72 results on '"Mirsalis JC"'
Search Results
2. Enhancement of sensitivity and quantification quality in the LC-MS/MS measurement of large biomolecules with sum of MRM (SMRM).
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Tang L, Swezey RR, Green CE, and Mirsalis JC
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- Animals, Feasibility Studies, Limit of Detection, Rats, Rats, Sprague-Dawley, Chromatography, Liquid methods, Tandem Mass Spectrometry methods
- Abstract
Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) provides a simple and efficient means for the measurement of analytes in biological matrices with high selectivity and specificity. LC-MS/MS plays an important role in the pharmaceutical industry and biomedical research, but it requires analytes to be in an ionized form in order to be detected. This can pose a challenge for large molecules such as proteins and peptides, because they can exist in multiple charged forms, and this will reduce the total analyte signal by distributing it into multiple ion peaks with a different number of charges in a mass spectrum. In conventional LC-MS/MS analysis of such macromolecules, one charged form is selected as the precursor ion which is then fragmented by collision-induced dissociation (CID) in MS/MS to generate product ions, a process referred to as multiple-reaction monitoring (MRM). The MRM method minimizes interference from endogenous molecules within biological matrices that share the same molecular weight of the precursor ion, but at the expense of signal intensity as compared to precursor ion intensity. We describe here an approach to boost detection sensitivity and expand dynamic range in the quantitation of large molecules while maintaining analytical specificity using summation of MRM (SMRM) transitions and LC separation technique. Protein image from PDB-101 (PDB101.rscb.org)., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2022
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3. Formulation, Stability, Pharmacokinetic, and Modeling Studies for Tests of Synergistic Combinations of Orally Available Approved Drugs against Ebola Virus In Vivo.
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Finch CL, Dyall J, Xu S, Nelson EA, Postnikova E, Liang JY, Zhou H, DeWald LE, Thomas CJ, Wang A, Xu X, Hughes E, Morris PJ, Mirsalis JC, Nguyen LH, Arolfo MP, Koci B, Holbrook MR, Hensley LE, Jahrling PB, Schmaljohn C, Johansen LM, Olinger GG, Schiffer JT, and White JM
- Abstract
Outbreaks of Ebola ebolavirus (EBOV) have been associated with high morbidity and mortality. Milestones have been reached recently in the management of EBOV disease (EVD) with licensure of an EBOV vaccine and two monoclonal antibody therapies. However, neither vaccines nor therapies are available for other disease-causing filoviruses. In preparation for such outbreaks, and for more facile and cost-effective management of EVD, we seek a cocktail containing orally available and room temperature stable drugs with strong activity against multiple filoviruses. We previously showed that (bepridil + sertraline) and (sertraline + toremifene) synergistically suppress EBOV in cell cultures. Here, we describe steps towards testing these combinations in a mouse model of EVD. We identified a vehicle suitable for oral delivery of the component drugs and determined that, thus formulated the drugs are equally active against EBOV as preparations in DMSO, and they maintain activity upon storage in solution for up to seven days. Pharmacokinetic (PK) studies indicated that the drugs in the oral delivery vehicle are well tolerated in mice at the highest doses tested. Collectively the data support advancement of these combinations to tests for synergy in a mouse model of EVD. Moreover, mathematical modeling based on human oral PK projects that the combinations would be more active in humans than their component single drugs.
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- 2021
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4. Selecting an anti-malarial clinical candidate from two potent dihydroisoquinolones.
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Chen Y, Zhu F, Hammill J, Holbrook G, Yang L, Freeman B, White KL, Shackleford DM, O'Loughlin KG, Charman SA, Mirsalis JC, and Guy RK
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- Animals, Antimalarials pharmacokinetics, Antimalarials toxicity, Biological Availability, Dogs, Hepatocytes drug effects, Heterocyclic Compounds, 4 or More Rings pharmacokinetics, Heterocyclic Compounds, 4 or More Rings pharmacology, Heterocyclic Compounds, 4 or More Rings toxicity, Humans, Isoquinolines pharmacokinetics, Isoquinolines toxicity, Mice, Microsomes, Liver drug effects, Rats, Antimalarials pharmacology, Isoquinolines pharmacology
- Abstract
Background: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described., Methods: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs., Results: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg., Conclusion: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators.
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- 2021
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5. In Vivo Activity of Amodiaquine against Ebola Virus Infection.
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DeWald LE, Johnson JC, Gerhardt DM, Torzewski LM, Postnikova E, Honko AN, Janosko K, Huzella L, Dowling WE, Eakin AE, Osborn BL, Gahagen J, Tang L, Green CE, Mirsalis JC, Holbrook MR, Jahrling PB, Dyall J, and Hensley LE
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- Animals, Disease Models, Animal, Drug Combinations, Female, Macaca mulatta, Male, Amodiaquine therapeutic use, Antiviral Agents therapeutic use, Artemisinins therapeutic use, Hemorrhagic Fever, Ebola drug therapy
- Abstract
During the Ebola virus disease (EVD) epidemic in Western Africa (2013‒2016), antimalarial treatment was administered to EVD patients due to the high coexisting malaria burden in accordance with World Health Organization guidelines. In an Ebola treatment center in Liberia, EVD patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. As artemether and artesunate are derivatives of artemisinin, the beneficial anti-Ebola virus (EBOV) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. Amodiaquine is a widely used antimalarial in the countries that experience outbreaks of EVD and, therefore, holds promise as an approved drug that could be repurposed for treating EBOV infections. We investigated the potential anti-EBOV effect of amodiaquine in a well-characterized nonhuman primate model of EVD. Using a similar 3-day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. However, the treatment regimen did not result in a survival benefit or decrease of disease signs in EBOV-infected animals. While amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral.
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- 2019
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6. In Vitro and In Vivo Activity of Amiodarone Against Ebola Virus.
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Dyall J, Johnson JC, Hart BJ, Postnikova E, Cong Y, Zhou H, Gerhardt DM, Michelotti J, Honko AN, Kern S, DeWald LE, O'Loughlin KG, Green CE, Mirsalis JC, Bennett RS, Olinger GG Jr, Jahrling PB, and Hensley LE
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- Amiodarone pharmacokinetics, Amiodarone therapeutic use, Animals, Chlorocebus aethiops, Female, Guinea Pigs, Hemorrhagic Fever, Ebola drug therapy, Male, Vero Cells, Amiodarone pharmacology, Ebolavirus drug effects
- Abstract
At the onset of the 2013-2016 epidemic of Ebola virus disease (EVD), no vaccine or antiviral medication was approved for treatment. Therefore, considerable efforts were directed towards the concept of drug repurposing or repositioning. Amiodarone, an approved multi-ion channel blocker for the treatment of cardiac arrhythmia, was reported to inhibit filovirus entry in vitro. Compassionate use of amiodarone in EVD patients indicated a possible survival benefit. In support of further clinical testing, we confirmed anti-Ebola virus activity of amiodarone in different cell types. Despite promising in vitro results, amiodarone failed to protect guinea pigs from a lethal dose of Ebola virus.
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- 2018
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7. Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.
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Curreli F, Belov DS, Kwon YD, Ramesh R, Furimsky AM, O'Loughlin K, Byrge PC, Iyer LV, Mirsalis JC, Kurkin AV, Altieri A, and Debnath AK
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- Anti-HIV Agents chemical synthesis, Anti-HIV Agents chemistry, Dose-Response Relationship, Drug, HIV Envelope Protein gp120 metabolism, Humans, Microbial Sensitivity Tests, Molecular Structure, Pyrroles chemical synthesis, Pyrroles chemistry, Structure-Activity Relationship, Anti-HIV Agents pharmacology, Computational Biology, HIV drug effects, HIV Envelope Protein gp120 antagonists & inhibitors, Pyrroles pharmacology
- Abstract
We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)
- Published
- 2018
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8. Efficacy of Tilorone Dihydrochloride against Ebola Virus Infection.
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Ekins S, Lingerfelt MA, Comer JE, Freiberg AN, Mirsalis JC, O'Loughlin K, Harutyunyan A, McFarlane C, Green CE, and Madrid PB
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- Animals, Antiviral Agents pharmacology, Caco-2 Cells, Cell Line, Tumor, Female, Humans, Male, Mice, Mice, Inbred BALB C, Microsomes, Liver drug effects, Ebolavirus drug effects, Hemorrhagic Fever, Ebola drug therapy, Tilorone pharmacology
- Abstract
Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was ∼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD., (Copyright © 2018 American Society for Microbiology.)
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- 2018
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9. Evaluation of Cardiac Toxicity Biomarkers in Rats from Different Laboratories.
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Kim K, Chini N, Fairchild DG, Engle SK, Reagan WJ, Summers SD, and Mirsalis JC
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- Animals, Cardiotoxicity, Drug Evaluation, Preclinical, Myocardium metabolism, Myocardium pathology, Rats, Biomarkers blood, Drug Discovery standards, Drug-Related Side Effects and Adverse Reactions blood, Heart drug effects, Laboratories standards
- Abstract
There is a great need for improved diagnostic and prognostic accuracy of potential cardiac toxicity in drug development. This study reports the evaluation of several commercially available biomarker kits by 3 institutions (SRI, Eli Lilly, and Pfizer) for the discrimination between myocardial degeneration/necrosis and cardiac hypertrophy as well as the assessment of the interlaboratory and interplatform variation in results. Serum concentrations of natriuretic peptides (N-terminal pro-atrial natriuretic peptide [NT-proANP] and N-terminal pro-brain natriuretic peptide [NT-proBNP]), cardiac and skeletal troponins (cTnI, cTnT, and sTnI), myosin light chain 3 (Myl3), and fatty acid binding protein 3 (FABP3) were assessed in rats treated with minoxidil (MNX) and isoproterenol (ISO). MNX caused increased heart-to-body weight ratios and prominent elevations in NT-proANP and NT-proBNP concentrations detected at 24-hr postdose without elevation in troponins, Myl3, or FABP3 and with no abnormal histopathological findings. ISO caused ventricular leukocyte infiltration, myocyte fibrosis, and necrosis with increased concentrations of the natriuretic peptides, cardiac troponins, and Myl3. These results reinforce the advantages of a multimarker strategy in elucidating the underlying cause of cardiac insult and detecting myocardial tissue damage at 24-hr posttreatment. The interlaboratory and interplatform comparison analyses also showed that the data obtained from different laboratories and platforms are highly correlated and reproducible, making these biomarkers widely applicable in preclinical studies.
