Kenneth H. Jones

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.

Cytotoxicity of tocopherols and their quinones in drug-sensitive and multidrug-resistant leukemia cells

Cytotoxicities of tocopherols (α-T, γ-T, δ-T), their para (α-TQ, γ-TQ, δ-TQ)-and ortho (Tocored)-quinone oxidation products, the synthetic quinone analog of γ-TQ containing a methyl group substituted for the phytyl side-chain (TMCQ) and the synthetic quinone analog of Tocored containing a methyl group substituted for the phytyl side-chain (PR) were measured in acute lymphoblastic leukemia cell lines that are drug-sensitive (CEM) and multidrug-resistant (CEM/VLB100). Among tocopherols, only δ-T exhibited cytotoxicity. Among para quinones, α-TQ showed no cytotoxicity, while γ-TQ and δ-TQ were highly cytotoxic in both CEM and CEM/VLB100 cell lines (LD50<10 μM). δ-TQ and γ-TQ were more cytotoxic than the widely studied chemotherapeutic agent doxorubicin, which also showed selective cytotoxicity to CEM cells. The orthoquinone Tocored was less cytotoxic than doxorubicin in drug-sensitive cells but more cytotoxic than doxorubicin in multidrug-resistant cells. Cytotoxicity was not a function of the phytyl side-chain since both TMCQ and PR were cytotoxic in leukemia cells. Cytotoxic para and ortho quinones were electrophiles that formed adducts with nucleophilic thiol groups in glutathione and 2-mercaptoethanol. Cytotoxicity was enhanced when the glutathione pool was depleted by preincubation with buthionine-[S,R]-sulfoximine, but cytotoxicity was diminished by the addition of N-acetylcysteine to cultures. α-T also diminished the cytotoxicity of para- and or-thoquinones. Buthionine-[S,R]-sulfoximine did not block the inhibitory effect of either N-acetylcysteine or α-T, showing that these agents did not act solely by maintaining the glutathione pool as an essential antioxidant system. In conclusion, tocopherylquinones represent a new class of alkylating electrophilic quinones that function as highly cytotoxic agents and escape multidrug resistance in acute lymphoblastic leukemia cell lines.

Antioxidant and cytotoxic tocopheryl quinones in normal and cancer cells.

We found previously that [d]-alpha-tocopherol (alpha-T) and [d]-gamma-tocopherol (gamma-T) are lipid antioxidants (thiobarbituric acid test) in model systems containing arachidonic acid (AA), cumene hydroperoxide, and Fe3+ and in smooth muscle cell (SMC) cultures challenged with AA. We now show that [d]-alpha-tocopherylquinone (alpha-TQ), [d]-delta-tocopherylquinone (delta-TQ), and [d]-gamma-tocopherylquinone (gamma-TQ) are antioxidants at low concentrations and prooxidants at high concentrations in the model system. Prooxidant activity is greater with gamma-TQ than either alpha-TQ or delta-TQ. Low concentrations of alpha-TQ, delta-TQ, and gamma-TQ are also antioxidants in SMC cultures challenged with AA. Unlike alpha-TQ, partially substituted gamma-TQ and glutathione (GSH) form a Michael adduct which has been purified and characterized. We found previously that alpha-T, gamma-T, and alpha-TQ are mitogenic in SMC. We now report that both delta-TQ and gamma-TQ but not alpha-TQ show concentration-dependent cytotoxicity (changes in morphology, propidium iodide stain) in SMC cultures. Cytotoxicity is greater with gamma-TQ than delta-TQ. An acute lymphoblastic leukemia (ALL) cell line shows greater chemosensitivity (MTT and Neutral Red assays) to gamma-TQ than to either doxorubicin (DOX) or vinblastine (VLB). An ALL cell line resistant to both DOX and VLB retains the same chemosensitivity to gamma-TQ as the drug-sensitive ALL cell line. ALL cell lines are unaffected by either alpha-TQ or the GSH Michael adduct of gamma-TQ. These data show that partially substituted tocopheryl quinones capable of forming Michael adducts are potential chemotherapeutic agents for multidrug-resistant cancer cells.

