James B. Mitchell

Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody.

Using a monoclonal antibody to bromodeoxyuridine (BUdR) and immunohistochemistry, we measured the incorporation of this thymidine analogue into the DNA of human normal and malignant cells exposed in vivo. BUdR given as a constant intravenous infusion for 12 or 24 h daily for up to 13 d resulted in a steady-state plasma level of 10(-6) M during the infusion. We demonstrated extensive incorporation of BUdR into both normal skin, normal bone marrow, and malignant melanoma cells. In addition, this infusion of BUdR was adequate to identify sister chromatid exchanges from human marrow chromosomes exposed in vivo. Using this constant infusion, significant but reversible (acute) toxicity was observed with myelosuppression and skin photosensitivity. These techniques, which are considerably less cumbersome and time-consuming than the use of radioactive isotopes of thymidine, can be used for further human studies of cell kinetics and chromosomal replication in both normal and malignant cells.

Cytotoxic studies of paclitaxel (Taxol ® ) in human tumour cell lines

The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM, however, resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,000 nM) had an increase in survival compared with cells treated with lower concentrations of the drug. Prolonging the time of exposure of cells to paclitaxel from 24 to 72 h increased cytotoxicity from 5 to 200 fold in different cell lines. Exponentially growing cells were more sensitive to paclitaxel than were cells in the plateau phase of growth. Cremophor EL, the diluent in which the clinical preparation of paclitaxel is formulated, antagonised paclitaxel at concentrations of 0.135% (v/v). These data suggest that paclitaxel will be most effective clinically when there is prolonged exposure of tumour to the drug. Further, it appears that modest concentrations (i.e., 50 nM) should be as effective as higher concentrations of paclitaxel. Finally, we have noted that Cremophor EL is a biologically active diluent and, at high concentrations (0.135% v/v), can antagonise paclitaxel cytotoxicity.

Viability measurements in mammalian cell systems

Abstract The term “cellular viability” and the diverse assays used to determine viability can strongly influence the interpretation of experimental results. With such one-component systems as purified enzymes, an effect usually can be assigned unambiguously to the modification of a specific enzymatic property. With a highly integrated and multicomponent system such as a cell, however, the assessment of a chemical effect may depend heavily on the specific assay used. Terms such as cell survival, cell killing, cytotoxicity, integrity, and toxicity are often arbitrarily applied, but in an effort to quantify and characterize the term “viability” a number of assays that correlate with either reproductive or functional capabilities have been developed. Not surprisingly, different disciplines have a tendency to favor a specific assay method to the exclusion of others. It is the purpose of this review to examine the merits of the major groups of currently available viability assays (Table 1) as they apply to mammalian cell systems. In addition, it is our intention to show that while some assays may be clearly superior in measuring a final endpoint, technical and practical considerations may limit their usefulness.

Mice Lacking Smad3 Are Protected Against Cutaneous Injury Induced by Ionizing Radiation

Transforming growth factor-beta (TGF-beta) plays a central role in the pathogenesis of inflammatory and fibrotic diseases, including radiation-induced fibrosis. We previously reported that mice null for Smad3, a key downstream mediator of TGF-beta, show accelerated healing of cutaneous incisional wounds with reduced inflammation and accumulation of matrix. To determine if loss of Smad3 decreases radiation-induced injury, skin of Smad3+/+ [wild-type (WT)] and -/- [knockout (KO)] mice was exposed to a single dose of 30 to 50 Gy of gamma-irradiation. Six weeks later, skin from KO mice showed significantly less epidermal acanthosis and dermal influx of mast cells, macrophages, and neutrophils than skin from WT littermates. Skin from irradiated KO mice exhibited less immunoreactive TGF-beta and fewer myofibroblasts, suggesting that these mice will have a significantly reduced fibrotic response. Although irradiation induced no change in the immunohistochemical expression of the TGF-beta type I receptor, the epidermal expression of the type II receptor was lost after irradiation whereas its dermal expression remained high. Primary keratinocytes and dermal fibroblasts prepared from WT and KO mice showed similar survival when irradiated, as did mice exposed to whole-body irradiation. These results suggest that inhibition of Smad3 might decrease tissue damage and reduce fibrosis after exposure to ionizing irradiation.

