Gold nanoparticles in combination with megavoltage radiation energy increased radiosensitization and apoptosis in colon cancer HT-29 cells

Purpose: Gold nanoparticles (GNP) act as a radiosensitizer in radiation therapy. However, recent studies have shown contradictory evidence in terms of radiosensitization in the presence of GNP combined with X-ray megavoltage energy (MV) on different cell types. In this study, the effect of GNP on radiosensitization enhancement of HT-29 human colorectal cancer cells at MV X-ray energy was evaluated.

Materials and methods: The cytotoxicity and radiosensitization of GNP were evaluated in HT-29 human colorectal cancer cells by MTS-assay and multiple MTS-assay, respectively. Cellular uptake was assayed using graphite furnace atomic absorption spectrometry (GFAAS). Apoptosis and cell cycle progression were determined by an Annexin V-FITC/propidium iodide (PI) kit and PI/RNase solution with flow cytometry, respectively.

Results: Results showed that the cell viability of the HT-29 cells was not influenced by exposure to different concentrations of GNP (10–100 μM). GNP alone did not affect the cell cycle progression and apoptosis. In contrast, GNP, in combination with radiation (9?MV), induced more apoptosis. The interaction of GNP with MV energy resulted in a significant radiosensitization enhancement compared with irradiation alone.

Conclusion: It was concluded that GNP may work as bio-inert material on HT-29 cancer cells and their enhancement of radiosensitization may be due to increase in the absorbed irradiation dose.     

A Comparison of the Intracellular Uptake and Radiosensitization Efficiency in Different Media of Uncharged 2-nitroimidazoles of Varying Lipophilicity

Summary

The effect of varying octanol: water partition coefficients, P, (range 0·026–260) on the uptake of uncharged 2-nitroimidazoles into Chinese hamster V79 379A cells has been studied. Average intracellular concentrations were measured by high performance liquid chromatography after centrifuging cells through oil or an aqueous medium. The ratio of intracellular concentration of radiosensitizer to extracellular concentration (C_i/C_e) for misonidazole (P = 0·43) was 0·85 for the oil method and 0·68 for the aqueous method. For values of P less than about 0·05 uptake was initially very slow and C_i was always less than C_e. When P ≧ 0·1 uptake was rapid and then remained unchanged for times up to 3 h; for P ≧ 10, C_i/C_e increased rapidly as P increased. Ro 31-1405 (P = 260) concentrated by a factor of 7 inside the cell. Although uptake was identical for cells suspended in full growth medium and PBS, radiosensitization was greater for cells in PBS: 1 mmol dm^?3 misonidazole produced an enhancement ratio of 1·6 in full growth medium and 1·9 in PBS. This increase in radiosensitization could not be accounted for by protein binding. However, measurements on cellular non-protein sulphydryl (NPSH) demonstrated the levels to be reduced to about 60 per cent for cells in PBS. Similar reductions in NPSH levels have previously been shown not to increase the radiosensitivity of control cells but to increase greatly the effectiveness of nitroimidazole radiosensitizers.     

Lesions in DNA of Hypoxic Mammalian Cells Irradiated in the Presence of TAN

Summary

TAN (triacetoneamine-N-oxyl) at a concentration of 10 mM gives a dose modifying factor (DMF) of 1·45 for the surviving fraction of γ-irradiated hypoxic mammalian cells. Under the same conditions, however, TAN produces little or no enhancement of single strand breaks (SSB) in DNA (DMF ? 1). In this study, enhancement of the type of DNA lesions exposed by an extract of M. luteus is reported. The DMF for this type of damage (MLS) is 1·5.

TAN is known to affect the size of DNA synthesized after irradiation, presumably by causing interruptions in synthesis at TAN–DNA adduct sites. The absolute number of M. luteus extract sensitive sites detected in this work corresponds to approximately one half of the number of sites of interrupted synthesis. In its sensitizing effect on DNA, the free radical TAN is different from the electron affinic drug, misonidazole.     

Enhancement of radiosensitivity of melanoma cells by pegylated gold nanoparticles under irradiation of megavoltage electrons

Purpose: Gold nanoparticles (GNP) have significant potential as radiosensitizer agents due to their distinctive properties. Several studies have shown that the surface modification of nanoparticles with methyl polyethylene glycol (mPEG) can increase their biocompatibility. However, the present study investigated the radiosensitization effects of mPEG-coated GNP (mPEG-GNP) in B16F10 murine melanoma cells under irradiation of 6?MeV Electron beam.

