David J. Grdina

Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003

Abstract Stone, H. B., Moulder, J. E., Coleman, C. N., Ang, K. K., Anscher, M. S., Barcellos-Hoff, M. H., Dynan, W. S., Fike, J. R., Grdina, D. J., Greenberger, J. S., Hauer-Jensen, M., Hill, R. P., Kolesnick, R. N., MacVittie, T. J., Marks, C., McBride, W. H., Metting, N., Pellmar, T., Purucker, M., Robbins, M. E., Schiestl, R. H., Seed, T. M., Tomaszewski, J., Travis, E. L., Wallner, P. E., Wolpert, M. and Zaharevitz, D. Models for Evaluating Agents Intended for the Prophylaxis, Mitigation and Treatment of Radiation Injuries. Report of an NCI Workshop, December 3–4, 2003. Radiat. Res. 162, 711–728 (2004). To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.

Radioprotectants: Current Status and New Directions

The ability to prevent radiotherapy-induced toxicity without affecting antitumor efficacy has the potential to enhance the therapeutic benefit for cancer patients without increasing their risk of serious adverse effects. Among the currently available cytoprotective agents capable of protecting normal tissue against damage caused by either chemo- or radiotherapy, only amifostine has been shown in clinical trials to reduce radiation-induced toxicity. Most notably, it reduces the incidence of xerostomia, which is a clinically significant long-term toxicity arising in patients undergoing irradiation of head and neck cancers. In vitro studies with the active metabolite of amifostine (WR-1065) have shown it to prevent both radiation-induced cell death and radiation-induced mutagenesis. The potential of this agent to prevent secondary tumors, as well as other radiation-induced toxicities is now the focus of ongoing research. Among other novel approaches to radioprotection being explored are methods to increase levels of the antioxidant mitochondrial enzyme manganese superoxide dismutase (MnSOD). In addition, the use of epoetin alfa, alone or in combination with cytoprotectants (e.g., amifostine), to treat radiation-induced anemia is also being investigated. The objective of developing newer cytoprotective therapies is to improve the therapeutic ratio by reducing the acute and chronic toxicities associated with more intensive and more effective anticancer therapies.

Amifostine: mechanisms of action underlying cytoprotection and chemoprevention.

Amifostine is an important drug in the new field of cytoprotection. It was developed by the Antiradiation Drug Development Program of the US Army Medical Research and Development Command as a radioprotective compound and was the first drug from that Program to be approved for clinical use in the protection of dose limiting normal tissues in patients against the damaging effects of radiation and chemotherapy. Its unique polyamine-like structure and attached sulfhydryl group give it the potential to participate in a range of cellular processes that make it an exciting candidate for use in both cytoprotection and chemoprevention. Amifostine protects against the DNA damaging effects of ionizing radiation and chemotherapy drug associated reactive species. It possesses anti-mutagenic and anti-carcinogenic properties. At the molecular level, it has been demonstrated to affect redox sensitive transcription factors, gene expression, chromatin stability, and enzymatic activity. At the cellular level it has important effects on growth and cell cycle progression. This review focuses on relating its unique chemical design to mechanisms of action that underlie its broad usefulness as both a cytoprotective and chemopreventive agent for use in cancer therapy.

Activation of NFκB and MnSOD gene expression by free radical scavengers in human microvascular endothelial cells

The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NFκB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNFα. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NFκB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however, were effective in activating NFκB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50–p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NFκB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NFκB activation by SH. Thus, while oxidative damage is known to activate NFκB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NFκB. There does not appear to be a significant role, if any, for the production of H2O2 as an intermediate step in the activation of NFκB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NFκB. In contrast to TNFα, exposure of cells to either 40 μM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure.