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- 2016
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10. Evaluation of the Activity of Lamivudine and Zidovudine against Ebola Virus.
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Cong Y, Dyall J, Hart BJ, DeWald LE, Johnson JC, Postnikova E, Zhou H, Gross R, Rojas O, Alexander I, Josleyn N, Zhang T, Michelotti J, Janosko K, Glass PJ, Flint M, McMullan LK, Spiropoulou CF, Mierzwa T, Guha R, Shinn P, Michael S, Klumpp-Thomas C, McKnight C, Thomas C, Eakin AE, O'Loughlin KG, Green CE, Catz P, Mirsalis JC, Honko AN, Olinger GG Jr, Bennett RS, Holbrook MR, Hensley LE, and Jahrling PB
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- Animals, Chlorocebus aethiops, Ebolavirus isolation & purification, Guinea Pigs, HeLa Cells, Hemorrhagic Fever, Ebola virology, Humans, Macrophages, Pilot Projects, Vero Cells, Virus Replication drug effects, Anti-HIV Agents pharmacology, Ebolavirus drug effects, Hemorrhagic Fever, Ebola drug therapy, Lamivudine pharmacology, Zidovudine pharmacology
- Abstract
In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD., Competing Interests: The SRI international affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.
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- 2016
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11. Preclinical Evaluations To Identify Optimal Linezolid Regimens for Tuberculosis Therapy.
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Brown AN, Drusano GL, Adams JR, Rodriquez JL, Jambunathan K, Baluya DL, Brown DL, Kwara A, Mirsalis JC, Hafner R, and Louie A
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- Cell Line, Cell Survival, Drug Evaluation, Preclinical, Humans, Microbial Viability, Models, Biological, Antitubercular Agents administration & dosage, Antitubercular Agents adverse effects, Linezolid administration & dosage, Linezolid adverse effects, Mycobacterium tuberculosis drug effects, Tuberculosis drug therapy
- Abstract
Unlabelled: Linezolid is an oxazolidinone with potent activity against Mycobacterium tuberculosis. Linezolid toxicity in patients correlates with the dose and duration of therapy. These toxicities are attributable to the inhibition of mitochondrial protein synthesis. Clinically relevant linezolid regimens were simulated in the in vitro hollow-fiber infection model (HFIM) system to identify the linezolid therapies that minimize toxicity, maximize antibacterial activity, and prevent drug resistance. Linezolid inhibited mitochondrial proteins in an exposure-dependent manner, with toxicity being driven by trough concentrations. Once-daily linezolid killed M. tuberculosis in an exposure-dependent manner. Further, 300 mg linezolid given every 12 hours generated more bacterial kill but more toxicity than 600 mg linezolid given once daily. None of the regimens prevented linezolid resistance. These findings show that with linezolid monotherapy, a clear tradeoff exists between antibacterial activity and toxicity. By identifying the pharmacokinetic parameters linked with toxicity and antibacterial activity, these data can provide guidance for clinical trials evaluating linezolid in multidrug antituberculosis regimens., Importance: The emergence and spread of multidrug-resistant M. tuberculosis are a major threat to global public health. Linezolid is an oxazolidinone that is licensed for human use and has demonstrated potent activity against multidrug-resistant M. tuberculosis. However, long-term use of linezolid has shown to be toxic in patients, often resulting in thrombocytopenia. We examined therapeutic linezolid regimens in an in vitro model to characterize the exposure-toxicity relationship. The antibacterial activity against M. tuberculosis was also assessed for these regimens, including the amplification or suppression of resistant mutant subpopulations by the chosen regimen. Higher exposures of linezolid resulted in greater antibacterial activity, but with more toxicity and, for some regimens, increased resistant mutant subpopulation amplification, illustrating the trade-off between activity and toxicity. These findings can provide valuable insight for designing optimal dosage regimens for linezolid that are part of the long combination courses used to treat multidrug-resistant M. tuberculosis., (Copyright © 2015 Brown et al.)
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- 2015
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12. Preclinical studies on the pharmacokinetics, safety, and toxicology of oxfendazole: toward first in human studies.
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Codd EE, Ng HH, McFarlane C, Riccio ES, Doppalapudi R, Mirsalis JC, Horton RJ, Gonzalez AE, Garcia HH, and Gilman RH
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- Administration, Oral, Animals, Anthelmintics adverse effects, Anthelmintics toxicity, Benzimidazoles adverse effects, Benzimidazoles toxicity, Cardiovascular System drug effects, Dogs, Dose-Response Relationship, Drug, Female, Leukemia L5178 genetics, Male, Mice, Micronucleus Tests, Mutagenicity Tests, Rats, Rats, Sprague-Dawley, Anthelmintics pharmacokinetics, Benzimidazoles pharmacokinetics
- Abstract
A 2-week study in rats identified target organs of oxfendazole toxicity to be bone marrow, epididymis, liver, spleen, testis, and thymus. Female rats had greater oxfendazole exposure and exhibited toxicities at lower doses than did males. Decreased white blood cell levels, a class effect of benzimidazole anthelmintics, returned to normal during the recovery period. The no observed adverse effect level was determined to be >5 but <25 mg/kg/d and the maximum tolerated dose 100 mg/kg/d. The highest dose, 200 mg/kg/d, resulted in significant toxicity and mortality, leading to euthanization of the main study animals in this group after 7 days. Oxfendazole did not exhibit genetic toxicology signals in standard Ames bacterial, mouse lymphoma, or rat micronucleus assays nor did it provoke safety concerns when evaluated for behavioral effects in rats or cardiovascular safety effects in dogs. These results support the transition of oxfendazole to First in Human safety studies preliminary to its evaluation in human helminth diseases., (© The Author(s) 2015.)
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- 2015
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13. Tenofovir disoproxil fumarate: toxicity, toxicokinetics, and toxicogenomics analysis after 13 weeks of oral administration in mice.
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Ng HH, Stock H, Rausch L, Bunin D, Wang A, Brill S, Gow J, and Mirsalis JC
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- Adenine blood, Adenine pharmacokinetics, Adenine toxicity, Administration, Oral, Animals, Anti-HIV Agents blood, Anti-HIV Agents pharmacokinetics, Cyclin-Dependent Kinase Inhibitor p21 genetics, Female, Gene Expression Profiling, Kidney anatomy & histology, Kidney metabolism, Liver metabolism, Liver pathology, Mice, Inbred BALB C, Oligonucleotide Array Sequence Analysis, Organophosphonates blood, Organophosphonates pharmacokinetics, Reverse Transcriptase Inhibitors blood, Reverse Transcriptase Inhibitors pharmacokinetics, Tenofovir, Toxicity Tests, Subchronic, Transcription, Genetic drug effects, Adenine analogs & derivatives, Anti-HIV Agents toxicity, Kidney drug effects, Liver drug effects, Organophosphonates toxicity, Reverse Transcriptase Inhibitors toxicity
- Abstract
Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir that exhibits activity against HIV and hepatitis B. The goals of this study were to evaluate the molecular mechanism of TDF-induced toxicity in mice after 13 weeks of daily oral administration (50-1000 mg/kg) by correlating transcriptional changes with plasma drug levels and traditional toxicology end points. Plasma levels and systemic exposure of tenofovir increased less than dose proportionally and were similar on days 1 and 91. No overt toxicity was observed following the completion of TDF administration. The kidneys of TDF-treated mice were histopathologically normal. This result is consistent with the genomic microarray results, which showed no significant differences in kidney transcriptional levels between TDF-treated animals and controls. In liver, after 4 and 13 weeks, cytomegaly was observed in mice treated with 1000 mg/kg of TDF, but mice recovered from this effect following cessation of administration. Analysis of liver transcripts on day 91 reported elevated levels of Cdkn1a in TDF-treated animals compared with controls, which may have contributed to the inhibition of liver cell cycle progression., (© The Author(s) 2015.)
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- 2015
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14. (+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium.
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Jiménez-Díaz MB, Ebert D, Salinas Y, Pradhan A, Lehane AM, Myrand-Lapierre ME, O'Loughlin KG, Shackleford DM, Justino de Almeida M, Carrillo AK, Clark JA, Dennis AS, Diep J, Deng X, Duffy S, Endsley AN, Fedewa G, Guiguemde WA, Gómez MG, Holbrook G, Horst J, Kim CC, Liu J, Lee MC, Matheny A, Martínez MS, Miller G, Rodríguez-Alejandre A, Sanz L, Sigal M, Spillman NJ, Stein PD, Wang Z, Zhu F, Waterson D, Knapp S, Shelat A, Avery VM, Fidock DA, Gamo FJ, Charman SA, Mirsalis JC, Ma H, Ferrer S, Kirk K, Angulo-Barturen I, Kyle DE, DeRisi JL, Floyd DM, and Guy RK
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- Antimalarials pharmacokinetics, Calcium-Transporting ATPases genetics, Cellular Senescence drug effects, Drug Discovery, Drug Resistance genetics, Erythrocytes drug effects, Flow Cytometry, Heterocyclic Compounds, 4 or More Rings pharmacokinetics, High-Throughput Screening Assays, Isoquinolines pharmacokinetics, Molecular Structure, Antimalarials pharmacology, Calcium-Transporting ATPases metabolism, Heterocyclic Compounds, 4 or More Rings pharmacology, Isoquinolines pharmacology, Malaria drug therapy, Models, Molecular, Plasmodium drug effects
- Abstract
Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.
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- 2014
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15. Evaluation of arylimidamides DB1955 and DB1960 as candidates against visceral leishmaniasis and Chagas' disease: in vivo efficacy, acute toxicity, pharmacokinetics, and toxicology studies.