Mutagenicity of Tocopheryl Quinones: Evolutionary Advantage of Selective Accumulation of Dietary a-Tocopherol

We have shown that phenolic antioxidant tocopherols are oxidized to nonarylating α-tocopheryl quinone (α-TQ) and arylating γ- and δ-TQ electrophiles. The arylating quinones stimulate apoptosis and are highly cytotoxic in mammalian cells. Some xenobiotic phenolic antioxidants are mutagens, and it has been suggested that their arylating quinone metabolites are the active agents in mutagenesis related to carcinogenesis. We found that neither α- nor γ-TQ was directly genotoxic in supercoiled-to-nicked circular DNA conversions, but these agents interacted with the cytomegalovirus reporter-driven plasmid and enhanced luciferase transfection, with γ-TQ >?α-TQ. The Ames test, using γ-TQ and a number of Salmonella strains, showed no evidence of bacterial mutagenesis. γ-TQ was highly cytotoxic and α-TQ slightly cytotoxic in eukaryocyte AS52 cells. A guanosine phosphoribosyltransferase gene assay showed that γ-TQ was highly mutagenic and α-TQ slightly mutagenic in AS52 cells. A review of the literature identified associations where a decrease in dietary γ-tocopherol (γ-T) diminishes and an increase in dietary γ-T and its quinone enhances carcinogenicity. Humans and other omnivores selectively accumulate α-tocopherol, even though γ-T is their principal dietary tocopherol. We suggest that this selectivity confers an evolutionary advantage by limiting tissue γ-T, a putative precursor of the mutagen γ-TQ.

γ-Tocopheryl quinone stimulates apoptosis in drug-sensitive and multidrug-resistant cancer cells

Chemotherapy-induced cell death is linked to apoptosis, and there is increasing evidence that multidrug-resistance in cancer cells may be the result of a decrease in the ability of a cell to initiate apoptosis in response to cytotoxic agents. In previous studies, we synthesized two classes of electrophilic tocopheryl quinones (TQ), nonarylating α-TQ and arylating γ-and δ-TQ, and found that γ-and δ-TQ, but not α-TQ, were highly cytotoxic in human acute lymphoblastic leukemia cells (CEM) and multidrug-resistant (MDR) CEM/VLB100. We have now extended these studies on tumor biology with CFM, HL60 and MDR HL60/MX2 human promyelocytic leukemia, U937 human monocytic leukemia, and ZR-75-1 breast adenocarcinoma cells. γ-TQ, but not α-TQ or tocopherols, showed concentration and incubation time-dependent effects on loss of plasma membrane integrity, diminished viable cell number, and stimulation of apoptosis. Its cytotoxicity exceeded that of doxorubicin in HL60/MX2 cells, which express MRP, an MDR-associated protein. Apoptosis was confirmed by TEM, TUNEL, and DNA gel electrophoresis. Kinetic studies showed that an induction period was required to initiate an irreversible multiphase process. γ-TQ released mitochondrial cytochrome c to the cytosol, induced the cleavage of poly(ADP-ribose)polymerase, and depleted intracellular glutathione. Unlike xenobiotic electrophiles, γ-TQ is a highly cytotoxic arylating electrophile that stimulates apoptosis in several cancer cell lines including cells that express MDR through both P-glycoprotein and MRP-associated proteins. The biological properties of arylating TQ electrophiles are closely associated with cytotoxicity and may contribute to other biological effects of these highly active agents.

Analysis of cellular heterogeneity in mouse thymus cultures

SummaryAnalysis of 5 to 6 d primary cultures of cells derived from murine thymus glands revealed a heterogeneous population of cells rather than “pure” reticuloepithelial cell cultures as was assumed previously by other investigators. The monolayer cultures consisted of at least three cell types: thymus epithelial cells, macrophagelike epithelioid cells, and fibroblasts. Surprisingly, about 50% of the cells had positive cytochemical staining reactions for acid phosphatase and nonspecific esterase. The same cells phagocytized carbon particles, latex beads, and yeast. Furthermore, these cells could be removed from the initial cell suspension by phagocytosis of carbonyl iron, followed by magnetic separation, but once they had adhered to the substratum they were resistant to trypsin removal. All of these findings supported the conclusion that about 50% of the cells in the monolayers were macrophages. The other cells present were thymus epithelial cells and a small number of fibroblasts. Both of the latter types of cell were cytochemically negative, did not phagocytize particulate material, and were not removed by carbonyl iron treatment, but were removed by treating the monolayer with trypsin. The findings in this report indicated that epithelioid morphology alone was inadequate to identify correctly the cell types found in thymus cultures and that the use of such cultures as a model to study in vitro the maturation of certain immunological functions has been based on assumptions here shown to be incorrect.

