Enhancement of DNA damage in mammalian cells upon bioreduction of the nitroimidazole-aziridines RSU-1069 and RSU-1131.

The induction of DNA double-(dsb) and single-(ssb) strand breaks by RSU-1069, RSU-1131 and misonidazole in V79 mammalian cells has been investigated using sedimentation in isokinetic sucrose gradients after incubation for various times (1-3 hr) at 310 K under both hypoxic and aerobic conditions. Double strand breaks are produced by RSU-1069 and RSU-1131 predominantly under hypoxic conditions. Comparison of the cellular DNA damage induced by these agents leads to the following facts: (1) the yield of ssb induced by these agents is substantially increased under hypoxia, (2) RSU-1069 and RSU-1131 are much more effective than misonidazole, on a concentration basis, at causing strand breakage both under hypoxic and aerobic conditions; and (3) RSU-1069 is more efficient on a concentration basis than RSU-1131 at inducing both ssb and dsb under both conditions. From these findings and molecular studies it is suggested that these 2-nitroimidazole aziridines act as monofunctional alkylating agents under aerobic conditions, a factor that governs their aerobic cytotoxicity. Under hypoxic conditions, it is suggested that the induction of dsb and crosslinks by these agents (bifunctional character) may play a major role in determining the ability of such agents to act as hypoxia-selective cytotoxins.

Induction of mutations in V79-4 mammalian cells under hypoxic and aerobic conditions by the cytotoxic 2-nitroimidazole-aziridines, RSU-1069 and RSU-1131. The influence of cellular glutathione.

Incubation of the 2-nitroimidazole-aziridine, RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridinyl)-2-propanol], and its monomethylaziridine analogue, RSU-1131 [1-(2-nitro-1-imidazolyl)-3-(1-(2-methylaziridinyl))-2-propanol], with V79-4 mammalian cells for 2 hr under aerobic or hypoxic conditions induces mutations as measured at the hypoxanthine phosphoribosyl transferase locus. The ability of these agents to induce mutations is increased by a factor of 12-14 under hypoxic conditions. The increased cytotoxicity of these agents under hypoxic conditions was confirmed following a 2 hr incubation period. Decreasing the glutathione (GSH) content of the cells with buthionine-(S,R)-sulphoximine to < 1% of the control generally results in an increase in the cytotoxicity and mutagenicity of these agents under both aerobic and hypoxic conditions. Since these agents do not modify the cellular GSH levels, it is inferred that the thiols partially detoxify through removal of a reactive metabolite of the agents, under hypoxic conditions, or removal of known DNA adducts, and not through their interaction with the agents themselves. Under aerobic conditions, the formation of mutations is consistent with the established monofunctional action of these agents whereas under hypoxic conditions the bifunctional action predominates for mutation induction, based upon the large differential aerobic:hypoxic effect. From a comparison of the number of mutations per lethal event, the effect of thiol depletion is more pronounced for cytotoxicity than for mutation induction by these agents. In summary, these agents are considered to be weak mutagens towards V79-4 cells under aerobic conditions when compared with other DNA alkylating agents, although they are more potent under anoxic conditions.

A comparison of the techniques of alkaline filter elution and alkaline sucrose sedimentation used to assess DNA damage induced by 2-nitroimidazoles.

The induction of DNA single-strand breaks (DNA-SSB) in Chinese hamster V79-379A lung fibroblasts by misonidazole or RSU-1069 under both aerobic and hypoxic conditions was examined following incubations for up to 4 hr at 310 degrees K using the technique of alkaline filter elution. Incubation with RSU-1069 induces DNA-SSB under both hypoxic and aerobic conditions, whereas incubation with misonidazole induces DNA-SSB only under hypoxia. The yield of breaks is dependent on both agent concentration and contact time. Following identical treatments with these agents, the yield of DNA-SSB (expressed in radiation dose equivalents) determined by alkaline filter elution is about one order of magnitude less than that previously determined by alkaline sucrose gradient sedimentation. In contrast to radiation induced DNA-SSB, alkaline elution is less sensitive than alkaline sucrose gradient sedimentation when determining DNA-SSB induced by RSU-1069 and misonidazole. During the filter elution assay, either increasing cell lysis from 2 to 4 hr, the pH of the lysing buffer from pH 8.7 to 12.5 or the elution buffer from pH 12.2 to 12.5 does not significantly effect the yield of DNA-SSB. Increasing the pH of the lysing or elution buffers to greater than pH 13 however results in considerable degradation of the DNA, whereby 50-85% of the total DNA passes through the filter with the lysing solution. This effect was similar for DNA from both control and chemically insulted cells. In conclusion, it is apparent that incubation with these agents results in the induction of DNA damage which is expressed as a DNA-SSB only after prolonged treatment under alkaline conditions. Further, the use of alkaline elution to study DNA-SSB damage induced chemically must be treated with caution in the light of these findings.