Materials and methods: The synthesized GNP were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy, and zeta potential. Enhancement of radiosensitization was evaluated by the clonogenic assay at different radiation doses of megavoltage electron beams.

Results: It was observed that mPEG-GNP with a hydrodynamic size of approximately 50?nm are almost spherical and cellular uptake occurred at all concentrations. Both proliferation efficiency and survival fraction decreased with increasing mPEG-GNP concentration. Furthermore, significant GNP sensitization occurred with a maximum dose enhancement factor of 1.22 at a concentration of 30 μM.

Conclusions: Pegylated-GNP are taken up by B16F10 cancer cells and cause radiosensitization in the presence of 6?MeV electrons. The radiosensitization effects of GNP may probably be due to biological processes. Therefore, the underlying biological mechanisms beyond the physical dose enhancement need to be further clarified.     

Radiosensitization of Chinese Hamster Cells by Oxygen and Misonidazole at Low X-ray Doses

Summary

The radiosensitization of Chinese hamster V79 cells in vitro by air and misonidazole at low X-ray doses (0·2–6·0 Gy) had been studied. These survival data, together with high-dose data, were fitted to the linear quadratic model ln S = ?(αD + βD²), deriving estimates of α and β by six different methods to illustrate the influence of the statistical treatment on the values so derived. This in vitro study clearly demonstrated that the survival parameters α and β are dependent to some degree on the method of analysis of the raw survival data; however, their ratios, the values of oxygen enhancement ratios (OERs) and radiosensitizer enhancement ratios (SERs) derived from the different methods, are similar. All methods of analysis give reduced OERs at low radiation doses for combined low- and high-dose X-ray data. However, the OERs are still appreciably high, ranging from 2·45 to 2·50 for an oxic dose of 2 Gy. All methods of analysis gave reduced SERs at low doses for combined low and high X-ray dose data for hypoxic cells irradiated in 1 mmol dm^?3 misonidazole. At survival levels corresponding to doses of 2 Gy in the presence of 1 mmol dm^?3 misonidazole and SERs ranged from 1·2 to 1·5.     

Biological Effects of Auger Processes of Bromine on Yeast Cells Induced by Monochromatic Synchrotron X-rays

Summary

The biological effects of inner-shell ionization in bromine atoms incorporated into DNA in the form of bromodeoxyuridine monophosphate (BrdUMP), induced by monochromatized synchrotron X-rays, were studied using a deoxythymidine monophosphate (dTMP)-permeable mutant of yeast, Saccharomyces cerevisiae. The BrdUMP-incorporated yeast cells were irradiated with monochromatic X-rays of 13·51 or 13·45 keV, between which the bromine K-absorption edge (13·47 keV) is located. The cells were 1·07 times more sensitive to irradiation by 13·51 keV X-rays than at 13·45 keV, while dTMP-incorporated cells did not show any difference in sensitivity. In the presence of a radioprotector during irradiation, BrdUMP-incorporated cells showed a larger enhancement (1·20). These enhancements observed in the bromine-incorporated cells cannot be explained only by an increase of the absorbed dose due to a substitution of CH₃ group of thymine by bromine. It may be concluded that a major part of the enhancement was caused by inner-shell photoionization, followed by an Auger cascade of the bromine in the DNA. The quantum yield of lethality caused by the photoabsorption of bromine K-shell is not affected by the presence of cysteamine, suggesting the biological enhancement by the Auger processes may not be influenced by chemical protection.     

Differences in Heat-induced Cell Killing as Determined in Three Mammalian Cell Lines Do Not Correspond with the Extent of Heat Radiosensitization

Summary

Three different cell lines, Ehrlich ascites tumour (EAT) cells, HeLa S₃ cells and LM mouse fibroblasts, were used to investigate whether or not the extent of heat killing (44°C) and heat radio-sensitization (44°C before 0–6 Gy X-irradiation) are related. Although HeLa cells were the most heat-resistant cell line and showed the least heat radiosensitization, we found that the most heat-sensitive EAT cells (D₀, EAT = 8·0 min; D₀, LM = 10·0 min; D₀ HeLa = 12·5 min) showed less radiosensitization than the more heat-resistant LM fibroblasts (TER_HeLa < TER_EAT < TER_LM). Therefore, it is concluded that the routes leading to heat-induced cell death are not identical to those determining heat radiosensitization. Furthermore the inactivation of DNA polymerase α and β activities by heat seemed not to correlate with heat survival alone but showed a positive relationship to heat radiosensitization. The possibility of these enzymes being a determinant in heat radiosensitization is discussed.     