Delayed radioprotection by NFκB-mediated induction of Sod2 (MnSOD) in SA-NH tumor cells after exposure to clinically used thiol-containing drugs

Abstract Murley, J. S., Kataoka, Y., Cao, D., Li, J. J., Oberley, L. W. and Grdina, D. J. Delayed Radioprotection by NFκB-Mediated Induction of Sod2 (MnSOD) in SA-NH Tumor Cells after Exposure to Clinically Used Thiol-Containing Drugs. Radiat. Res. 162, 536–546 (2004). The ability of thiol-containing reducing agents to activate transcription factors leading to changes in gene expression and enzyme activities provides an additional mechanism to potentially protect against radiation-induced cell killing. Manganese superoxide dismutase (Sod2) is one such gene whose expression levels have been shown to be elevated after exposure to the thiol compounds WR-1065 and N-acetyl-l-cysteine (NAC), resulting in an increase in radiation resistance. To further characterize this effect, SA-NH sarcoma cells, both wild-type and a clone stably transfected with a plasmid containing an IκBα gene mutated at serines 32 and 36, which prevents the inducible phosphorylation of these residues and the subsequent activation of NFκB (SA-NH+mIκBα1), were grown to confluence and then exposed to amifostines free thiol WR-1065 at a concentration of 4 mM for 30 min. Effects of thiol exposure on NFκB activation in SA-NH+mIκBα1 cells were determined by a gel shift assay, and changes in Sod2 protein levels in these cells 24 h after exposure to 40 μM or 4 mM WR-1065 were measured by Western blot analysis and compared with wild-type cells exposed to the NFκB inhibitor BAY 11-7082. Changes in radiation response, measured immediately after thiol exposure or 24 h later, were determined using a colony-forming assay and were correlated with NFκB activation and Sod2 protein levels. The effects of captopril, mesna and NAC, each at a dose of 4 mM, on radiation response were also determined and contrasted with those of WR-1065. Only WR-1065 and captopril protected SA-NH cells when present during irradiation, i.e. 1.57 and 1.31 times increase in survival at 2 Gy, respectively. All four thiols were protective if irradiation with 2 Gy occurred 24 h later; i.e. increases in survival of 1.40, 1.22, 1.35, and 1.25 times were found for WR-1065, captopril, mesna and NAC, respectively. This delayed radioprotective effect correlated with elevated Sod2 protein levels in wild-type SA-NH tumor cells but was not observed in SA-NH+mIκBα1 cells, indicating that interference with thiol-induced NFκB activation abrogates this delayed radioprotective effect. Because the delayed radioprotective effect is readily demonstrable at a radiation dose of 2 Gy 24 h after exposure to clinically approved thiol-containing drugs such as amifostine, captopril, mesna and NAC, it suggests a new potential concern regarding the issue of tumor protection and the use of these agents in cancer therapy.

Amifostine reduces lung vascular permeability via suppression of inflammatory signalling

Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. The present study investigated the effect of pre-treatment with amifostine, a thiol antioxidant compound, on lung endothelial barrier dysfunction induced by Gram-negative bacteria wall-lipopolysaccharide (LPS). Endothelial permeability was monitored by changes in transendothelial electrical resistance. Cytoskeletal remodelling and reactive oxygen species (ROS) production was examined by immunofluorescence. Cell signalling was assessed by Western blot. Measurements of Evans blue extravasation, cell count and protein content in bronchoalveolar lavage fluid were used as in vivo parameters of lung vascular permeability. Hydrogen peroxide, LPS and interleukin-6 caused cytoskeletal reorganisation and increased permeability in the pulmonary endothelial cells, reflecting endothelial barrier dysfunction. These disruptive effects were inhibited by pre-treatment with amifostine and linked to the amifostine-mediated abrogation of ROS production and redox-sensitive signalling cascades, including p38, extracellular signal regulated kinase 1/2, mitogen-activated protein kinases and the nuclear factor-κB pathway. In vivo, concurrent amifostine administration inhibited LPS-induced oxidative stress and p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and neutrophil recruitment to the lungs. The present study demonstrates, for the first time, protective effects of amifostine against lipopolysaccharide-induced lung vascular leak in vitro and in animal models of lipopolysaccharide-induced acute lung injury.

radiation-induced DNA double-strand break frequencies in human squamous cell carcinoma cell lines of different radiation sensitivities

