Edward R. Epp

The role of thiols in cellular response to radiation and drugs.

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.

Biochemistry of reduction of nitro heterocycles.

Misonidazole is a metabolically active drug. Its addition to cells causes an immediate alteration in cellular electron transfer pathways. Under aerobic conditions the metabolic alterations can result in futile cycling with electron transfer to oxygen and production of peroxide. Thiol levels are extremely important in protecting the cell against the peroxide formation and potentially hazardous conditions for hydroxyl radical production. Nevertheless such electron shunting out of cellular metabolism will result in alterations in pentose cycle, glycolysis and cellular capacity to reduce metabolites to essential intermediates needed in DNA metabolism (i.e. deoxyribonucleotides). Glutathione must be depleted to very low levels before toxic effects of misonidazole and other nitro compounds are manifested in cell death via peroxidative damage. Under hypoxic conditions misonidazole also diverts the pentose cycle via its own reduction; however, unlike the aerobic conditions, there are a number of reductive intermediates produced that react with non-protein thiols such as GSH as well as protein thiols. The reaction with protein thiols results in the inhibition of glycolysis and other as yet undetermined enzyme systems. The consequences of the hypoxic pretreatment of cells with nitro compounds are increased vulnerability to radiation and chemotherapeutic drugs such as L-PAM, cis-platinum and bleomycin. The role that altered enzyme activity has in the cellular response to misonidazole and chemotherapeutic agents remains to be determined. It is also clear that the GSH depleted state not only makes cells more vulnerable to oxidative stress but also to hypoxic intermediates produced by the reduction of misonidazole beyond the one electron stage. The relevancy of the present work to the proposed use of thiol depletion in vivo to enhance the radiation or chemotherapeutic response of tumor tissue lies with the following considerations. Apparently, spontaneous peroxidative damage to normal tissue such as liver can occur with GSH depletion to 10-20% of control and with other normal tissue when GSH reaches 50% of control. This situation can obviously become more critical if peroxide producing drugs are administered. The only advantage to such combined drug treatments would lie in the possibility that tumors vary in their catalase and peroxidase activity and consequently may be more vulnerable to oxidative stress (cf. review by Meister. Our tumor model, the A549 human lung carcinoma cell in vitro, appears to be an exception because it has catalase, peroxidase and a high content of GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Nonprotein Thiols and the Radiation Response of A549 Human Lung Carcinoma Cells

Glutathione (GSH)-depletion by buthionine sulphoximine (BSO) altered both the aerobic and anaerobic radiation response of A549 human lung cancer cells grown in vitro. The oxygen enhancement ratio (o.e.r) was increased slightly from 3.0-3.3. The lack of an effect of GSH-depletion on o.e.r. reduction, provides a system whereby the mechanism of action of the thiol reactive reagent diethylmaleate (DEM) can be investigated. Pretreatment of cells with DEM, under non-toxic concentrations, removed 13 per cent of the intracellular NPSH and resulted in an o.e.r. of 2. When BSO followed by DEM was used, so that both GSH and NPSH were reduced to zero, an o.e.r. of 1.5 was obtained. Cells treated with 1 mM BSO for 24 hours contained 10 per cent NPSH and no GSH. When these cells were exposed to 0.5 or 1 mM DEM briefly, during irradiation, the o.e.r. was 2.4 and 1.7 respectively. In some cases altered o.e.r.s occurred in combination with increased aerobic responses. This was especially true for aerobic irradiations of BSO-treated cells in the presence or absence of DEM. However, the increased aerobic response was offset by a more dramatic increase in the hypoxic response. These results indicate (a) that GSH plays a significant role in aerobic radiation response but is not a principal factor in o.e.r.-reduction, and (b) that reduction of the o.e.r. by DEM is not due primarily to GSH-removal. The preferential radiosensitization of hypoxic cells by DEM may involve reactions of this compound with NPSH or protein SH, or may be related to the ability of DEM to mimic oxygen as a hypoxic cell radiosensitizer.

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.

Underdosing of lesions resulting from lack of electronic equilibrium in upper respiratory air cavities irradiated by 10 mv X-ray beams

Abstract When a high energy photon beam, such as from a linear accelerator or cobalt unit, is used to treat lesions located in the upper respiratory air passages, the beam often must traverse an air cavity before it reaches the lesion. Because of this traversal of air, it is not clear that the surface layers of the lesion forming the air-tumor tissue interface will be in a state of near electronic equilibrium; if they are not, underdosing of these layers could result. This problem has been investigated for 10 MV X-ray beams which are becoming widely available for radiotherapy with linear accelerators. The build-up of ionization in a small parallel plate chamber was measured when the chamber was located in a phantom air cavity, the size of which could be varied in three dimensions over the anatomical range as the irradiating field was changed. A significant effect was measured and was determined to be very dependent on field size and on air cavity dimension. It was found that this non-electronic equilibrium effect at the surface of an air cavity is more severe for the higher energy photon beam than that previously measured for 60 Co radiation. This problem is of clinical concern when small fields are used to irradiate lesions through air cavities in the upper respiratory tract.