Flow Cytometric Analysis of the Effects of 0·4 MeV Fission Neutrons on Mouse Spermatogenesis

Summary

(C57B1/Cne × C3H/Cne)F₁ male mice were irradiated with single acute doses of 0·4 MeV neutrons ranging from 0·05 to 2 Gy, and testis cell suspensions were prepared for cytometric analysis of the DNA content 2–70 days after irradiation. Various cell subpopulations could be identified in the control histogram including mature and immature spermatids, diploid spermatogonia and spermatocytes, tetraploid cells and cells in the S-phase. Variations in the relative proportions of different cell types were detected at each dose and time, reflecting lethal damage induced on specific spermatogenetic stages. The reduction of the number of elongated spermatids 28 days after irradiation was shown to be a particularly sensitive parameter for the cytometrical assessment of the radiosensitivity of differentiating gonia. A D₀ value of 0·13 Gy was calculated and compared with data obtained after X-irradiation, using the same experimental protocol. In the latter case a biphasic curve was obtained over the dose range from 0·25 to 10 Gy, possibly reflecting the existence of some cell population heterogeneity. RBE values were estimated at different neutron doses relative to the radiosensitive component of the X-ray curve, and ranged from 3·3 to 4, in agreement with data in the literature. Genotoxic effects were monitored 7 days after irradiation by a dose-dependent increase of the coefficient of variation (CV) values of the round spermatid peak, reflecting the induction of numerical and structural chromosome aberrations, and 14 or 21 days after irradiation by the detection of diploid elongated spermatids, probably arising from a radiation-induced complete failure of the first or second meiotic division.     

Effect of Ploidy on the Response of V79 Cells to Ionizing Radiation

Summary

The responses of diploid, tetraploid and near-hexaploid V79 cells to X-irradiation or DNA-associated ¹²⁵I-decay were compared. When cell killing, following X-irradiation, was plotted against the induced level of DNA double-strand breakage (dsb) per unit length of DNA, there was no significant difference between the relationships for each cell line. This suggested that the number of X-ray-induced DNA dsb per cell required to produce a lethal lesion was proportional to ploidy. Consistent with the X-ray results, tetraploid cells required 121 ± 4 and diploid cells 60 ± 1 ¹²⁵I-decays to produce a lethal lesion. However, the hexaploid cells deviated from this relationship and required 137 ± 5 decays. The relationship between relative elution and ¹²⁵I decays/cell reflected cellular DNA content. It is concluded that current models of radiation action are unable to explain these findings satisfactorily.     

Is sensitization with nicotinamide and carbogen dependent on nicotinamide concentration at the time of irradiation?

Purpose: To determine whether tumour radiosensitization and the therapeutic benefit of administering carbogen with nicotinamide depend upon irradiating at the time of peak drug concentration.

Materials and methods: Local tumour control of CaNT tumours in CBA mice and acute skin reactions in albino WHT mice were assessed after treatment with 10 X‐ray fractions in air, carbogen alone or combined with 0.1, 0.2 or 0.5?mg?g^?1 nicotinamide, injected 15, 30 or 60?min before irradiation. Plasma and tumour drug pharmacokinetics were performed.

Results: Nicotinamide was rapidly taken up into tumours; a six‐ and threefold higher concentration was obtained with 0.5?mg?g^?1 compared with 0.1 and 0.2?mg?g^?1, respectively. Tumour, but not skin, radiosensitization increased as the dose of nicotinamide increased (p=0.03), but at each dose level there was no significant difference in radiosensitivity when irradiations were done at or after the time of peak concentration. An almost eightfold increase in plasma levels increased tumour enhancement ratios from 1.74 to 1.92 (p<0.0001). In tumours all schedules gave significant enhancement relative to carbogen alone (p≤0.04).