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Zhu X, Liu Q, Yang S, Parman T, Green CE, Mirsalis JC, de Nazaré Correia Soeiro M, Mello de Souza E, da Silva CF, da Gama Jaen Batista D, Stephens CE, Banerjee M, Farahat AA, Munde M, Wilson WD, Boykin DW, Wang MZ, and Werbovetz KA
- Subjects
- Amidines therapeutic use, Animals, Antiprotozoal Agents pharmacology, Disease Models, Animal, Female, Furans therapeutic use, Male, Mice, Mice, Inbred BALB C, Parasitemia drug therapy, Solubility, Trypanosoma cruzi drug effects, Trypanosoma cruzi pathogenicity, Antiprotozoal Agents pharmacokinetics, Antiprotozoal Agents therapeutic use, Chagas Disease drug therapy, Leishmaniasis, Visceral drug therapy
- Abstract
Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels.
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- 2012
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16. Metabolism, pharmacokinetics, tissue distribution, and stability studies of the prodrug analog of an anti-hepatitis B virus dinucleoside phosphorothioate.
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Coughlin JE, Pandey RK, Padmanabhan S, O'Loughlin KG, Marquis J, Green CE, Mirsalis JC, and Iyer RP
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- Administration, Oral, Animals, Biotransformation, Chromatography, High Pressure Liquid, Drug Design, Drug Stability, Female, Gastric Juice chemistry, Humans, In Vitro Techniques, Injections, Intravenous, Intestinal Secretions chemistry, Male, Mass Spectrometry, Microsomes, Liver enzymology, Microsomes, Liver metabolism, Models, Biological, Molecular Structure, Rats, Rats, Sprague-Dawley, Stereoisomerism, Tissue Distribution, Antiviral Agents chemistry, Antiviral Agents metabolism, Antiviral Agents pharmacokinetics, Hepatitis B virus drug effects, Phosphorothioate Oligonucleotides chemistry, Phosphorothioate Oligonucleotides metabolism, Phosphorothioate Oligonucleotides pharmacokinetics, Prodrugs chemistry, Prodrugs metabolism, Prodrugs pharmacokinetics
- Abstract
The alkoxycarbonyloxy dinucleotide prodrug R(p), S(p)-2 is an orally bioavailable anti-hepatitis B virus agent. The compound is efficiently metabolized to the active dinucleoside phosphorothioate R(p), S(p)-1 by human liver microsomes and S9 fraction without cytochrome P450-mediated oxidation or conjugation. The conversion of R(p), S(p)-2 to R(p), S(p)-1 appears to be mediated by liver esterases, occurs in a stereospecific manner, and is consistent with our earlier reported studies of serum-mediated hydrolytic conversion of R(p), S(p)-2 to R(p), S(p)-1. However, further metabolism of R(p), S(p)-1 does not occur. The presence of a minor metabolite, the desulfurized product 10 was noted. The prodrug R(p), S(p)-2 was quite stable in simulated gastric fluid, whereas the active R(p), S(p)-1 had a half-life of <15 min. In simulated intestinal fluid, the prodrug 2 was fully converted to 1 in approximately 3 h, whereas 1 remained stable. To ascertain the tissue distribution of the prodrug 2 in rats, the synthesis of (35)S-labeled R(p), S(p)-2 was undertaken. Tissue distribution studies of orally and intravenously administered radiolabeled [(35)S]2 demonstrated that the radioactivity concentrates in the liver, with the highest liver/plasma ratio in the intravenous group at 1 h being 3.89 (females) and in the oral group at 1 h being 2.86 (males). The preferential distribution of the dinucleotide 1 and its prodrug 2 into liver may be attributed to the presence of nucleoside phosphorothioate backbone because phosphorothioate oligonucleotides also reveal a similar tissue distribution profile upon intravenous administration.
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- 2012
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17. Biodistribution, toxicology, and radiation dosimetry of 5-HT1A-receptor agonist positron emission tomography ligand [11C]CUMI-101.
- Author
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Kumar DJ, Bai B, Ng HH, Mirsalis JC, Erlandsson K, Milak MS, Majo VJ, Prabhakaran J, Mann JJ, and Parsey RV
- Subjects
- Animals, Carbon Radioisotopes, Female, Ligands, Male, Papio, Piperazines toxicity, Positron-Emission Tomography, Radiometry, Rats, Rats, Sprague-Dawley, Serotonin 5-HT1 Receptor Agonists toxicity, Tissue Distribution, Triazines toxicity, Piperazines pharmacokinetics, Serotonin 5-HT1 Receptor Agonists pharmacokinetics, Triazines pharmacokinetics
- Abstract
Sprague Dawley rats (10/sex/group) were given a single intravenous (iv) dose of CUMI-101 to determine acute toxicity of CUMI-101 and radiation dosimetry estimations were conducted in baboons with [(11)C]CUMI-101. Intravenous administration of CUMI-101 did not produce overt biologically or toxicologically significant adverse effects except transient hypoactivity immediately after dose in the mid- and high-dose groups, which is not considered to be a dose-limiting toxic effect. No adverse effects were observed in the low-dose group. The no observed adverse effect level (NOAEL) is considered to be 44.05 µg/kg for a single iv dose administration in rats. The maximum tolerated dose (MTD) was estimated to be 881 µg/kg for a single iv dose administration. The Medical Internal Radiation Dose (MIRDOSE) estimates indicate the maximum permissible single-study dosage of [(11)C]CUMI-101 in humans is 52 mCi with testes and urinary bladder as the critical organ for males and females, respectively.
- Published
- 2011
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18. WITHDRAWN. Neurological assessments after treatment with the antimalarial β-arteether in neonatal and adult rats.
- Author
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Erickson RI, Defensor EB, Fairchild DG, Mirsalis JC, and Steinmetz KL
- Abstract
The World Health Organization currently recommends combinatorial treatment including artemisinins as first-line therapy against drug-resistant Plasmodium falciparum malaria. Although highly efficacious, artemisinin and its derivatives, including β-arteether (βAE), are associated with ototoxicity, tremors, and other autonomic and motor impairments in the clinic. Similar neurological symptoms, as well as brainstem lesions, have been observed in adult laboratory species (mice, rats, dogs, and non human primates) following acute treatment with βAE; however, few long-term, nonclinical studies have been conducted. Furthermore, the majority of deaths attributed to malarial infection occur in children under age five, yet no laboratory studies have been initiated in neonatal or juvenile animals. In the current study, neonatal 7-day-old rats were administered intramuscular doses of 1-90mg/kg βAE in sesame oil for up to eight treatment cycles (one cycle=7days treatment+7days without treatment). Neonates were tested for changes in sensorimotor function, and the same animals were tested as adults in the Functional Observational Battery, for motor activity, and in the 8-arm radial maze. Pups receiving a single cycle of 60 or 90mg/kg died within a week of treatment but had few behavioral changes and no brainstem pathology. In the long-term study, behavioral and motor changes and brainstem lesions were observed in a dose- and time-related manner. Rats given repeated cycles of 1 or 5mg/kg βAE showed subtle motor abnormalities (e.g., slight loss of righting reflex) while repeated cycles of 10mg/kg βAE treatment resulted in obvious motor and behavioral changes. Rats receiving 1mg/kg βAE had no brainstem lesions whereas some rats treated with 5mg/kg βAE and all rats treated with 10mg/kg βAE had brainstem lesions. Brainstem lesions were observed after as few as five cycles and were characterized by gliosis, satellitosis and progressive necrosis in motor neurons of the trapezoid, vestibular, and olivary nuclei. This study shows that repeated treatment with clinically relevant doses of βAE causes motor deficits associated with brainstem damage in rodents and suggests that repeated treatment with βAE in children may elicit neurological damage., (Copyright © 2011. Published by Elsevier B.V.)
- Published
- 2011
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19. Neurological assessments after treatment with the antimalarial β-arteether in neonatal and adult rats.
- Author
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Erickson RI, Defensor EB, Fairchild DG, Mirsalis JC, and Steinmetz KL
- Subjects
- Age Factors, Aging, Animals, Animals, Newborn, Antimalarials administration & dosage, Artemisinins administration & dosage, Brain Stem pathology, Dose-Response Relationship, Drug, Drug Administration Schedule, Injections, Intramuscular, Maze Learning drug effects, Motor Activity drug effects, Necrosis, Neurologic Examination, Neurotoxicity Syndromes pathology, Neurotoxicity Syndromes psychology, Rats, Rats, Sprague-Dawley, Risk Assessment, Antimalarials toxicity, Artemisinins toxicity, Behavior, Animal drug effects, Brain Stem drug effects, Neurotoxicity Syndromes etiology
- Abstract
The World Health Organization currently recommends combinatorial treatment including artemisinins as first-line therapy against drug-resistant Plasmodium falciparum malaria. Although highly efficacious, artemisinin and its derivatives, including β-arteether (βAE), are associated with ototoxicity, tremors, and other autonomic and motor impairments in the clinic. Similar neurological symptoms, as well as brainstem lesions, have been observed in adult laboratory species (mice, rats, dogs, and non human primates) following acute treatment with βAE; however, few long-term, nonclinical studies have been conducted. Furthermore, the majority of deaths attributed to malarial infection occur in children under age five, yet no laboratory studies have been initiated in neonatal or juvenile animals. In the current study, neonatal 7-day-old rats were administered intramuscular doses of 1-90 mg/kg βAE in sesame oil for up to eight treatment cycles (one cycle=7 days treatment+7 days without treatment). Neonates were tested for changes in sensorimotor function, and the same animals were tested as adults in the Functional Observational Battery, for motor activity, and in the 8-arm radial maze. Pups receiving a single cycle of 60 or 90 mg/kg died within a week of treatment but had few behavioral changes and no brainstem pathology. In the long-term study, behavioral and motor changes and brainstem lesions were observed in a dose- and time-related manner. Rats given repeated cycles of 1 or 5mg/kg βAE showed subtle motor abnormalities (e.g., slight loss of righting reflex) while repeated cycles of 10mg/kg βAE treatment resulted in obvious motor and behavioral changes. Rats receiving 1mg/kg βAE had no brainstem lesions whereas some rats treated with 5mg/kg βAE and all rats treated with 10 mg/kg βAE had brainstem lesions. Brainstem lesions were observed after as few as five cycles and were characterized by gliosis, satellitosis and progressive necrosis in motor neurons of the trapezoid, vestibular, and olivary nuclei. This study shows that repeated treatment with clinically relevant doses of βAE causes motor deficits associated with brainstem damage in rodents and suggests that repeated treatment with βAE in children may elicit neurological damage., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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20. Development of a new generation of 4-aminoquinoline antimalarial compounds using predictive pharmacokinetic and toxicology models.