Intraoperative irradiation: A pilot study combining external beam photons with “boost” dose intraoperative electrons

Intraoperative “boost” dose electron beam therapy given in combination with 4500‐5000 rad (45–50 Gray) external beam irradiation has been demonstrated as a practical therapeutic modality at the MGH. This procedure has been employed thus far in 58 patients; the results in the initial 36 are analyzed in detail in this paper. Thirty‐four of the 36 patients had locally advanced lesions—unresectable, recurrent, or residual disease. Results achieved to date are in full agreement with our expectations: high radiation doses have been delivered to the primary intra‐abdominal and pelvic tumors, excluding the sensitive structures from irradiation. This has been accomplished by a truly multidisciplinary effort comprising surgery, anesthesiology, OR nursing, administration, engineers, physicists, therapy technologists, and radiation therapists. Although follow‐up is not yet sufficient to judge ultimate efficacy, acute and chronic severe morbidity is low and local control is good. There is justified enthusiasm for continuing the procedure.

The Oxygen Effect in Bacterial Cells Irradiated with High-Intensity Pulsed Electrons

The oxygen effect has been studied in Escherichia coli B/r irradiated with high-intensity pulsed electrons as a function of oxygen concentration. The dose to the cells was delivered in single pulse...

Neutrons from high-energy x-ray medical accelerators: An estimate of risk to the radiotherapy patient

The problem of neutrons produced by many of the high-energy x-ray therapy machines (10 MV and above) is reviewed, and the possible risk their presence poses to radiotherapy patients is estimated. A review of the regulatory background containing a summary of the recommendations of the U.S. Council of State Governments (USCSG), and of the International Electro-Technical Commission (IEC), as well as an indication that recommendations will be forthcoming from the National Council on Radiation Protection (NCRP) and the International Commission of Radiological Protection (ICRP) is presented. The neutrons in question are produced by high-energy photons (x rays) incident on the various materials of the target, flattening filter, collimators, and other essential components of the equipment. The neutron yield (per treatment dose) increases rapidly as the megavoltage is increased from 10 to 20 MV, but remains approximately constant above this. Measurements and calculations of the quantity, quality, and spatial distribution of these neutrons and their concomitant dose are summarized. Values of the neutron dose are presented as entrance dose, midline dose (10-cm depth), and integral dose, both within and outside of the treatment volume. These values are much less than the unavoidable photon doses which are largely responsible for treatment side effects. For typical equipment, the average neutron integral dose from accelerator-produced neutrons is about 4-7 g cGy (per treatment cGy), depending on the treatment plan. This translates into an average dose of neutrons [averaged over the body of a typical 70-kg (154 lb) patient] of 0.06-0.10 cGy for a treatment of 1000 cGy. Using these neutron doses and the best available neutron risk coefficients, it is estimated that 50 X 10(-6) fatal malignancies per year due to the neutrons may follow a typical treatment course of 5000 rads of 25-MV x rays. This is only about 1/60th of the average incidence of malignancies for the general population. Thus, the cancer risk to the radiotherapy patient from accelerator-produced neutrons poses an additional risk to the patient that is negligible in comparison.

Oxygen Sensitization of Mammalian Cells under Different Irradiation Conditions

The oxygen dependence of the radiosensitivity of cultured CHO cells was examined in detail with particular attention paid to avoiding possible artifacts due to radiolytic oxygen depletion. Two methods of gas equilibration and irradiation were used. In the first approach, cells were irradiated with 50-kVp X rays in a thin-layer geometry which offered maximum interchange between the cells and the surrounding gas. The second technique employed 280-kVp X irradiation of cells under full-medium conditions with mechanical agitation to minimize the effect of radiochemical oxygen consumption by promoting rapid oxygen replenishment. With these techniques oxygen radiosensitization was clearly resolved at an oxygen concentration of 0.03% in the gas phase. The oxygen K curves measured by these two methods were similar in shape over a wide range of oxygen concentration.

Dosimetry, field shaping and other considerations for intra-operative electron therapy

Abstract In the early part of 1978, a pilot program of intra-operative radiotherapy was initiated at the Massachusetts General Hospital (MGH), using beams of high energy electrons. A technique has been employed to irradiate the tumor bearing area using a sterilized acrylic resin cone that slides into a metal holder attached to the head of the accelerator. The acrylic resin cone is inserted into the patient directly over the tumor; the patient couch is adjusted until the cone is correctly aligned inside the holder. The dosimetry for this procedure has been investigated as a function of the primary collimator setting of the linear accelerator. A fixed setting was chosen as a compromise between increased bremsstrahlung, low effective electron dose rate observed with narrower settings, and more rounded beam profiles together with somewhat poorer depth dose characteristics found with larger settings. Field shaping and blocking of critical organs was achieved using sterile lead sheets that are cut to the appropriate size. Consideration has been given to improved beam design by increasing the incident electron dose rate and by improving the depth dose at each energy. The design of a dedicated intra-operative facility, using a high energy linear accelerator, is presented with respect to shielding requirements for the machine and the room.