Electrophile tocopheryl quinones in apoptosis and mutagenesis: Thermochemolysis of thiol adducts with proteins and in cells

Electrophile tocopheryl quinones from the phenolic antioxidants γ-tocopherol and δ-tocopherol form Michael adducts with the thiol nucleophile glutathione. These tocopheryl quinones are involved in cytotoxicity, apoptosis, and mutagenesis, and their biologic properties are associated with the depletion of intracellular thiols. We now show that both proteins and tissues treated with the electrophile γ-tecopheryl quinone (γ-TQ) form thiol adducts. The monoglutathion-S-yl derivative of γ-TQ was subjected to thermochemolysis with the strong methylating base tetramethylammonium hydroxide. GC/MS showed four signature peaks and a fragmentation pattern characteristic of the thiol adduct. Similarly, pure monoglutathion-S-yl and diglutathion-S-yl derivatives of δ-TQ were subjected to thermochemolysis, and GC/MS showed characteristic fragmentation patterns for thiol adducts. The four signature peaks were identified when pure proteins with accessible thiol groups (hemoglobin and histone), FBS, and tissue culture medium and cell preparations were treated with γ-TQ. Signature peaks in both complete medium and washed cells showed the presence of both soluble and insoluble thiol adducts. The effective or free arylating electrophile concentration in complete medium should always be evaluated in tissue culture studies. γ-TQ is a mutagen but not a genotoxin; therefore, the histone adduct may be a previously unrecognized histone modification involved in chromatin dynamics leading to mutagenesis.

Surgical Clinical Correlates in Anatomy: Design and implementation of a first‐year medical school program

Medical students state the need for a clinically oriented anatomy class so to maximize their learning experience. We hypothesize that the first‐year medical students, who take the Surgical Clinical Correlates in Anatomy program, will perform better than their peers in their anatomy course, their surgical clerkships and ultimately choose surgical residencies. We designed and recently implemented this program for first‐year medical students. It consisted of General Surgical Knowledge, Orthopedic Surgery, Plastic Surgery, Urology, Cardiothoracic Surgery, General Surgery, Vascular Surgery, and Ear, Nose, and Throat (ENT) sessions. Each session had defined learning objectives and interactive cadaveric operations performed by faculty members and students. The program was elective and had 25 participants randomly chosen. An evaluative questionnaire was completed before and after the program. Comparative analysis of the questionnaires, first‐year anatomy examination results, clinical surgical rotation scores, and residency match results will be completed. The positive opinions of surgeons increased for all medical students from the pre‐evaluation to the post‐evaluation, and there was a greater increase in positive opinions for our participants. Our participants also had the highest average overall for all combined anatomy examinations. A need exists among medical students to develop a clinically correlated anatomy program that will maximize their learning experience, improve their performance and allow them to make moreinformed career choices. The recent implementation of this Surgical Clinical Correlates in Anatomy program fulfills this need. Anat Sci Educ 2: 265‒272, 2009.

Development of a fluid-filled catheter system for dynamic pressure measurement in soft-tissue trauma

Abstract In victims of motor vehicle crashes, rapid increases in internal fluid pressure may play a role in causing injury to solid abdominal organs such as the liver. The objective of this study was to develop a practical and cost-effective technique to measure impact-induced pressure changes within physiologically pressurised porcine liver specimens. This technique employs instrumentation that is remote from the impacted organ and could be applied to fluid-filled abdominal components of crash-test dummies. A fluid-filled catheter (FFC) pressure-measurement device was modelled as an under-damped second-order linear system. A transfer function was defined to correct the characteristic distortion of pressure waveforms in the FFC pressure-measurement system. Linear regression analysis was employed to evaluate the accuracy of transfer function-corrected pressure measurements in impact tests of a simplified model system and of physiologically pressurised ex vivo porcine livers. Results demonstrated a very high correlation between transfer function-corrected FFC pressure measurements and reference pressures in model system impacts (R = 0.95–0.97) and in ex vivo porcine liver impacts (R = 0.91–0.96). A near one-to-one relationship between the magnitudes of the corrected FFC pressures and the reference pressures was demonstrated by calculating the slopes of the regression equations (0.821 ± 0.016 to 1.085 ± 0.005, p < 0.05). The FFC technique has the potential to be a valuable tool in future studies requiring dynamic measurements of extremely high intravascular pressures associated with impact loading of soft tissues such as the liver. Information about the relationship between pressure and injury in solid abdominal organs could be applied to improve the design of crash-test dummies.