Conclusions: Tumour and skin radiosensitivity was independent of time of nicotinamide administration. Higher drug concentrations were not mirrored by proportionally higher enhancement ratios. Lower plasma levels than previously suggested significantly enhanced tumour radiosensitivity relative to carbogen alone. The clinical implications of these findings are discussed.     

The Relationship between the Magnitude of the Effect and the Oxygen-concentration in Irradiation Experiments on Tradescantia Pollen-tube Chromosomes

Summary

The enhancement of killing by γ-irradiation, which is seen when E. coli K 1060 are cooled below the transition temperature of their membrane lipids, is blocked by procaine-HCl. These data are consistent with the hypothesis that increased killing associated with irradiation at 0°C is the result of membrane microviscosity increases, since procaine is known to fluidize membranes.

A cooling enhancement ratio (c.e.r.) is defined as the ratio of radiation D₀ at 22°C to its value at 0°C. The c.e.r. for oxygen-bubbled cells is 1·5 and for nitrogen-bubbled cells is 2·1. In the presence of 25 mM procaine the respective c.e.r. values are 1·08 and 1·29.

The oxygen enhancement ratio (o.e.r.) at 22°C is 3·43 and at 0°C is 2·45. The addition of procaine does not change the o.e.r. Thus, the temperature effect on o.e.r. does not appear to be related to membrane fluidity.     

Hypoxia-selective Radiosensitization of Mammalian Cells by Nitracrine,an Electron-affinic DNA Intercalator

Summary

The radiosensitizing ability of the 1-nitroacridine nitracrine (NC) is of interest since it is an example of a DNA intercalating agent with an electron-affinic nitro group. NC radiosensitization was evaluated in Chinese hamster ovary cell (AA8) cultures at 4°C in order to suppress the rapid metabolism and potent cytotoxicity of the drug. Under hypoxic conditions, submicromolar concentrations of NC resulted in sensitization (SER = 1·6 at 1 µmol dm^?3). Sensitization was also seen under aerobic conditions but a concentration more than 10-fold higher was required. In aerobic cultures NC radiosensitization was independent of whether cells were exposed before and during, or after, irradiation. Postirradiation sensitization was not observed under hypoxic conditions. The time dependence of NC uptake and the development of radiosensitization were similar (maximal at 30 min at 4°C under hypoxia) suggesting that sensitization, unlike cytotoxicity, is due to unmetabolized drug. NC is about 1700 times more potent as a radiosensitizer than misonidazole. This high potency is adequately accounted for by the electron affinity of NC (E(1) value at pH 7 of ?275mV versus NHE) and by its accumulation in cells to give intracellular concentrations approximately 30 times greater than in the medium. However, concentrations of free NC appear to be low in AA8 cells, presumably because of DNA binding. If radiosensitization by NC is due to bound rather than free drug, it suggests that intercalated NC can interact very efficiently with DNA target radicals. This is despite a binding ratio in the cell estimated as less than 1 NC molecule/400 base pairs under conditions providing efficient sensitization. This work suggests a new approach in the search for more effective clinical radiosensitizers, and poses questions on the means by which intercalated drugs can interact with DNA damage.     

The Influence of Thiols on the Pre-irradiation Incubation Effect of Nitroimidazoles in E. Coli Cells

Summary

The increase in the degree of radiosensitization of Escherichia coli cells following prolonged pre-irradiation incubation with nitroimidazoles is not correlated with the loss of intracellular non-protein thiols (NPSH) alone. The rates of reduction of the nitro compounds and the NPSH removal do not show strong dependencies on the lipophilicities of the nitroimidazoles whereas the highly lipophilic compound RGW-609 effects an increase in radiosensitization in a much shorter incubation time than the other nitroimidazoles. Exogenous dithiothreitol (DTT) increased the rate of reduction of misonidazole in the cells but did not alter the fraction converted to the amine. Added DTT (0·15 mmol dm^?3) completely protected against the pre-irradiation incubation effect of misonidazole (2·5 mmol dm^?3) when added at the start of the incubation but only partially protected when added before irradiation. It is suggested that NPSH can intercept metabolite(s) (or their precursors) of nitroimidazoles which can potentiate cell killing by radiation.     