- Author
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Ray S, Madrid PB, Catz P, LeValley SE, Furniss MJ, Rausch LL, Guy RK, DeRisi JL, Iyer LV, Green CE, and Mirsalis JC
- Subjects
- Aminoquinolines therapeutic use, Animals, Antimalarials pharmacology, Drug Evaluation, Preclinical, Half-Life, Mice, Pharmacokinetics, Plasmodium falciparum drug effects, Small Molecule Libraries, Toxicology, Aminoquinolines pharmacology, Antimalarials chemistry
- Abstract
Among the known antimalarial drugs, chloroquine (CQ) and other 4-aminoquinolines have shown high potency and good bioavailability. Yet complications associated with drug resistance necessitate the discovery of effective new antimalarial agents. ADMET prediction studies were employed to evaluate a library of new molecules based on the 4-aminoquinolone-related structure of CQ. Extensive in vitro screening and in vivo pharmacokinetic studies in mice helped to identify two lead molecules, 18 and 4, with promising in vitro therapeutic efficacy, improved ADMET properties, low risk for drug-drug interactions, and desirable pharmacokinetic profiles. Both 18 and 4 are highly potent antimalarial compounds, with IC(50) values of 5.6 and 17.3 nM, respectively, against the W2 (CQ-resistant) strain of Plasmodium falciparum (for CQ, IC(50) = 382 nM). When tested in mice, these compounds were found to have biological half-lives and plasma exposure values similar to or higher than those of CQ; they are therefore desirable candidates to pursue in future clinical trials.
- Published
- 2010
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21. Biodistribution, toxicity and radiation dosimetry studies of the serotonin transporter radioligand 4-[18F]-ADAM in rats and monkeys.
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Huang YY, Ma KH, Tseng TW, Chou TK, Ng H, Mirsalis JC, Fu YK, Chu TC, Huang WS, and Shiue CY
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- Adult, Animals, Benzylamines chemistry, Benzylamines metabolism, Female, Humans, Male, Radiation Dosage, Radioligand Assay, Radiometry, Rats, Tissue Distribution, Benzylamines pharmacokinetics, Benzylamines toxicity, Fluorine Radioisotopes chemistry, Haplorhini, Serotonin Plasma Membrane Transport Proteins metabolism
- Abstract
Purpose: 4-[(18)F]-ADAM is a potent serotonin transport imaging agent. We studied its toxicity in rats and radiation dosimetry in monkeys before human studies are undertaken., Methods: Single and multiple-dosage toxicity studies were conducted in Sprague-Dawley rats. Male and female rats were injected intravenously with 4-F-ADAM as a single dose of 1,023.7 microg/kg (1,000 times the human dose) or as five consecutive daily doses of 102.37 microg/kg (100 times the human dose). PET/CT scans were performed in seven Formosa Rock monkeys (four males and three females) using a Siemens Biograph scanner. After injection of 4-[(18)F]-ADAM (182+/-8 MBq), a low dose CT scan and a series of eight whole-body PET scans were performed. Whole-body images were acquired in 3-D mode. Time-activity data of source organs were used to calculate the residence times and estimate the absorbed radiation dose using OLINDA/EXM software., Results: In the rats neither the single dose nor the five daily doses of 4-F-ADAM produced overt adverse effects clinically. In the monkeys the radiation doses received by most organs ranged between 7.1 and 35.7 microGy/MBq, and the urinary bladder was considered to be the critical organ. The effective doses extrapolated to male and female adult humans were 17.4 and 21.8 microSv/MBq, respectively., Conclusion: Toxicity studies in Sprague-Dawley rats and radiation dosimetry studies in Formosa Rock monkeys suggested that 4-[(18)F]-ADAM is safe for use in human PET imaging studies.
- Published
- 2010
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22. Gene expression analysis reveals new possible mechanisms of vancomycin-induced nephrotoxicity and identifies gene markers candidates.
- Author
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Dieterich C, Puey A, Lin S, Swezey R, Furimsky A, Fairchild D, Mirsalis JC, and Ng HH
- Subjects
- Alkaline Phosphatase analysis, Analysis of Variance, Animals, Anti-Bacterial Agents administration & dosage, Blood Urea Nitrogen, Cluster Analysis, Female, Gene Expression drug effects, Inflammation metabolism, Injections, Intraperitoneal, Injections, Intravenous, Kidney chemistry, Kidney pathology, Mice, Mice, Inbred BALB C, Oligonucleotide Array Sequence Analysis, Oxidative Stress drug effects, Signal Transduction drug effects, Vancomycin administration & dosage, gamma-Glutamyltransferase analysis, Anti-Bacterial Agents toxicity, Biomarkers metabolism, Kidney Diseases chemically induced, Kidney Diseases metabolism, Vancomycin toxicity
- Abstract
Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription-polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury.
- Published
- 2009
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23. Microarray evaluation of the Listeria monocytogenes infection and amoxicillin treatment in mice.
- Author
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Dieterich C, Iglehart D, Riccio E, Mirsalis JC, and Ng HH
- Subjects
- Animals, Female, Gene Expression Profiling, Genes, Bacterial, Genetic Markers, Listeria monocytogenes genetics, Mice, Mice, Inbred BALB C, Amoxicillin therapeutic use, Anti-Bacterial Agents therapeutic use, Listeriosis drug therapy, Listeriosis genetics, Oligonucleotide Array Sequence Analysis
- Abstract
By using Affymetrix Mouse Genome Arrays and 20 biological replicates per experimental condition, the predictive value of liver and blood gene expression profiles previously identified was validated as predictive of Listeria monocytogenes infection severity (lethal and nonlethal infection). The ability of these genes to predict the outcome of antibiotic treatment was also assessed. Lethally infected BALB/c mice were treated with amoxicillin at 10 or 20 mg/kg; only the higher dose prevented death. The liver genes predicted that 70% of the animals treated at 10 mg/kg, but only 25% of the mice treated at 20 mg/kg, belonged to the lethal infection group, and this prediction was similar to the ultimate mortality outcome. These results confirm the value of microarrays as tools to predict host response to infection and efficacy of antibacterial therapy. These results might lead to applications that would help clinicians to adjust antibiotic dosages for efficient treatment but yet without toxicity.
- Published
- 2008
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24. Evaluation of chemopreventive agents for genotoxic activity.
- Author
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Doppalapudi RS, Riccio ES, Rausch LL, Shimon JA, Lee PS, Mortelmans KE, Kapetanovic IM, Crowell JA, and Mirsalis JC
- Subjects
- Animals, CHO Cells, Chemoprevention adverse effects, Chromosome Aberrations chemically induced, Cricetinae, Cricetulus, Escherichia coli drug effects, Escherichia coli genetics, Female, Humans, In Vitro Techniques, Leukemia L5178, Male, Mice, Micronucleus Tests, Mutagenicity Tests methods, Salmonella typhimurium drug effects, Salmonella typhimurium genetics, Anticarcinogenic Agents toxicity, Mutagens toxicity
- Abstract
We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay, for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, and for micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate (BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4'-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid (alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7alpha-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative in the Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenic response in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the 8 compounds that were tested, 4'-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positive in the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomal aberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.
- Published
- 2007
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25. Preclinical acute toxicity studies and dosimetry estimates of the novel sigma-1 receptor radiotracer, [18F]SFE.
- Author
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Waterhouse RN, Zhao J, Stabin MG, Ng H, Schindler-Horvat J, Chang RC, and Mirsalis JC
- Subjects
- Animals, Cardiovascular System diagnostic imaging, Cardiovascular System drug effects, Dogs, Drug Evaluation, Preclinical, Female, Male, Maximum Tolerated Dose, Piperidines adverse effects, Positron-Emission Tomography methods, Rabbits, Radiation Dosage, Radioactive Tracers, Tissue Distribution, Sigma-1 Receptor, Piperidines administration & dosage, Piperidines toxicity, Receptors, sigma metabolism
- Abstract
[(18)F]1-(2-Fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]SFE) is a novel, selective, high-affinity sigma-1 receptor radioligand that has been preclinically well characterized in rodents. To support an investigational new drug (IND) application for the first evaluation of [(18)F]SFE in humans, single-organ and whole-body radiation adsorbed doses associated with [(18)F]SFE injection were estimated from rat distribution data. In addition, single- and multiple-dose toxicity studies were conducted in rabbits and in dogs. Multiple-dose toxicity studies in rabbits and single-dose toxicity studies in beagles suggest at least a 100-fold safety margin for humans studies at a mass dose limit of 4.0 mug per intravenous injection, based on the combined no observable adverse effect levels (NOAEL, mg/m(2)) measured in these species. Radiation dosimetry estimates obtained from rat biodistribution analyses of [(18)F]SFE suggest that most tissues would receive about 0.010-0.020 mGy/MBq, while the adrenal glands, brain, bone, liver, lungs, and spleen would receive slightly higher doses (0.024-0.044 mGy/MBq). The adrenal glands were identified as the critical organ, because they received the highest adsorbed radiation dose. The total exposure resulting from a 5 mCi administration of [(18)F]SFE is well below the FDA-defined limits for yearly cumulative and per-study exposures to research participants. These combined results support the expectation that [(18)F]SFE will be safe for use in human positron emission tomography (PET) imaging studies with the administration of 5 mCi and a mass dose equal to or less than 4.0 mug SFE per injection.
- Published
- 2006
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26. Gene expression profiling of mouse host response to Listeria monocytogenes infection.
- Author
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Ng HH, Frantz CE, Rausch L, Fairchild DC, Shimon J, Riccio E, Smith S, and Mirsalis JC
- Subjects
- Alanine Transaminase blood, Analysis of Variance, Animals, Aspartate Aminotransferases blood, Cluster Analysis, DNA Primers, Female, Listeriosis pathology, Liver metabolism, Liver pathology, Mice, Mice, Inbred BALB C, Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Genes genetics, Listeria monocytogenes, Listeriosis blood, Listeriosis metabolism
- Abstract
The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.
