR 72701, USA.

CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increases linearly at low BD exposures and then saturates at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC50 studies, EB was the strongest inhibitor (IC50 54μM, mouse; 98μM, rat), BD-diol considerably weaker (IC50 1200μM, mouse; 1000μM, rat), and DEB inhibition nonexistent (IC50>25mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC50 studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis.
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http://dx.doi.org/10.1016/j.toxlet.2013.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831829PMC
November 2013

Potential role of UGT1A4 promoter SNPs in anastrozole pharmacogenomics.

Drug Metab Dispos 2013 Apr 31;41(4):870-7. Epub 2013 Jan 31.

Division of Medical Genetics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

Anastrozole belongs to the nonsteroidal triazole-derivative group of aromatase inhibitors. Recently, clinical trials demonstrated improved antitumoral efficacy and a favorable toxicity with third-generation aromatase inhibitors, compared with tamoxifen. Anastrozole is predominantly metabolized by phase I oxidation with the potential for further phase II glucuronidation. It also, however, is subject to direct N-glucuronidation by UDP-glucuronosyltransferase 1A4 (UGT1A4). Anastrozole pharmacokinetics vary widely among patients, but pharmacogenomic studies of patients treated with anastrozole are sparse. In this study, we examined individual variability in the glucuronidation of anastrozole and its association with UGT1A4 promoter and coding region polymorphisms. In vitro assays using liver microsomal preparations from individual subjects (n = 96) demonstrated 235-fold variability in anastrozole glucuronidation. Anastrozole glucuronidation was correlated (r = 0.99; P < 0.0001) with lamotrigine glucuronidation (a diagnostic substrate for UGT1A4) and with UGT1A4 mRNA expression levels in human liver microsomes (r = 0.99; P < 0.0001). Recombinant UGT1A4 catalyzed anastrozole glucuronidation, which was inhibited by hecogenin (IC50 = 15 µM), a UGT1A4 specific inhibitor. The promoter region of UGT1A4 is polymorphic, and compared with those homozygous for the common allele, lower enzymatic activity was observed in microsomes from individuals heterozygous for -163G
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http://dx.doi.org/10.1124/dmd.112.048157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608453PMC
April 2013

Metabolism of R- and S-warfarin by CYP2C19 into four hydroxywarfarins.

Drug Metab Lett 2012 Sep;6(3):157-64

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 516. Little Rock, AR 72205, USA.

Coumadin (R/S-warfarin) is a highly efficacious and widely used anticoagulant; however, its highly variable metabolism remains an important contributor to uncertainties in therapeutic responses. Pharmacogenetic studies report conflicting findings on the clinical relevance of CYP2C19. A resolution to this controversy is impeded by a lack of de tailon the potential role of CYP2C19 in warfarin metabolism. Consequently, we assessed the efficiency of CYP2C19 metabolism of R- and S-warfarin and explored possible contributions in the liver using in vitro methods. Recombinant CYP2C19 metabolized R- and S-warfarin mainly to 6-, 7-, and 8-hydroxywarfarin, while 4'-hydroxywarfarin was a minormetabolite. Over all R-warfarin metabolism was slightly more efficient than that for S-warfarin. Metabolic pathways thatproduce R-6-, 7-, and 8-hydroxywarfarin in human liver microsomal reactions correlated strongly with CYP2C19 Smephenytoinhydroxylase activity. Similarly, CYP1A2 activity toward phenacetin correlated with formation of R-6 and 7-hydroxywarfarin such that R-8-hydroxywarfarin seems unique to CYP2C19 and possibly a biomarker. In following, CYP2C19 likely impacts R-warfarin metabolism and patient response to therapy. Intriguingly, CYP2C19 may contributeto S-warfarin metabolism in patients, especially when CYP2C9 activity is compromised due to drug interactions orgenetic polymorphisms.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713490PMC
http://dx.doi.org/10.2174/1872312811206030002DOI Listing
September 2012

Cooperative effects for CYP2E1 differ between styrene and its metabolites.

Xenobiotica 2013 Sep 18;43(9):755-64. Epub 2013 Jan 18.

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

Cooperative interactions are frequently observed in the metabolism of drugs and pollutants by cytochrome P450s; nevertheless, the molecular determinants for cooperativity remain elusive. Previously, we demonstrated that steady-state styrene metabolism by CYP2E1 exhibits positive cooperativity. We hypothesized that styrene metabolites have lower affinity than styrene toward CYP2E1 and limited ability to induce cooperative effects during metabolism. To test the hypothesis, we determined the potency and mechanism of inhibition for styrene and its metabolites toward oxidation of 4-nitrophenol using CYP2E1 Supersomes® and human liver microsomes. Styrene inhibited the reaction through a mixed cooperative mechanism with high affinity for the catalytic site (67 µM) and lower affinity for the cooperative site (1100 µM), while increasing substrate turnover at high concentrations. Styrene oxide and 4-vinylphenol possessed similar affinity for CYP2E1. Styrene oxide behaved cooperatively like styrene, but 4-vinylphenol decreased turnover at high concentrations. Styrene glycol was a very poor competitive inhibitor. Among all compounds, there was a positive correlation with binding and hydrophobicity. Taken together, these findings for CYP2E1 further validate contributions of cooperative mechanisms to metabolic processes, demonstrate the role of molecular structure on those mechanisms and underscore the potential for heterotropic cooperative effects between different compounds.
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http://dx.doi.org/10.3109/00498254.2012.760764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979932PMC
September 2013

Exposure profiling of reactive compounds in complex mixtures.

Toxicology 2013 Nov 3;313(2-3):145-150. Epub 2012 Dec 3.

Department of Environmental and Occupational Health, The Winthrop P. Rockefeller Cancer Institute at The University of Arkansas for Medical Sciences, Little Rock, AR, United States.

Humans are constantly exposed to mixtures, such as tobacco smoke, exhaust from diesel, gasoline or new bio-fuels, containing several 1000 compounds, including many known human carcinogens. Covalent binding of reactive compounds or their metabolites to DNA and formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal dose of reactive compounds or their metabolites and are an integral part of science-based risk assessment. However, technical limitations have prevented comprehensive detection of a broad spectrum of adducts simultaneously. Therefore, most studies have focused on measurement of abundant individual adducts. These studies have produced valuable insight into the metabolism of individual carcinogens, but they are insufficient for risk assessment of exposure to complex mixtures. To overcome this limitation, we present herein proof-of-principle for comprehensive exposure assessment, using N-terminal valine adduct profiles as a biomarker. The reported method is based on our previously established immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with modification to enrich all N-terminal valine alkylated peptides. The method was evaluated using alkylated peptide standards and globin reacted in vitro with alkylating agents (1,2-epoxy-3-butene, 1,2:3,4-diepoxybutane, propylene oxide, styrene oxide, N-ethyl-N-nitrosourea and methyl methanesulfonate), known to form N-terminal valine adducts. To demonstrate proof-of-principle, the method was successfully applied to globin from mice treated with four model compounds. The results suggest that this novel approach might be suitable for in vivo biomonitoring.
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http://dx.doi.org/10.1016/j.tox.2012.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868061PMC
November 2013
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