低温等离子体联合辐射对三种癌细胞系放射增敏效应的研究

目的 探讨低温等离子体对人肝癌细胞系HepG2、非小细胞肺癌细胞系A549及人宫颈癌细胞系HeLa的放射增敏作用及其机制。方法 应用克隆形成实验观察低温等离子体对3种细胞的放射增敏作用;流式细胞仪分析3种细胞周期分布、凋亡率及活性氧含量;Western blot法检测单纯照射(R组)、单纯等离子体作用(P组)及其联合辐射(P+R组)对3种细胞中Bcl-2、Caspase-3蛋白表达的影响。结果 低温等离子体对HepG2、A549及HeLa细胞均有放射增敏作用,放射增敏比(SER_D0)分别为1.28、1.32、1.29。HepG2、A549及HeLa细胞P+R组G₂/M期比例、凋亡率及活性氧含量与R组比较均明显增高(t_G2/M期=9.52、8.24、9.53,P<0.05;t_凋亡率=10.67、38.56、6.74,P<0.05;t_{活性氧含量}=9.41、15.42、13.53,P<0.05)。HepG2和A549细胞P+R组G₂/M期比例、凋亡率及活性氧含量与P组比较均明显升高(t_G2/M期=8.75、20.37,P<0.05;t_凋亡率=8.43、9.99,P<0.05;t_{活性氧含量}=4.82、5.27,P<0.05)。3种癌细胞中P+R组Bcl-2蛋白表达较R组降低,而Caspase-3蛋白表达升高。结论 低温等离子体可提高HepG2、A549及HeLa细胞系的放射敏感性,其对HepG2及A549细胞系的放射增敏机制可能与抑制亚致死损伤修复,使细胞周期阻滞在G₂/M期,以及提高细胞内ROS水平诱导细胞凋亡有关。     

Influence of the 100% w/v Perfluorooctyl Bromide (PFOB) Emulsion Dose on Tumour Radiosensitivity

Summary

The radiosensitizing effect of a 100% w/v emulsion of a fluorocarbon, PFOB, which carries 4 times more oxygen than does Fluosol-DA 20% emulsion, was studied on two human tumour xenografts (HRT18 and HT29) and the murine tumour EMT6. This effect was compared with that obtained with carbogen alone. The fluorocrit (amount of fluorocarbon in the blood) and haematocrit remained unchanged from 7 to 65 min post-injection of the emulsion (8 ml/kg). Tumour-bearing mice were pretreated with 100% w/v PFOB emulsion doses ranging from 2 to 15 ml/kg in the presence of carbogen for 30 min prior to and during irradiation. The fluorocrit increased from 1·5% to 9·5% as the dose of 100% w/v PFOB emulsion increased from 2 to 15 ml/kg. The haematocrit remained the same for all the fluorocarbon emulsion doses used. Tumour radiosensitization varied with the fluorocarbon emulsion dose. Clinically relevant doses (2–4 ml/kg) of the 100% w/v PFOB emulsion plus carbogen produced significantly more radiosensitization than carbogen alone, with sensitizing enhancement ratios of 1·4 for EMT6 and 1·7 for HRT18. The radiosensitivity of HRT18 cells was thus very close to that obtained with normally oxygenated cells. For higher doses (8–15 ml/kg) the radiosensitizing effect of 100% w/v PFOB emulsion plus carbogen becomes comparable to that of carbogen alone. These experiments show that clinically useful doses of 100% w/v PFOB plus carbogen produced tumour radiosensitization only at relatively low fluorocrits. Thus the fluorocrit, and hence the fluorocarbon's oxygen-carrying capacity, is not the only factor involved in radiosensitizing tumour cells by oxygen-carrying fluorocarbon emulsions.     

Enhancement of Radiosensitivity of Cultured Mammalian Cells by Neocarzinostatin. I. Inhibition of the Repair of Sublethal Damage

Summary

The enhancement of radiosensitivity by neocarzinostatin (NCS), an antitumour drug, was studied using three strains of cultured mammalian cells with different repair capabilities for sublethal damage. NCS enhanced the radiosensitivity of the cells when applied both during and after X-irradiation under aerobic conditions. The enhancement ratios of NCS during X-irradiation were 1·25, 1·27 and 1·38 for mouse lymphoma L5178Y, Chinese hamster V79 and mouse mammary tumour FM 3A cells, respectively. The corresponding ratios after X-irradiation were 1·18, 1·27 and 1·38, respectively. These ratios were proportional to the repair capabilities of the cells for sublethal damage. NCS completely inhibited the repair of sublethal damage regardless of the repair capabilities of the cells for sublethal damage. NCS was equally effective for hypoxic cells. These results suggested that NCS enhanced the radiosensitivity of the cells probably by interacting with the residual damage after X-irradiation, thereby converting the sublethal damage or potentially lethal damage into lethal damage.     