DNA neutral (pH 9.6) filter elution was used to measure radiation-induced DNA double-strand break (dsb) frequencies in eight human squamous cell carcinoma cell lines with radiosensitivities (D0) ranging from 1.07 to 2.66 Gy and D values ranging from 1.46 to 4.08 Gy. The elution profiles of unirradiated samples from more radiosensitive cell lines were all steeper in slope than the profiles from resistant cells. The shapes of the dsb induction curves were curvilinear and there was some variability from cell line to cell line in the dose-response for the induction of DNA dsb after exposures to 5-100 Gy 60Co gamma-rays. There was no relation between the shapes of the survival curves and the shapes of the dose-responses for the induction of DNA dsb. At low doses (5-25 Gy), three out of four of the more sensitive cell lines (D less than 2.5 Gy) had larger initial break frequencies than the more resistant lines (D greater than 3.0 Gy). Although the low-dose (5-25 Gy) elution results were variable, they do suggest that DNA neutral elution will detect differences between sensitive and resistant tumour cells in initial DNA dsb frequencies.

A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed.

Protective effects of 2-[(aminopropyl)amino] ethanethiol against bleomycin and nitrogen mustard-induced mutagenicity in V79 cells

This study examines the effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on bleomycin (BLM) and nitrogen mustard- (HN2) induced cytotoxicity, DNA damage, and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells. The anti-mutagenic effect of WR-1065 on cis-diamminedichloroplatinum (cis-DDP) and radiation- (XRT) induced HGPRT mutations was also evaluated for comparative purposes. WR-1065 (4 mM) was added prior to exposure of cells to therapy agents. All exposure times were 30 min. and both cell survival and mutagenesis were assayed. WR-1065 was effective in protecting against both effects. The induction of mutants corrected for background by BLM, HN2, cis-DDP, or XRT was linear in all cases. Mutation frequencies without WR-1065 were 78 X 10(-6) per unit BLM, 66 X 10(-7) per microgram HN2, 25 X 10(-7) per microgram cis-DDP; and 87 X 10(-7) per Gy of XRT. With WR-1065, these were reduced to 37 X 10(-6) per unit BLM, 40 X 10(-7) per microgram HN2, 1 X 10(-7) per microgram cis-DDP, and 44 X 10(-7) per Gy of XRT. Mutation protection factors (MPF), a ratio of the corresponding slopes of the mutation induction curves, with and without WR-1065 were: BLM, MPF = 2.8; HN2, MPF = 3.4; cis-DDP, MPF = 7.1; and XRT, MPF = 5.1. Single-strand-break (SSB) formation in DNA by BLM or HN2, assayed by alkaline elution, was protected against by WR-1065. WR-1065 did not induce SSB in control cells. The reduction of the mutagenic effects of agents used in radiation and chemotherapy by radioprotectors may be an important additional benefit for consideration in their use in cancer treatment.

Measurement of differences in pO2 in response to perfluorocarbon/carbogen in FSa and NFSa murine fibrosarcomas with low-frequency electron paramagnetic resonance oximetry.

We have used very low-frequency electron paramagnetic resonance (EPR) oximetry to measure the change in oxygen concentration (delta pO2) due to change in breathing atmosphere in FSa and NFSa fibrosarcomas implanted in the legs of C3H mice infused with perfluoro-octylbromine (PFOB). Measurements in each tumor were made before and after the administration of the high-density (47% v/v) perfluorocarbon PFOB, perflubron (Alliance Pharmaceutical Corporation, San Diego, CA). Measurements in each tumor were also made, after the administration of the PFOB, both before (PFOB/air) and after the administration of carbogen (95% O2 + 5% CO2, PFOB/carbogen). Large changes (delta p02) relative to PFOB/air oxygenation were seen with the administration of PFOB/carbogen. No significant difference in oxygen concentration was seen between air-breathing mice with and without PFOB. The mean delta pO2 for FSa tumors was 13 +/- 6 torr, while the mean for NFSa fibrosarcomas was 28 +/- 7 torr. There were such large intertumor differences that the trend toward a smaller change in the more hypoxic FSa tumors was not significant (P = 0.13). This paper describes a novel method of measuring differences in oxygenation in tumor tissues. The results of such measurements indicate large differences in pO2 response to different breathing atmospheres in PFOB-infused tumors of similar histology. The intertumor delta pO2 differences may correlate with differences in radiation response.