- Published
- 2005
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27. Evaluation of mutant frequencies of chemically induced tumors and normal tissues in lambda/cII transgenic mice.
- Author
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Mirsalis JC, Shimon JA, Johnson A, Fairchild D, Kanazawa N, Nguyen T, de Boer J, Glickman B, and Winegar RA
- Subjects
- Animals, Clone Cells, Female, Gene Frequency, Liver ultrastructure, Liver Neoplasms, Experimental chemically induced, Liver Neoplasms, Experimental pathology, Lung ultrastructure, Lung Neoplasms chemically induced, Lung Neoplasms pathology, Male, Mice, Mice, Transgenic, Mutation, Oxazepam, Phenobarbital, Carcinogens, Diethylnitrosamine, Liver Neoplasms, Experimental genetics, Lung Neoplasms genetics
- Abstract
Genomic instability has been implicated as an important component in tumor progression. Evaluation of mutant frequencies (MFs) in tumors of transgenic mice containing nontranscribed marker genes should be useful for quantitating mutation rates in tumors as the physiologically inactive transgene provides neither a positive nor a negative selective pressure on the tumor. We have conducted long-term carcinogenicity studies in lambda/cII transgenic B6C3F1 mice using a variety of genotoxic and nongenotoxic test agents and have evaluated the mutant frequencies in both tumors and normal tissues from these animals. Mice were administered diethylnitrosamine (DEN) as three intraperitoneal injections of 15 mg/kg; phenobarbital (PB) or oxazepam (OXP) provided ad libitum at 0.1% or 0.25% in the diet, respectively; DEN initiation plus PB in the diet; or urethane (UTH) provided ad libitum at 0.2% in the drinking water. Normal tissues and tumors were isolated at various times over a 2-year period and half of each tissue/tumor was evaluated histopathologically and the other half was evaluated for MF in the cII transgene. Approximately 20 mutants from each of 166 individual tissues (tumor and nontumor) were sequenced to determine whether increases in MF represented unique mutations or were due to clonal expansion. UTH produced significant increases in MF in normal liver and lung. DEN either with or without PB promotion produced significant increases in MF in liver and correction of MF for clonality produced little change in the overall MF in these groups. PB produced a twofold increase in liver MF over controls after 27 weeks of treatment, but a similar increase was not observed with longer dosing times; at later time points, the MF in the PB groups was lower than that of the control group, suggesting that PB is not producing direct DNA damage in the liver. OXP failed to produce an increase in MF over controls, even after 78 weeks of treatment. Selected cases of genomic instability were observed in tumors from all treatments except OXP, with individual liver tumors showing very high MF values even after clonal correction. One rare and interesting finding was noted in a single mouse treated with UTH, where a mammary metastasis had an MF approximately 10-fold greater than the parent tumor, with 75% of the mutations independent, providing strong evidence of genomic instability. There was no clear correlation between tumor phenotype and MF except that pulmonary adenomas generally had higher MFs than normal lung in both genotoxic and nongenotoxic treatment groups. Likewise, there was no correlation between tumor size and MF after correction for clonality. The results presented here demonstrate that individual tumors can show significant genomic instability, with very significant increases in MF that are not attributed to clonal expansion of a single mutant cell., (2004 Wiley-Liss, Inc.)
- Published
- 2005
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28. Pharmacokinetics of the antimalarial drug, AQ-13, in rats and cynomolgus macaques.
- Author
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Ramanathan-Girish S, Catz P, Creek MR, Wu B, Thomas D, Krogstad DJ, De D, Mirsalis JC, and Green CE
- Subjects
- Administration, Oral, Animals, Antimalarials administration & dosage, Antimalarials metabolism, Area Under Curve, Biological Availability, Blood Proteins metabolism, Female, Humans, Injections, Intravenous, Macaca fascicularis, Male, Metabolic Clearance Rate, Protein Binding, Quinolines administration & dosage, Quinolines metabolism, Rats, Rats, Sprague-Dawley, Antimalarials blood, Quinolines blood
- Abstract
The purpose of this study was to evaluate the bioavailability and pharmacokinetics of a new antimalarial drug, AQ-13, a structural analog of chloroquine (CQ) that is active against CQ-resistant Plasmodium species, in rats and cynomolgus macaques. Sprague-Dawley rats (n = 4/sex) were administered a single dose of AQ-13 intravenously (i.v.) (10 mg/kg) or orally (20 or 102 mg/kg). Blood and plasma samples were collected at several timepoints. AQ-13 achieved C(max) after oral administration at approximately 3 to 4 h and could be detected in blood for 2 to 5 days after oral administration. The ratio of area under the curve (AUC) values at the high and low dose for AQ-13 deviated from an expected ratio of 5.0, indicating nonlinear kinetics. A metabolite peak was noted in the chromatograms that was identified as monodesethyl AQ-13. Oral bioavailability of AQ-13 was good, approximately 70%. The pharmacokinetics of AQ-13 was also determined in cynomolgus macaques after single (i.v., 10 mg/kg; oral, 20 or 100 mg/kg) and multiple doses (oral loading dose of 50, 100, or 200 mg/kg on first day followed by oral maintenance dose of 25, 50, or 100 mg/kg, respectively, for 6 days). The AUC and C(max) values following single oral dose administration were not dose proportional; the C(max) value for AQ-13 was 15-fold higher following an oral dose of 100 mg/kg compared to 20 mg/kg. Monodesethyl AQ-13 was a significant metabolite formed by cynomolgus macaques and the corresponding C(max) values for this metabolite increased only 3.8-fold over the dose range, suggesting that the formation of monodesethyl AQ-13 is saturable in this species. The bioavailability of AQ-13 in cynomolgus macaques following oral administration was 23.8% for the 20-mg/kg group and 47.6% for the 100-mg/kg group. Following repeat dose administration, high concentrations of monodesethyl AQ-13 were observed in the blood by day 4, exceeding the AQ-13 blood concentrations through day 22. Saturation of metabolic pathways and reduced metabolite elimination after higher doses are suggested to play a key role in AQ-13 pharmacokinetics in macaques. In summary, the pharmacokinetic profile and metabolism of AQ-13 are very similar to that reported in the literature for chloroquine, suggesting that this new agent is a promising candidate for further development for the treatment of chloroquine-resistant malaria.
- Published
- 2004
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29. In vivo transgenic mutation assays.
- Author
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Thybaud V, Dean S, Nohmi T, de Boer J, Douglas GR, Glickman BW, Gorelick NJ, Heddle JA, Heflich RH, Lambert I, Martus HJ, Mirsalis JC, Suzuki T, and Yajima N
- Subjects
- Animals, Animals, Genetically Modified, Female, Male, Mice, Mice, Transgenic, Rats, Biological Assay standards, DNA Mutational Analysis standards
- Abstract
Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.
- Published
- 2003
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30. Toxicity of a quinocarmycin analog, DX-52-1, in rats and dogs in relation to clinical outcome.
- Author
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Mirsalis JC, Schindler-Horvat J, Hill JR, Green CE, Mitoma C, Tomaszewski JE, Tyson CA, and Donohue SJ
- Subjects
- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents pharmacology, Bone Marrow drug effects, Bone Marrow pathology, Digestive System drug effects, Digestive System pathology, Dogs, Dose-Response Relationship, Drug, Infusions, Intravenous, Injections, Intravenous, Isoquinolines administration & dosage, Isoquinolines pharmacology, Kidney drug effects, Kidney pathology, Rats, Rats, Inbred F344, Antineoplastic Agents toxicity, Isoquinolines toxicity
- Abstract
Purpose: Quinocarmycin analog DX-52-1 is a cyanated derivative of quinocarmycin, a compound isolated from cultures of Streptomyces melanovinaceus. DX-52-1 was selected for preclinical development because it showed efficacy against melanoma cell lines in the NCI human tumor cell screen and melanoma xenografts in mice. This report describes studies in rats and dogs to determine the maximum tolerated dose (MTD) and identify dose-limiting toxicities (DLT) in each species in different regimens to establish a safe starting dose and potential target organs of DX-52-1 for phase I clinical trials., Methods: DX-52-1 was administered to Fischer 344 rats using repeated intravenous (i.v.) slow bolus injections following q3hx3 and q3hx3,q7dx3 regimens, and to beagle dogs using a single injection, 6-h continuous i.v. infusion (c.i.v.) and weekly 6-h c.i.v. for 3 weeks. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues., Results: The MTD of DX-52-1 was a total dose of 18 mg/m(2) body surface area for q3hx3 administration in rats and 30 mg/m(2) for a single c.i.v. administration in dogs. The total dose MTD for rats on a weekly (q3hx3,q7dx3) regimen was 54 mg/m(2), and for dogs on the weekly x3 (6-h c.i.v.) infusion was 60 mg/m(2). In rats, significant elevations in blood urea nitrogen and creatinine were observed together with acute renal tubular necrosis histologically. Modest increases in liver enzymes were also observed, as were decreases in reticulocytes that were unaccompanied by histologic changes in liver and bone marrow. In dogs, adverse signs included vomiting/retching, diarrhea, and transient hypothermia; also red blood cells, hemoglobin, hematocrit, and lymphocytes were decreased. Histologic evaluation of tissues from dogs revealed necrosis and cellular depletion of the bone marrow, and extensive damage to the entire gastrointestinal tract, including marked cellular necrosis of the mucosa and lymphoid necrosis of the gastrointestinal associated lymphoid tissue. Destruction of the mucosal lining of the intestinal tract was likely responsible for dehydration, toxemia, septicemia, and shock seen in moribund dogs., Conclusions: The MTD values were comparable between rats and dogs given roughly similar dose regimens (single dose or weekly) and both species tolerated a higher total dose with weekly administration. However, the principal target organ responsible for DLT in rats was the kidney, whereas in dogs, the most severe effects were on the gastrointestinal tract and bone marrow. Both renal and gastrointestinal toxicities were reported in patients after 6-h c.i.v. infusions in a limited phase I clinical trial, indicating that neither animal model alone was predictive of DX-52-1-induced toxicity in humans, and that both species were required to define human toxicity.