The repair of DNA damage induced in V79 mammalian cells by the nitroimidazole-aziridine, RSU-1069. Implications for radiosensitization.

The induction and repair of single (ssb) and double (dsb) strand breaks in DNA under aerobic or hypoxic conditions have been determined using sucrose sedimentation techniques following incubation of V79 mammalian cells with RSU-1069 or misonidazole, representative of a conventional 2-nitroimidazole radiosensitizer, for 1-1.5 hr at either 293 or 277 degrees K and subsequent irradiation at 277 degrees K. In all cases, the dose dependences for the induction of strand breaks are linear and consistent with an enhancement in the yield of DNA damage induced by the 2-nitroimidazoles under hypoxic conditions. With RSU-1069 at 293 degrees K, the dose dependence of ssb is displaced reflecting DNA damage induced during pre-incubation. From these dependences, it is evident that the enhanced radiosensitization by RSU-1069 may not be accounted for in terms of accumulation of the agent at DNA. From the repair studies, DNA breaks induced by RSU-1069 in the absence of radiation have been shown to persist for at least 3 hr. With a combination of RSU-1069 and radiation under hypoxic conditions, the repair timescale of the induced breaks is significantly longer and an increase in the residual yields of both ssb and dsb (at 2-3 hr) was observed when compared with the observation in the presence of misonidazole or oxygen. From these studies, it is inferred that the enhanced radiosensitization of RSU-1069 at 293 degrees K is a consequence of the formation of non-repairable DNA damage together with a modification of the repairability of the radiation-induced DNA breaks.

Contribution of base lesions to radiation-induced clustered DNA damage: implication for models of radiation response.

Biophysical modeling of radiation-induced DNA damage shows that significant yields of clustered DNA damage are formed after energy deposition by a single radiation track. To date, the majority of studies on radiation-induced DNA damage in cells have concentrated on determination of the yields of single- and double-strand breaks (DSBs), the latter representing one type of clustered DNA damage. It was recognized, however, that clustered DNA damage, which does not contain a DSB, might contain a combination of DNA base lesions and single-strand breaks in proximity. This mini-review discusses some of the recent experimental data confirming the induction of non-DSB, clustered DNA damage by radiation.

Induction and rejoining of DNA double-strand breaks in Chinese hamster V79-4 cells irradiated with characteristic aluminum K and copper L ultrasoft X rays.

Characteristic aluminum K (AlK) (energy of 1.5 keV) and copper L (CuL) (energy of approximately 0.96 keV) ultrasoft X rays have been used to investigate the effectiveness of the numerous low-energy secondary electrons produced by low-linear energy transfer (LET) ionizing radiation. Cellular inactivation and induction and rejoining of DNA double-strand breaks (DSBs) in Chinese hamster V79-4 cells irradiated as monolayers with these ultrasoft X radiations have been studied under aerobic and anaerobic conditions. The mean cell thickness, determined by confocal laser scanning fluorescence microscopy, was used to calculate the mean dose to the nucleus of the irradiated cells. Relative to 60Co gamma rays, the relative biological effectiveness (RBE) for cellular inactivation at 10% survival is 1.7 +/- 0.1 and 2.3 +/- 0.3 for AIK and CuL ultrasoft X rays, respectively. The RBE values for induction of DSBs of 2.5 +/- 0.2 and 3.0 +/- 0.3 for AlK and CuL X rays, respectively, were determined after irradiation at 277 K using the technique of pulsed-field gel electrophoresis. Induction of DSBs is linearly dependent on dose. Oxygen enhancement ratios of 1.9 and 2.1 for cellular inactivation and DSB induction, respectively, were obtained with AIK X rays. These values are less than those for 60Co gamma radiation. The repair kinetics for rejoining of DSBs after a dose of 15 Gy is similar for both X-ray energies and 60Co gamma rays with a first half-life of 18-22 +/- 5 min. From these studies, it is suggested that induction of DSBs by low-LET radiations such as 60Co gamma rays reflects clustered damage produced predominantly by low-energy electron "track ends," which represent about 30% of the total dose.

