Hollow Bragg waveguides fabricated by controlled buckling of Si/SiO2 multilayers.

We describe integrated air-core waveguides with Bragg reflector claddings, fabricated by controlled delamination and buckling of sputtered Si/SiO2 multilayers. Thin film deposition parameters were tailored to produce a desired amount of compressive stress, and a patterned, embedded fluorocarbon layer was used to define regions of reduced adhesion. Self-assembled air channels formed either spontaneously or upon heating-induced decomposition of the patterned film. Preliminary optical experiments confirmed that light is confined to the air channels by a photonic band-gap guidance mechanism, with loss ~5 dB/cm in the 1550 nm wavelength region. The waveguides employ standard silicon processes and have potential applications in MEMS and lab-on-chip systems.

Thermal tuning of hollow waveguides fabricated by controlled thin-film buckling.

We describe the thermal tuning of air-core Bragg waveguides, fabricated by controlled formation of delamination buckles within a multilayer stack of chalcogenide glass and polymer. The upper cladding mirror is a flexible membrane comprising high thermal expansion materials, enabling large tuning of the air-core dimensions for small changes in temperature. Measurements on the temperature dependence of feature heights showed good agreement with theoretical predictions. We applied this mechanism to the thermal tuning of modal cutoff conditions in waveguides with a tapered core profile. Due to the omnidirectional nature of the cladding mirrors, these tapers can be viewed as waveguide-coupled, tunable Fabry-Perot filters.

Chip-scale spectrometry based on tapered hollow Bragg waveguides.

We describe a micro-spectrometer that exploits out-of-plane radiation at mode cutoff in a tapered leaky waveguide clad by omnidirectional Bragg reflectors. The device can be viewed as a side-coupled, tapered Fabry-Perot cavity. An effective-index transfer-matrix model reveals that optimal resolution is dependent on the reduction or mitigation of back-reflection and standing waves leading up to the cutoff point. We address this by insertion of low numerical aperture optics between the taper and the detector, and demonstrate an experimental resolution as small as approximately 1 nm and operating bandwidth >100 nm in the 1550 nm range, from a tapered waveguide with footprint approximately 50 microm x 500 microm. The device combines the small size of a Fabry-Perot instrument with the detector array compatibility and fixed optics of a grating-based instrument.

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate.

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.

Effect of varying the concentration of damage restituting species in the radical repair model.

From analytical expressions derived for the radical-repair (competition) model describing the relationship between cellular radiosensitivity and oxygen concentration, "K-curve" behavior has been quantified as a function of the concentration of the species S which restitutes the radiation-induced radicals to their original molecular configuration. If these species are identified with thiols, K-curves modified by fractionally depleting [S] through calculation can be compared with experimental data where cells have their thiols depleted using various means, for example, by chemical agents or by the use of cells with decreased thiols because of genetic deficiency. Families of curves have been calculated related both to the S-depleted and the non-S-depleted hypoxic control, the latter of which is used to calculate enhancement ratios. Comparison of the model with experimental data is made.

Role of glutathione in the aerobic radiation response.

We will review the relationships between glutathione (GSH), protein thiols, and cellular responses to radiation, peroxides, and peroxide-producing drugs. Our primary interest involves the behavior of sulfhydryls as electron and hydrogen carriers, and their capacity to protect various target molecules against radiation and peroxidative damage. We used reagents such as L-buthionine sulfoximine (LBSO), alone and in combination with N-ethyl maleimide (NEM), diamide, and dimethylfumarate, to decrease GSH so that it could no longer participate in the electron transfer reactions. Our results indicate that aerobic sensitization produced by GSH depletion can be further enhanced if electron-accepting agents, such as tertiary butyl hydroperoxide (t-BOOH), are present during irradiation. Hydroperoxide is a substrate for glutathione peroxidase and diverts electrons and hydrogen away from target molecules during its reduction. Sensitivity to radiation seems to be due to the inhibition of the mitochondria's capacity to reduce hydroperoxide. We will also report the mitochondria's ability to reduce the oxygen radicals produced by radiation and drugs. Data also indicate that t-BOOH oxidizes protein thiols which are enzymatically involved in repair of DNA damage.

Therapeutic gain factors for fractionated radiation treatment of spontaneous murine tumors using fast neutrons, photons plus O2(1) or 3 ATA, or photons plus misonidazole.