- Published
- 2003
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31. Skin irritation, basal epithelial cell proliferation, and carcinogenicity evaluations of a representative specialty acrylate and methacrylate.
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Van Miller JP, Garman RH, Hermansky SJ, Mirsalis JC, and Frederick CB
- Subjects
- Administration, Cutaneous, Animals, Carcinogenicity Tests, Cell Division drug effects, Epidermal Cells, Epithelial Cells cytology, Immunohistochemistry, Male, Mice, Mice, Inbred C3H, Proliferating Cell Nuclear Antigen metabolism, Skin cytology, Skin Irritancy Tests methods, Acrylates toxicity, Epithelial Cells drug effects, Polyethylene Glycols toxicity, Polymethacrylic Acids toxicity, Skin drug effects
- Abstract
Specialty acrylates and methacrylates (SAM) comprise a large family of industrial monomers. In the late 1980s, the United States EPA and the industry SAM Panel collaborated to evaluate the potential effects, particularly carcinogenesis, of this family of chemicals. As part of this arrangement, the SAM Panel, with EPA input and approval, conducted four studies with a representative acrylate, triethyleneglycol diacrylate (TREGDA), and methacrylate, triethyleneglycol dimethacrylate (TREGDMA). All studies used unoccluded skin application to male mice as follows: Study 1, evaluation of skin irritation compared to cell proliferation in the basal epithelium (BE) following 7 or 14 days of treatment; Study 2, 14-day dose range-finding study; Study 3, 90-day subchronic toxicity study; and Study 4, chronic bioassays employing the EPAs draft guidelines for dermal chronic bioassays. BE cell proliferation was determined in subchronic and carcinogenicity studies (Studies 1, 3, and 4). Organ weight changes (Studies 3 and 4) and increased mortality (Study 4) were observed for the highest dose of TREGDMA. However, there was no related histopathology. Both chemicals induced cell proliferation (7 days through 78 weeks) that correlated with acute and chronic inflammation of the skin. No skin tumors were observed in this study. TREGDA resulted in skin lesions at doses approximately 20-fold lower than TREGDMA. Most of the skin lesions showed similar patterns of microscopic cutaneous alteration suggestive of nonspecific irritation for both chemicals. However, the high concentration TREGDA group in the 78-week study also had evidence of epidermal cell necrosis. In contrast to earlier studies with acrylates, dose selection was based on careful examination of skin irritation and cell proliferation to avoid excessive skin damage. Under these conditions, TREGDA and TREGDMA were not carcinogenic., (Copyright 2003 Elsevier Science (USA))
- Published
- 2003
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32. Genotoxicity and carcinogenicity studies of soy isoflavones.
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Misra RR, Hursting SD, Perkins SN, Sathyamoorthy N, Mirsalis JC, Riccio ES, and Crowell JA
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- Administration, Oral, Animals, Bone Marrow Cells drug effects, Carcinogenicity Tests, Escherichia coli drug effects, Escherichia coli genetics, Female, Lymphoma chemically induced, Lymphoma pathology, Male, Mice, Mice, Knockout, Micronucleus Tests, Mutation, Neoplasms, Experimental chemically induced, Neoplasms, Experimental pathology, Salmonella typhimurium drug effects, Salmonella typhimurium genetics, Tumor Cells, Cultured, Tumor Suppressor Protein p53 genetics, Carcinogens toxicity, Genistein toxicity, Lymphoma genetics, Mutagens toxicity, Neoplasms, Experimental genetics
- Abstract
The potential cancer preventive efficacy of soy isoflavones is being investigated in preclinical and phase 1 clinical studies sponsored by the U.S. National Cancer Institute. Although 90-day oral toxicity studies with PTI G-2535 (an investigational soy isoflavone drug product) in rats and dogs, as well as teratology studies, indicated no signs of toxicity, there remains a mechanistic concern associated with the ability of isoflavones (i.e., genistein) to inhibit topoisomerase, possibly leading to DNA strand breaks. The present report describes results from two in vitro genotoxicity studies, one in vivo genotoxicity study, and a single carcinogenicity study conducted in p53 knockout mice. Bacterial mutagenesis experiments using six tester strains without metabolic activation revealed no evidence that PTI G-2535 was mutagenic. In similar experiments with exogenous metabolic activation there were statistically significant increases in revertants, but less than twofold, in a single (Salmonella typhimurium TA100) tester strain. Mouse lymphoma cell mutagenesis experiments conducted with and without metabolic activation revealed statistically significant increases in mutation frequency at PTI G-2535 concentrations > or = 0.8 and 12 microg/ml, respectively; such increases were dose related and increases in the frequency of both small and large colonies were observed. A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was also seen 24 hours after treatment in male, but not female, mice who received 500 and 1000 mg/kg body weight PTI G-2535; however,such increases were small, were not dose related, and were not observed 48 hours after treatment. In contrast, dietary genistein had no effect on survival, weight gain, or the incidence or types of tumors that developed in cancer-prone rodents lacking the p53 tumor suppressor gene, p53 knockout mice. The apparent risk/benefit of isoflavone ingestion may ultimately depend on the dose and developmental timing of exposure.
- Published
- 2002
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33. Genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, AQ-13.
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Riccio ES, Lee PS, Winegar RA, Krogstad DJ, De D, and Mirsalis JC
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- Animals, Chloroquine analogs & derivatives, Mice, Mutagenicity Tests, Rats, Rats, Sprague-Dawley, Salmonella typhimurium genetics, Toxicity Tests, Tumor Cells, Cultured, Antimalarials toxicity, Chloroquine toxicity, Quinolines toxicity
- Abstract
AQ-13 ([N1-(7-chloro-quinolin-4yl)-3-(N3,N3-diethylamino)propylamine] dihydrochloride trihydrate) is an aminoquinoline antimalarial drug that is effective against chloroquine-resistant strains of Plasmodium falciparum. It is structurally similar to the widely used chloroquine diphosphate (CQ). We evaluated these drugs in the three assays currently recommended by the International Conference on Harmonization (ICH): bacterial mutagenesis in Salmonella typhimurium and Escherichia coli, mammalian cell mutagenesis in L5178Y mouse lymphoma cells, and micronucleus induction in rat bone marrow. A small but statistically significant increase in revertant colonies was produced by CQ with Salmonella tester strain TA98 without metabolic activation (MA) and by AQ-13 with strain TA1537 both with and without MA. In L5178Y cells, testing of CQ and AQ-13 up to cytotoxic concentrations with and without MA produced no increase in mutant colonies and no increase in the numbers of small colonies. Slight decreases in the ratio of polychromatic erythrocytes (PCE) to red blood cells (RBC) were observed in male and female rats treated with CQ and in females only treated with AQ-13; however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of CQ or AQ-13. Although both CQ and AQ-13 showed weak bacterial mutagenicity, this mutagenic effect was not confirmed in either the mouse lymphoma mutagenesis assay or the micronucleus assay. These results indicate that CQ and AQ-13 should pose minimal risk of genotoxic damage in human populations being administered these drugs., (Copyright 2001 Wiley-Liss, Inc.)
- Published
- 2001
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34. Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo.
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MacGregor JA, Hamilton CM, Kubicek JE, and Mirsalis JC
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- Animals, Body Weight drug effects, Carcinogens toxicity, Cell Nucleus drug effects, Cell Nucleus ultrastructure, Cytoplasm drug effects, Cytoplasm ultrastructure, Dimethylnitrosamine toxicity, Male, Mice, Mice, Inbred Strains, DNA Repair drug effects, Hepatocytes drug effects, Pyridines pharmacology
- Abstract
Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.
- Published
- 2000
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35. Vaccination with a recombinant vaccinia vaccine containing the B7-1 co-stimulatory molecule causes no significant toxicity and enhances T cell-mediated cytotoxicity.
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Freund YR, Mirsalis JC, Fairchild DG, Brune J, Hokama LA, Schindler-Horvat J, Tomaszewski JE, Hodge JW, Schlom J, Kantor JA, Tyson CA, and Donohue SJ
- Subjects
- Alanine Transaminase blood, Alkaline Phosphatase blood, Animals, Antibodies, Antinuclear blood, B7-1 Antigen genetics, Blood Cell Count, Blood Urea Nitrogen, Female, Immunity, Cellular, Mice, Mice, Inbred C57BL, Vaccinia virus, B7-1 Antigen immunology, Cytotoxicity, Immunologic, Lymphocyte Activation immunology, T-Lymphocytes immunology, Vaccines, Synthetic immunology, Vaccines, Synthetic toxicity
- Abstract
B7-1 is a co-stimulatory molecule that provides a second signal for T-cell activation. Several studies have demonstrated that vaccination with a vector containing genes encoding B7-1 and an antigen appears to be efficacious at promoting immune responsiveness to the antigen. To evaluate the safety of such a protocol and determine the effect of the B7-1 vector on immune responsiveness, female C57BL/6 mice were administered Wyeth wild-type vaccinia virus (V-WT) or V-WT containing the gene for B7-1 (rV-B7-1) as a single s. c. injection or 3 monthly s.c. injections. Immunologic parameters were evaluated in half of the mice and general toxicity in the other half. Immunologic end points included determination of splenic lymphocyte phenotypes, mitogen-induced T- and B-cell proliferation, T-cell proliferation in response to alloantigens, cell-mediated cytotoxicity (CMC), natural killer cell activity and serum anti-nuclear antibody (ANA) titers. No significant signs of general toxicity were noted. The primary immunologic effect was an increase in the ability of spleen cells to lyse allogeneic targets and to proliferate in response to allogeneic stimulation. Numbers of splenic CD8(+) cells were also increased. These effects were more pronounced after 3 vaccinations than after a single vaccination. Minimal differences in ANA were observed between mice immunized with V-WT and rV-B7-1. In addition, no serum antibodies against B7-1 were detected in any mice. The data suggest that vaccination with rV-B7-1 augments CMC with minimal toxicity., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
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36. In vivo transgenic mutation assays.