Effects of Fractionated X-irradiation on Subsequent Response to Acute X-irradiation in Two Human Tumour Cell Lines in Vitro

Summary

The exposure of two human tumour cell lines, one derived from a squamous cell carcinoma of the tongue (HN-1) and the other from an adenocarcinoma of the breast (MCF-7), to fractionated X-irradiation in vitro, resulted in altered sensitivity to subsequent acute X-irradiation exposure in the former but not the latter tumour cell type. The X-ray-pretreated HN-1 cells, designated HN-1/DXR₁₁ cells, showed a significantly increased sensitivity to X-irradiation with a D₀ of 0·97 Gy, compared with a figure of 1·39 Gy for the parental cells. No significant changes were noted in a number of basic cell kinetic or biological parameters in the X-ray-pretreated cells. However, this enhanced X-ray sensitivity in the HN-1/DXR₁₁ cells was associated with decreased cellular levels of total intracellular glutathione. These findings are consistent with the theory that intracellular thiols are involved in protection from radiation damage. This is one of the first observations that prior exposure to X-irradiation can modify subsequent responses to acute X-irradiation treatment in human tumour cells.     

Differential Radiosensitization of Human Tumour Cells by 3-aminobenzamide and Benzamide: Inhibitors of Poly(ADP-ribosylation)

Summary

The effect of 3-aminobenzamide (3AB) and benzamide (BZ) (inhibitors of poly(ADP-ribose) synthetase) on radiosensitivity was investigated in normal human fibroblasts and three human cell lines established from tumours with varying degrees of clinical radiocurability. The human tumour cell lines selected were: (1) Ewing's sarcoma, a bone tumour usually considered radiocurable with moderate radiation doses; (2) lung adenocarcinoma, a tumour considered radiocurable with high doses of radiotherapy; and (3) osteosarcoma, a very resistant tumour which is rarely controlled by standard doses of radiotherapy. Poly(ADP-ribose) synthetase inhibitors were added to cultures 2h prior to irradiation and removed 24h after. Inhibitors were used at doses producing little or no toxicity in cells.

In the presence of these inhibitors, a differential radiosensitization was observed. Ewing's sarcoma cells and normal human fibroblasts were sensitized to an equal extent by either 8 mm 3AB or 4 mm BZ. However, no sensitization was observed at these concentrations in the lung adenocarcinoma cells or osteosarcoma cells. The degree of radiosensitization in vitro by 3AB and BZ correlates well with the clinical radiocurability of these tumours in vivo.     

Radiation Chemical and Physical Mechanisms of Radiosensitization of Single Cell Systems by Iothalamate

Summary

The radiosensitizing effect of iothalamate (ITA) has been investigated in bacterial and mammalian cells in order to obtain a better understanding of the physical and radiation chemical mechanisms of sensitization displayed by the drug. In order to distinguish between the two, Escherichia coli B/r cells were irradiated with 9 MeV electrons, which allow only the radiation chemical mechanism to operate, and V79 cells with 250 KVp X-rays, which instead make possible the occurrence of both mechanisms.

It has been shown that: (a) Maximum sensitization already occurs in bacteria with 10^?2 mol dm^?3 ITA (enhancement ratio (ER) 11·2 in oxygen, 2·7 in nitrogen), while in mammalian cells a concentration higher by a factor of 10 is required (ER 2·2 both in air and nitrogen). (b) ITA sensitization is inhibited when bacteria are irradiated in growth medium instead of buffer. Such inhibition does not occur with V79 cells. (c) Cysteine and glycerol completely cancel the sensitizing effect of ITA on bacterial cells in both gas phases. Dimethylsulphoxide (DMSO) does the same in nitrogen, while in oxygen it only reduces ITA sensitization to about 50 per cent of the level observed in control conditions. With mammalian cells, all the three scavengers do not modify significantly the enhancement produced by ITA, either in air or in nitrogen. The experimental results are consistent with both postulated mechanisms of sensitization.