Biserka Nagy

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.

The effect of 2-[(aminopropyl)amino] ethanethiol (WR1065) on radiation-induced DNA damage and repair and cell progression in V79 cells

The radioprotector 2-[(aminopropyl)amino] ethanethiol (WR1065) was investigated with respect to its ability to affect radiation-induced DNA damage and repair in V79 cells. Studies were performed to evaluate the protector under conditions in which it is known to be effective in reducing the cytotoxic and mutagenic effects of gamma-irradiation. At a concentration of 4 mM, WR1065 protected against the formation of single strand breaks (SSB), as determined by the method of alkaline elution, when it was present during irradiation. The protector appeared, however, to inhibit the subsequent postirradiation repair or rejoining of SSB. While repair was complete within 24 h, the protector reduced the rate of repair by a factor of 3. This inhibitory effect on the rate of repair did not correlate with either measured differences in cell survival or mutagenesis. The radioprotector was also investigated with respect to its ability to affect cell cycle progression. WR1065 present in the growth medium inhibited the progression of cells through S-phase, and cell-doubling time following a 3 h exposure to the protector was increased from 11 to 18 h. These data are consistent with the well characterized property of thiols to inhibit DNA polymerase activity. It was concluded that, while the presence of WR1065 during irradiation reduced SSB-DNA damage, its effect on the subsequent rejoining of these breaks could not be correlated with its observed effect on protecting against radiation-induced mutagenesis. It may be that the inhibition of cell-cycle progression by the protector allowed more time to enhance the fidelity of repair as measured by the protectors ability to protect against radiation-induced mutagenesis.

Radioprotectors in treatment therapy to reduce risk in secondary tumor induction.

Radiation and chemotherapy are two effective modalities in the arsenal of cancer therapy. Because of increased awareness by the general public and earlier detection and improved diagnosis, more and more patients are being effectively treated and cured of cancer. Unfortunately, most of these therapeutic agents are, for the most part, extremely mutagenic and/or carcinogenic. When multiple agents such as combined radiation and chemotherapy are used, the carcinogenic potential may rise sharply. For example, patients treated for Hodgkins disease appear to exhibit a relatively high risk for therapy-induced acute myelogenous leukemia (Bartolucci et al . , 1983) and non-Hodgkins lymphoma (Jacquillat et al . , 1984). Thus, the risk of therapy-induced secondary tumors in patients having potentially curable disease is becoming a significant health problem (Rowley et al . , 1981; Penn, 1982; Chak et al . , 1984; Jacobs and Gale, 1984; Dorr and Coltman, 1985). Considerable effort has been devoted to developing treatment protocols that maximize therapeutic gain. Emphasis has been directed toward improving the ability of patients to withstand the acute deleterious effects of these therapeutic agents. The intent has been to attempt to enhance a sparing or differential protective effect to dose-limiting normal tissues. It is this rationale that has prompted considerable study of a class of chemical agents collectively referred to as radioprotectors. Specifically, the compound S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721, ethiofos) has been investigated because of its reported ability to differentially protect normal tissues compared with neoplastic tissues (Yuhas et al. , 1980; Phillips, 1980). Evaluation of this compound for use in radiation therapy is currently under way at a number of institutions. Recently, however, investigators have reported that WR-2721 can also significantly protect against the induction of tumors in rodents by ionizing radiation (Milas et al . , 1984). This observation is especially intriguing since it suggests that this class of agents might also be effective fi~r use in reducing the risk of late-effects damage such as the formation of therapy-induced secondary tumors.

Effect of an Aminothiol (WR-1065) on Radiation-Induced Mutagenesis and Cytotoxicity in Two Repair-Deficient Mammalian Cell Lines

WR-2721 and its free thiol WR-1065 have been found to effectively protect against radiation- and/or chemotherapy-induced mutagenesis (1-4), transformation (3) and carcinogenesis (6,7). With respect to the antimutagenic effect, WR-1065 significantly reduced the frequency of HGPRT mutants even when it was administered up to three hours following exposure of cells to radiation (1,4).

Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.

Protection by WR-2721 and WR-151327 against late effects of gamma rays and neutrons

Two thiophosphoroate compounds WR-2721 and WR-151327 were assessed for their ability to modify the deleterious effects (life shortening and carcinogenesis) of fission-spectrum neutrons (kerma-weighted mean energy of 0.85 MeV) or gamma rays on B6CF1 hybrid mice. Male and female mice, 200 of each sex per experimental group, were irradiated individually at 110 days of age. Radioprotectors (400 mg/kg of WR-2721 or 580 mg/kg of WR-151327) were administered intraperitoneally 30 min prior to irradiation. Neutron doses were 10 cGy or 40 cGy and gamma ray doses were 206 cGy or 417 cGy. Animals were housed five to a cage; cage locations in the holding rooms were randomized by computer. Animals were checked daily and all deceased animals were necropsied. WR-2721 afforded protection against both neutron- and gamma-ray-induced carcinogenesis and subsequent life shortening. Cumulative survival curves for unirradiated mice of either sex were unaffectecd by protectors. WR-2721 protected irradiated groups against life shortening by approximately 10 cGy of neutrons or 100 cGy of gamma rays. WR-151327 was as effective as WR-2721 against neutron irradiation.

JANUS neutron irradiation of a mouse cell line containing a shuttle vector plasmid

Recent reports indicate that high-LET neutrons are significantly more toxic to biological systems than low-LET radiation (1). On a unit dose basis, neutrons are much more effective in transforming cultured cells than are X or gamma rays (2). Also, they are 2.7 times more effective in producing chromosomal aberrations (3) and 10 times more efficient in inducing mutations in mammalian cells than are gamma rays (4). In contrast, certain sources of neutrons are no more effective than gamma rays at producing double-strand DNA breaks (DSB) and are actually less effective at producing single strand DNA breaks (SSB) (5). For example, the relative biological effectiveness (RBE) for JANUS neutron-induced DSBs is 1, while the RBEs for chromosomal aberrations, mutations, cellular transformation, and carcinogenesis are in the range of 4 to 20.

Plasminogen activator activity in clonogenic cell populations separated from a murine fibrosarcoma

Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that converts the zymogen plasminogen into the trypsin-like enzyme plasmin. Several studies indicate that tumor cell invasion is accompanied by proteolysis and that PA, generated by highly malignant cells, is by far the most ubiquitous protease associated with malignant transformation. Subpopulations of FSa cells were isolated by using density gradient centrifugation and the ability of these populations to form lung colonies was compared with their associated levels of PA production. Five populations of cells from a murine fibrosarcoma were separated in continuous gradients of Renografin in the density range 1-05–1·18 g/cm2. The PA activities of an unseparated control cell lysate and cell lysates of the five separated populations were determined by using [125I]fibrin as a substrate in a reaction between cell lysate and plasminogen. The assay was based on the release of digested [125I]fibrin from the surface of Petri dishes into the supernatant solution, and the results were expressed as a percentage of the total radioactivity. The cell populations collected at densities of 1-05 and 109 (B1, B2) were the more clonogenic with relative clonogenic efficiencies of 2·6 and 3·3 times that of the unseparated tumor population, respectively. Analysis for PA demonstrated that enzyme formation was restricted mostly to these two populations. Cells from populations 4 and 5 did not secrete increased amounts of PA and had reduced clonogenic efficiencies compared with the unseparated FSa control population. These results are consistent with the hypothesis that PA activity is correlated with the clonogenicity of tumor subpopulations isolated from a heterogeneous and complex tumor system such as the FSa.