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Heddle JA, Dean S, Nohmi T, Boerrigter M, Casciano D, Douglas GR, Glickman BW, Gorelick NJ, Mirsalis JC, Martus HJ, Skopek TR, Thybaud V, Tindall KR, and Yajima N
- Subjects
- Animals, Animals, Genetically Modified, Mice, Mice, Transgenic, Rats, Rats, Inbred F344, Specimen Handling, Mutagenicity Tests
- Abstract
Transgenic rodent gene mutation models provide quick and statistically reliable assays for mutations in the DNA from any tissue. For regulatory applications, assays should be based on neutral genes, be generally available in several laboratories, and be readily transferable. Five or fewer repeated treatments are inadequate to conclude that a compound is negative but more than 90 daily treatments may risk complications. A sampling time of 35 days is suitable for most tissues and chemicals, while shorter sampling times might be appropriate for highly proliferative tissues. For phage-based assays, 5 to 10 animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3 x 10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (PFU or CFU) per tissue. Data should be generated for two dose groups but three should be treated, at the maximum tolerated dose (MTD), two-thirds the MTD, and one-third the MTD. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a block design and the total number of PFUs or CFUs and the MF for each tissue and animal reported. Sequencing data would not normally be required but might provide useful additional information in specific circumstances. Statistical tests used should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is statistically nonsignificant with all mean MF within two standard deviations of the control., (Copyright 2000 Wiley-Liss, Inc.)
- Published
- 2000
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37. Toxicity of dolastatin 10 in mice, rats and dogs and its clinical relevance.
- Author
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Mirsalis JC, Schindler-Horvat J, Hill JR, Tomaszewski JE, Donohue SJ, and Tyson CA
- Subjects
- Anemia chemically induced, Animals, Antineoplastic Agents administration & dosage, Bone Marrow drug effects, Bone Marrow pathology, Depsipeptides, Dogs, Female, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells pathology, Humans, Injections, Intravenous, Leukocyte Count drug effects, Male, Mice, Mice, Inbred Strains, Oligopeptides administration & dosage, Rats, Rats, Inbred F344, Reticulocyte Count drug effects, Weight Gain drug effects, Weight Loss, Antineoplastic Agents toxicity, Oligopeptides toxicity
- Abstract
Purpose: Dolastatin 10 (DOL 10), an oligopeptide isolated from the sea hare Dolabella auricularia, has been shown to be a highly potent cytotoxic agent in a variety of human tumor cell lines. The purpose of this study was to conduct preclinical toxicity evaluations to determine the target organ(s) of toxicity and its reversibility, the dose-limiting toxicity and the maximum tolerated dose (MTD), and to use this information for arriving at a safe starting dose and dose schedule for phase I clinical trails., Methods: DOL10 was administered as a single intravenous bolus dose to CD2F1 mice, Fischer-344 rats and beagle dogs. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues., Results: The MTD (i. e. the highest dose that did not cause lethality but produced substantial toxicity) was approximately 1350 microg/m(2) body surface area (450 microg/kg) in mice, 450 microg/m(2) (75 microg/kg) in rats and =400 microg/m(2) (=20 microg/kg) in dogs. Adverse signs were observed at doses >/=1350 microg/m(2) in mice, >/=150 microg/m(2) in rats and >/=400 microg/m(2) in dogs. Decreased weight gain or actual weight loss was observed at doses >/=1350 microg/m(2) in mice, >/=600 microg/m(2) in rats and >/=450 microg/m(2) in dogs. In all three species, the primary target organ of toxicity was the bone marrow, as indicated by decreases in the numbers of erythroid cells, myeloid cells, and megakaryocytes in the femoral bone marrow and by decreased white blood cell (WBC) and reticulocyte counts in peripheral blood. Marked neutropenia (i.e. >50% decrease compared to control animal or baseline values) was the principal effect on WBCs and occurred within a week of dosing. A mild anemia was evident 1 week after administering the drug to rats and dogs. The hematologic effects were transient and reversed by study termination. Other lesions at the MTD levels were cellular depletion and necrosis in lymphoid organs (rats and dogs), marked depletion of extramedullary hematopoietic cellular elements in the spleen (rats), thymic atrophy (mice and dogs), and minimal cellular necrosis in the ileum (rats). More extensive and severe pathology was observed in animals sacrificed in a moribund condition or found dead., Conclusions: Myelotoxicity was dose-limiting in all three species with mice being the least sensitive. In a phase I clinical trial, granulocytopenia was dose-limiting. Moreover, the MTD of DOL10 for rats and dogs is comparable to the human MTD. Therefore, the results from the preclinical toxicology studies correctly predicted a safe starting dose, the dose-limiting toxicity, and the MTD in humans.
- Published
- 1999
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38. Mutational spectrum of dimethylnitrosamine in the liver of 3- and 6-week-old lacI transgenic mice.
- Author
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de Boer JG, Mirsalis JC, and Glickman BW
- Subjects
- Animals, DNA genetics, Lac Repressors, Mice, Mice, Transgenic, Bacterial Proteins genetics, Dimethylnitrosamine pharmacology, Escherichia coli Proteins, Liver drug effects, Mutagens pharmacology, Mutation, Repressor Proteins genetics
- Abstract
We determined the spectrum of mutations in the lacI gene in the liver of Big Blue(R) transgenic mice after exposure to five daily doses of 2 mg/kg dimethylnitrosamine (DMN) at 3 and 6 weeks of age. This dose has been reported to increase the mutant frequency 9-fold when the animals are 3 weeks old. The lacI mutations recovered when treated at 3 weeks consist of mainly G:C --> A:T transitions, predominantly at non-CpG sites, and thus are consistent with mutagenesis by DMN. No increase in mutant frequency was reported when the mice were treated at 6 weeks of age. As we have previously shown that changes in mutational spectrum can be detected even when no statistically significant increase in mutant frequency is seen, we also examined the spectrum after treatment at 6 weeks. No changes from the spontaneous spectrum were detected. The comparison of the outcome of DMN treatment at 3 and 6 weeks confirms a change in metabolic activation, adduct removal, or mutation fixation between 3 and 6 weeks of age., (Copyright 1999 Wiley-Liss, Inc.)
- Published
- 1999
39. Evaluation of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) following treatment of rats and mice with p-dichlorobenzene.
- Author
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Sherman JH, Nair RS, Steinmetz KL, Mirsalis JC, Nestmann ER, and Barter JA
- Subjects
- Animals, Carcinogenicity Tests, Cell Division drug effects, Cells, Cultured, Chlorobenzenes administration & dosage, Chlorobenzenes metabolism, Female, Kidney drug effects, Liver drug effects, Male, Mice, Mutagenicity Tests, Rats, Rats, Inbred F344, Chlorobenzenes toxicity, DNA biosynthesis, DNA Repair, Insecticides toxicity
- Abstract
p-Dichlorobenzene (PDCB) has been reported to produce tumors in the male and female mouse liver and in the male rat kidney in 2-year gavage studies (NPT, 1987). To elucidate the possible mechanisms of carcinogenicity more fully, UDS and RDS were evaluated in B6C3F1 mouse hepatocytes and F-344 rat kidney cells by autoradiography following in vivo administration of PDCB. All corn oil gavage doses of PDCB (300, 600, and 1,000 mg/kg) and the negative control resulted in < 0 net grains/nucleus (NG) in the mouse liver and rat kidney, indicating that PDCB does not induce UDS in either tissue. Compared to controls with < or = 0.29% hepatocytes in S-phase (%S), treatment of mice induced 0.46, 1.90, and 1.52 %S (males) and 2.61, 1.18, and 4.45 %S (females), which indicates that PDCB acts as an inducer of cell proliferation in the liver. In male rat kidney cells, the same doses produced 0.87, 0.67, and 1.01 %S (0.38% in controls) and in females 0.48, 0.43, and 0.32 %S (0.52% in controls), indicating that PDCB induces cell replication in the male but not the female rat kidney. Therefore, these data demonstrate that PDCB is not genotoxic in the mouse liver or rat kidney at single oral doses comparable to the daily doses given in the National Toxicology Program (NTP) bioassay (NTP, 1987). Furthermore, the increases in RDS support the hypotheses that mouse liver tumor formation occurs via stimulation of hepatocyte proliferation and male rat kidney carcinogenesis via increased renal cell proliferation.
- Published
- 1998
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40. Analysis of the mutagenic potential of ENU and MMS in germ cells of male C57BL/6 lacI transgenic mice.
- Author
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Winegar RA, Carr G, and Mirsalis JC
- Subjects
- Animals, DNA drug effects, DNA isolation & purification, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Seminiferous Tubules cytology, Ethylnitrosourea toxicity, Methyl Methanesulfonate toxicity, Mutagenicity Tests, Mutagens toxicity, Spermatozoa drug effects
- Abstract
Mutant frequencies in male germ cells were determined in mice 3 days after exposure to saline, methylmethane sulfonate (MMS), or ethylnitrosourea (ENU). DNA was isolated from seminiferous tubules by a modified version of the drop dialysis method. A 5-fold increase in mutant frequency was observed in mice treated with ENU. No statistically significant increase was observed in mice treated with MMS.
- Published
- 1997
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41. Evaluation of positive controls for the in vitro unscheduled DNA synthesis assay using hepatocytes from induced (Aroclor 1254) and uninduced male cynomolgus monkey.
- Author
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Hamilton CM, Dabbs JE, Cunningham GD, Vernetti LA, Mirsalis JC, and Snyder RD
- Subjects
- 9,10-Dimethyl-1,2-benzanthracene toxicity, Animals, Benzo(a)pyrene toxicity, Biotransformation, Dimethylnitrosamine toxicity, Enzyme Induction, Liver, Macaca fascicularis, Male, Methyl Methanesulfonate toxicity, Aroclors pharmacology, DNA Repair, Mutagenicity Tests methods
- Abstract
We have evaluated the use of four different positive control compounds for assessing UDS in monkey hepatocytes and have found three of these, methylmethanesulfonate, benzo[a]pyrene, and dimethylbenz[a]anthracene, to produce strong positive responses in vitro. Dimethylnitrosamine induced only weak responses. We also report that the strength of the response induced by procarcinogens was not enhanced in hepatocytes taken from Aroclor 1254-pretreated monkeys, even though substantial induction of cytochrome P450 enzymes was demonstrated in these cells. These studies raise the question of the utility of employing an in vivo induction system to enhance the monkey UDS assay.