Chemical biology of nitric oxide: Regulation and protective and toxic mechanisms

Publisher Summary This chapter discusses the important aspects of the solution chemistry of nitrogen oxide (NO) and reactive nitrogen oxide species (RNOS), biochemical targets of NO and intermediates in the autoxidation (NO X ), and the effect of NO in the presence of other toxic molecules, such as reactive oxygen species (ROS). There are two types of nitric-oxide synthase: constitutive (cNOS) and inducible (iNOS). Since cNOS generates low levels of NO, direct effects rather than indirect effects of NO would be particularly relevant. In case of iNOS, considerably higher concentrations of NO are formed for longer periods of time; therefore, both direct and indirect effects could be relevant. This chapter discusses, from a chemical perspective, those processes that are involved in the interactions with key cellular components as well as detoxification and control of NO in vivo . Defining the chemical, biochemical, and cellular pathways of NO quantitatively can provide insights into the role that NO plays in the etiology of various diseases that in turn can provide a basis for the development of new therapeutic agents. The chemical biology of NO will provide the understanding as to how NO can be regulatory, toxic, and protective in biological systems.

Radioprotectors and Mitigators of Radiation-Induced Normal Tissue Injury

The article reviews agents in clinical use or in development as radioprotectors and mitigators of radiation-induced normal tissue injury.

Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity

The antioxidant tempol reduces obesity in mice. Here we show that tempol alters the gut microbiome by preferentially reducing the genus Lactobacillus and its bile salt hydrolase (BSH) activity leading to the accumulation of intestinal tauro-β-muricholic acid (T-β-MCA). T-β-MCA is an farnesoid X receptor (FXR) nuclear receptor antagonist, which is involved in the regulation of bile acid, lipid and glucose metabolism. Its increased levels during tempol treatment inhibit FXR signalling in the intestine. High-fat diet-fed intestine-specific Fxr-null (Fxr(ΔIE)) mice show lower diet-induced obesity, similar to tempol-treated wild-type mice. Further, tempol treatment does not decrease weight gain in Fxr(ΔIE) mice, suggesting that the intestinal FXR mediates the anti-obesity effects of tempol. These studies demonstrate a biochemical link between the microbiome, nuclear receptor signalling and metabolic disorders, and suggest that inhibition of FXR in the intestine could be a target for anti-obesity drugs.

Overhauser enhanced magnetic resonance imaging for tumor oximetry: Coregistration of tumor anatomy and tissue oxygen concentration

An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2–7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO2 (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg = 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O2/5% CO2) inhalation. The pO2 measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO2 Eppendorf electrode. This work illustrates that anatomically coregistered pO2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO2 sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO2 measurements of the same animal with useful spatial (≈1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.

Ionizing Radiation-Induced Oxidative Stress Alters miRNA Expression

Background MicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H2O2). Methodology/Principal Findings Normal human fibroblasts were exposed to radiation, H2O2, or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H2O2 treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation. Conclusions These results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H2O2, and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.

Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic, Tempol

Stable nitroxide radicals have been previously shown to function as superoxide dismutase (SOD)2 mimics and to protect mammalian cells against superoxide and hydrogen peroxide-mediated oxidative stress. These unique characteristics suggested that nitroxides, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), might protect mammalian cells against ionizing radiation. Treating Chinese hamster cells under aerobic conditions with 5, 10, 50, and 100 mM Tempol 10 min prior to X-rays resulted in radiation protection factors of 1.25, 1.30, 2.1, and 2.5, respectively. However, the reduced form of Tempol afforded no protection. Tempol treatment under hypoxic conditions did not provide radioprotection. Aerobic X-ray protection by Tempol could not be attributed to the induction of intracellular hypoxia, increase in intracellular glutathione, or induction of intracellular SOD mRNA. Tempol thus represents a new class of non-thiol-containing radiation protectors, which may be useful in elucidating the mechanism(s) of radiation-induced cellular damage and may have broad applications in protecting against oxidative stress.