Therapeutic gain factors (TGFs) have been determined for three spontaneous tumors of the C3H mouse treated by photons + normobaric oxygen (O2(1) ATA), photons + hyperbaric oxygen (O2 3 ATA), photons + misonidazole, or fast neutrons. The tumors were early generation isotransplants of spontaneous tumors: MCaIV, a mammary carcinoma; FSaII, a fibrosarcoma; and SCCVII, a squamous cell carcinoma. The tumors, transplanted to the right leg, were 6 mm at start of treatment. Normal tissue responses studied were acute reaction of normal skin (all treatment modalities) and LD50 following irradiation of the upper abdomen (in test of photons + O2 at 1 or 3 ATA). Thus both the tumor and normal tissues would be classified as "acute responding." All subject tissues were at congruent to 34.5-35 degrees C at irradiation. Treatments were based on d(25)Be or p(43)Be fast neutron beams, 60Co and 137Cs photon beams. Treatments were given in 5 or 15 equal doses in 5 days. For photon treatments, TGFs (air/O2 3 ATA) were substantially and significantly larger than 1 for all three tumor systems treated at small or large doses per fraction when related to skin or abdominal tissue responses. The TGFs (air/O2 1 ATA) were greater than 1 at small doses per fraction for MCaIV and FSaII for skin as the normal tissue; the TGFs for all three tumors and at all doses per fraction would be greater than 1 when related to upper abdominal tissues. TGFs (O2 1 ATA/O2 3 ATA) for photon irradiation greater than 1 were found only for SCCVII and that obtained for both large and small doses per fraction. Misonidazole achieved impressive TGFs (air/air + miso or air/O2 1 ATA + miso); the drug was tested only at 10-12 Gy/fraction and relative to skin. RBEs(FN) for the three tumors were lower at 1.5-2 Gy(FN)/fraction than at 5-6 Gy(FN)/fraction, i.e. the opposite to that reported for normal tissue (RBE increases with decreasing dose per fraction). A TGF (relative to skin reaction) greater than 1 for fast neutron therapy was found only for SCCVII when treated at large doses/fraction; this was true for air or O2 1 ATA conditions.

Effect of dimethyl fumarate on the radiation sensitivity of mammalian cells in vitro.

Dimethylfumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction which may be mediated by GSH-S-transferase. In Chinese hamster ovary cells this depletion is rapid; e.g., 0.5 mM DMF depletes GSH to less than 10% of control in 5 min at room temperature. DMF is a very effective hypoxic cell radiosensitizer, with an enhancement ratio (ER) of about 3 obtained by a 5-min exposure of cells at room temperature to 5 mM DMF, without significant toxicity. At this same concentration of drug, there is a small enhancement of aerobic cells (ER = 1.3), but the 5 mM DMF in hypoxia results in nearly a complete collapse of the hypoxic dose-response curve to the same level as seen in air with DMF. It has been suggested previously that DMF sensitizes cells via electron affinic mechanisms. However, this appears not to be the case in this study, as shown by the fact that cells pretreated with DMF and then washed free of the drug remained equally radiosensitive as cells irradiated in the presence of the drug. This large enhancement of radiation sensitivity appears to be related to the drug's ability to deplete thiols; i.e., thiols appear to be a major factor responsible for radioresistance of hypoxic cells.

The role of glutathione in the aerobic radioresponse. I. Sensitization and recovery in the absence of intracellular glutathione.

The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549. Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG. Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased. Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min). However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected. These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization. Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.

Enhancement in the aerobic toxicity of misonidazole and SR-2508 by buthionine sulfoximine and 4-hydroxypyrazole: the role of hydrogen peroxide.

Chronic aerobic exposure of A549 human lung carcinoma cell cultures to 0.1 mM L-buthionine-S,R-sulfoximine and 1 mM misonidazole, or 1 mM SR-2508 results in inhibition of cell growth and decreased clonogenic survival. These patterns are not apparent with the individual drug treatments. Both parameters demonstrate maximum toxicity after 72 hr in culture, which parallels the time required to deplete A549 cells of glutathione with 0.1 mM L-BSO under these growth conditions. Toxicity appears to be related to hydrogen peroxide-produced during 1 electron reduction of the nitro compounds in the presence of oxygen. A549 cells have a lowered capacity to reduce peroxide due to the effect of thiol depletion on the enzymes GSH-peroxidase and GSH-S-transferase, which require the tripeptide as a substrate. The addition of catalase, another important enzyme involved in peroxide reduction, partially reverses the observed toxicity. 4-Hydroxypyrazole, which inhibits endogenous catalase activity, causes an increase in the observed cytotoxicity. Similar effects of L-BSO and 4-hydroxypyrazole are seen for toxicity due to hydrogen peroxide being added directly to cell cultures.

The effect of L-buthionine sulfoximine on the aerobic radiation response of A549 human lung carcinoma cells.

Our data show that A549 cells are increasingly radiosensitive with prolonged exposure to L-BSO. The resulting glutathione and protein thiol depleted cells show both loss of shoulder and slope modification. Furthermore, there is an increase in single strand DNA breaks and irrepairable cross-linking. The aerobic radiation damage in the thiol depleted state appears to be different from that obtained with hypoxic cells. Any postulated role for GSH in reducing or preventing peroxidative radiation damage must also include protection against single strand DNA breaks as well as involvement in repairing DNA-protein cross-links. The latter effect may be related to decreased protein thiol content as reflected in a decreased enzyme capacity to repair DNA damage.

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)

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy.

We have measured the rate of GSH resynthesis in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with either 0.25 mM or 0.5 mM DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. The 24 hr BSO + 2 hr DEM treatment sensitizes hypoxic cells to a greater degree than either agent alone but does not increase the aerobic response more than is seen with BSO alone. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The decreased cell survival following aerobic irradiation, after prolonged treatment with BSO or acute exposure to BSO + DEM, may be in part due to inhibition of glutathione peroxidases. For example, glutathione-S-transferase, known to have glutathione peroxidase activity (non-selenium), is nearly completely inhibited by the BSO treatments. In addition, cellular capacity to react with peroxide (glutathione peroxidase, selenium containing) was also inhibited. We suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased.

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.

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.

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.