- Published
- 1997
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42. Mutagenic response to benzene and tris(2,3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis.
- Author
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Provost GS, Mirsalis JC, Rogers BJ, and Short JM
- Subjects
- Animals, Bacterial Proteins drug effects, Carcinogens toxicity, Dose-Response Relationship, Drug, Female, Lac Repressors, Male, Mice, Mice, Inbred C3H, Mice, Inbred C57BL, Mutagenicity Tests methods, Mutagens toxicity, Repressor Proteins drug effects, Tissue Distribution, Bacterial Proteins genetics, Benzene toxicity, Escherichia coli Proteins, Mice, Transgenic genetics, Mutation, Organophosphates toxicity, Repressor Proteins genetics
- Abstract
The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay. Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations. Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals. Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice. Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays. In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested.
- Published
- 1996
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43. Spectrum of mutations in kidney, stomach, and liver from lacI transgenic mice recovered after treatment with tris(2,3-dibromopropyl)phosphate.
- Author
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de Boer JG, Mirsalis JC, Provost GS, Tindall KR, and Glickman BW
- Subjects
- Animals, Bacterial Proteins drug effects, Gastric Mucosa metabolism, Kidney metabolism, Lac Repressors, Liver drug effects, Liver metabolism, Male, Mice, Mice, Transgenic, Repressor Proteins drug effects, Sequence Analysis, DNA, Bacterial Proteins genetics, Escherichia coli Proteins, Kidney drug effects, Mutation drug effects, Organophosphates toxicity, Repressor Proteins genetics, Stomach drug effects
- Abstract
The flame retardant tris(2,3-dibromopropyl)phosphate (TDBP), once used in cotton sleep wear for children, is presently banned from commerce. It produces tumors in rodents in both a sex- and tissue-specific manner. The kidney is the main target for tumor formation in male and female rats, as well as in male mice. In contrast, tumors are formed in the liver of female animals. We have used lacI transgenic male B6C3F1 mice (Big Blue) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days) of TDBP. At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01). A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28). Sequence analysis of the recovered mutants showed a TDBP-specific change in mutation spectrum in kidney, which was not observed in liver and stomach. In kidney, a dose-dependent decrease in G:C-->A:T transitions, including at 5'-CpG-3' sites, was observed. This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15%. These results illustrate both the sensitivity and specificity of the lacI transgenic system in the analysis of tissue-specific mutation. This study also reinforces the importance of examining mutational spectra when mutant induction levels are low.
- Published
- 1996
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44. A strategy for the application of transgenic rodent mutagenesis assays.
- Author
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Gorelick NJ and Mirsalis JC
- Subjects
- Animals, Bacterial Proteins genetics, Bone Marrow drug effects, Carcinogenicity Tests, Germ Cells drug effects, Guidelines as Topic, Humans, Lac Repressors, Liver drug effects, Mice, Mice, Transgenic, Mutagens administration & dosage, Mutagens pharmacokinetics, Mutagens toxicity, Rats, Repressor Proteins genetics, Tissue Distribution, beta-Galactosidase genetics, Animals, Genetically Modified genetics, Escherichia coli Proteins, Mutagenicity Tests methods
- Abstract
The past several years have seen an enormous increase in the development and use of transgenic animal models to measure mutations in specific inserted reporter genes. These systems provide gene mutation data in vivo in a wide range of relevant tissues. Numerous laboratories are now using these systems with consistent results. This paper describes the unique niche that transgenic mutagenesis systems can fill in product development and registration strategies. In addition to tissue-specific mechanistic studies, transgenic assays are available to follow up mutagenic effects demonstrated in Salmonella, Escherichia coli, mouse lymphoma (L5178Y) cells, or other in vitro systems.
- Published
- 1996
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45. Chromium (VI) at plausible drinking water concentrations is not genotoxic in the in vivo bone marrow micronucleus or liver unscheduled DNA synthesis assays.
- Author
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Mirsalis JC, Hamilton CM, O'Loughlin KG, Paustenbach DJ, Kerger BD, and Patierno S
- Subjects
- Animals, Bone Marrow ultrastructure, Female, Liver metabolism, Male, Mice, Micronucleus Tests, Rats, Rats, Inbred F344, Bone Marrow drug effects, Chromium toxicity, DNA Repair, Liver drug effects, Mutagens toxicity, Water Supply
- Published
- 1996
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46. Transgenic models for detection of mutations in tumors and normal tissues of rodents.
- Author
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Mirsalis JC
- Subjects
- Animals, Liver Neoplasms, Experimental chemically induced, Mice, Mice, Inbred C57BL, Mice, Transgenic, Liver Neoplasms, Experimental genetics, Mutation
- Abstract
Transgenic rodents that contain easily retrievable target genes allow the rapid quantitation of mutations in any tissue from which DNA can be isolated. We are using the Stratagene Big Blue transgenic mouse system that contains a lacI target and an alpha lacZ reporter gene to study the parameters that affect mutations. We have evaluated a number of chemicals to determine mutant frequency (MF) in specific target tissues of C57Bl/6 and B6C3F1 mice. The correlation between mutagenesis and carcinogenesis in this system is excellent. For example, the liver carcinogen dimethylnitrosamine produces significant increases in MF in mouse liver, whereas the nonhepatocarcinogenic mutagen methylmethane sulfonate does not. We have also evaluated the induction of mutations by radiation and demonstrated that this system is suitable for the study of agents that produce deletion mutations. This system is also useful for studying changes in MF in developing tumors. We have used an initiation-promotion protocol to induce hepatocellular carcinomas, and we then measured MF in normal liver, tumors, and metastases from these mice. Animals initiated with diethylnitrosamine maintain an elevated MF in normal liver, even 1 year after initiation. This MF increases exponentially in developing liver tumors, possibly owing to a breakdown in the fidelity of DNA replication and DNA repair in tumors. This system offers a unique tool for the study of mutations induced in specific target tissues of rodents and should become an important assay for evaluating the mutagenic risk of drugs and chemicals.
- Published
- 1995
- Full Text
- View/download PDF
47. Transgenic animal models for detection of in vivo mutations.
- Author
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Mirsalis JC, Monforte JA, and Winegar RA
- Subjects
- Animals, Carcinogens toxicity, Lac Operon, Mutagenesis, Rodentia, Animals, Genetically Modified, Mutagenicity Tests methods
- Abstract
Transgenic rodent models for measuring mutations provide a tool for assessing tissue-specific mutations following in vivo treatment. These systems are based on the insertion into the rodent genome Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from tissues of interest, and the target gene is screened for mutations using either lambda-phage packaging or isolation of the target gene with magnetic affinity capture. In this paper we review the various experimental methods used in the conduct of transgenic mutation assays and discuss critical factors that affect the interpretations of results of these assays.
- Published
- 1995
- Full Text
- View/download PDF
48. Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice.
- Author
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Winegar RA, Lutze LH, Hamer JD, O'Loughlin KG, and Mirsalis JC
- Subjects
- Animals, Base Sequence, Bone Marrow radiation effects, Bone Marrow Cells, DNA Mutational Analysis, DNA Transposable Elements, Dose-Response Relationship, Radiation, Gene Deletion, Genetic Vectors, Male, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Micronucleus Tests, Molecular Sequence Data, Point Mutation, Spleen cytology, Spleen radiation effects, Gamma Rays, Lac Operon radiation effects, Mice, Transgenic genetics, Mutagenesis, Site-Directed, Mutation
- Abstract
Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis systems has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei. C57Bl/6 transgenic mice were irradiated with 137Cs gamma-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 gamma-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the alpha lacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue lacI transgenic mouse system in sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene affects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57Bl/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57Bl/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.
- Published
- 1994
- Full Text
- View/download PDF
49. Transgenic animals in toxicology.
- Author
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Goldsworthy TL, Recio L, Brown K, Donehower LA, Mirsalis JC, Tennant RW, and Purchase IF
- Subjects
- Animals, Genes, p53 physiology, Mice, Mutagenesis, Neoplasms, Experimental genetics, Risk Factors, Mice, Transgenic, Toxicology methods
- Abstract
Recent advances have been made in the characterization of a number of transgenic animal models. These animal models have provided a powerful toxicological tool for studying in vivo chemical effects and have increased our understanding of the role of specific genetic alterations as predisposing factors for chemical carcinogenesis. The goal of this symposium was to introduce the development of transgenic animals and the utilization of transgenics in toxicology research focusing on understanding tissue-specific mutation, chemical effects, and cancer. The production of transgenic animals, including gene insertions and gene knockouts, and the utilization of transgenic technology for studying multistage carcinogenesis and tumor suppressor genes are described. Data on the application and implications of transgenics as a genetic endpoint are also discussed. The use of transgenic animals in toxicology should improve our understanding of the role of specific genetic alterations in the carcinogenic process and lead to improved estimations of human health risks.
- Published
- 1994
- Full Text
- View/download PDF
50. Transgenic animal models for measuring mutations in vivo.
- Author
-
Mirsalis JC, Monforte JA, and Winegar RA
- Subjects
- Animals, Animals, Genetically Modified genetics, Mutagenesis physiology, Mutation physiology
- Abstract
Transgenic animal models for measuring mutations provide a powerful tool for rapidly assessing tissue-specific mutations following in vivo treatment. These models are based on the insertion into the rodent genome of the Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations. Following in vivo treatment of animals, genomic DNA is isolated from various tissues and the target gene is packaged into lambda-phage heads; the lambda-phage are used to infect E. coli in order to produce plaques. Mutations in the target gene are then detected using colorimetric or selective procedures. In this review methods are discussed for producing these transgenic models, the target genes used, gene rescue techniques, sequencing of isolated mutants, and parameters that affect dosing regimens and design of studies. We also present a summary of data published to date with these systems and present our conclusions and proposed directions for future research.
- Published
- 1994
- Full Text
- View/download PDF
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