Induction of DNA-protein crosslinks in Chinese hamster V79-4 cells exposed to high- and low-linear energy transfer radiation.

The induction of DNA-protein crosslinks (DPCs) in Chinese hamster V79-4 cells after irradiation under hypoxic and aerobic conditions at 277 K with 60Co gamma rays, 238Pu alpha particles and aluminum K (Al(K)) ultrasoft X rays has been determined using a nitrocellulose filter binding assay. The dose dependences for the induction of DPCs, which involves covalent linkage, are linear over the absorbed dose range used (0-400 Gy with alpha-particle and gamma radiation, 0-600 Gy with Al(K) X rays). The yield of DPCs induced under hypoxic conditions is 55, 51 and 25 DPCs per gray per cell for 60Co gamma rays, alpha particles and Al(K) X rays, respectively. The yield of DPCs is significantly reduced in the presence of oxygen by 20, 50 and 79% for 60Co gamma rays, alpha particles and Al(K) X rays, respectively. Since the mean size of the DNA attached to the protein is uniform for 60Co gamma rays and alpha particles, variations in the DNA size do not influence the yields of DPCs. Although a DPC may be considered as a complex lesion combining two macromolecules, the dependence of the yield of DPCs on LET does not reflect the ionizing density of the radiations used. Further, this dependence on LET and the effect of oxygen do not reflect the corresponding dependences determined for a variety of biological responses. From these findings and knowledge of the radiation tracks, it is proposed that DPCs induced particularly under aerobic conditions with 60Co gamma rays are formed mainly in the sparsely ionizing segments of the radiation track.

The effect of dimethyl sulfoxide on the induction of DNA double-strand breaks in V79-4 mammalian cells by alpha particles.

The present study was undertaken to assess the protective effect of dimethyl sulfoxide (DMSO) against the induction and rejoining of DNA double-strand breaks (DSBs) and inactivation of V79-4 Chinese hamster cells by both high- and low-linear energy transfer (LET) radiations. The cells were exposed under aerobic conditions as monolayers to either low-LET photons (60Co gamma rays) or high-LET alpha particles (238Pu) at 277 K. The initial yield of DSBs, determined by elution under nondenaturing conditions, is linearly dependent on dose. When the irradiation was carried out in the presence of DMSO (0-0.6 mol dm-3), the initial yields of DSBs induced by both gamma and alpha-particle irradiation decrease. With gamma irradiation at [DMSO] > 0.6 mol dm-3, a further decrease in the yield of DSBs occurs. DMSO (0.5 mol dm-3) reduces the initial yield of DSBs by 50 +/- 5% and 32 +/- 4% for photons and alpha particles, respectively. DMSO protects more effectively against cellular inactivation and DSB induction at low LET compared with alpha-particle irradiation with protection factors of 1.7 and 1.4, respectively, for survival and 2.0 and 1.5, respectively, for DSBs. After incubation of the irradiated cells for 3 h at 310 K after high-LET irradiation, the residual yield of DSBs is reduced by < 13% when the irradiations were carried out in the presence of 0.5 mol dm-3 DMSO. With gamma irradiation in the presence of 0.5 mol dm-3 DMSO, 90% of the DSBs are rejoined by 3 h incubation at 310 K. Therefore, the nonscavengeable DSBs induced by alpha particles are not significantly rejoined within 3 h, in contrast to rejoining of the majority of the nonscavengeable DSBs induced by gamma irradiation. From comparison of the data on DSBs and survival for alpha-particle irradiation, it is inferred that the severity of damage is reduced by DMSO through minimizing the formation of OH-induced sugar/base modifications in the vicinity of nonscavengeable DSBs.

Dependence of the yield of DNA double-strand breaks in Chinese hamster V79-4 cells on the photon energy of ultrasoft X rays.

Induction of DNA DSBs by low-LET radiations reflects clustered damage produced predominantly by low-energy, secondary electron "track ends". Cell inactivation and induction of DSBs and their rejoining, assayed using pulsed-field gel electrophoresis, were determined in Chinese hamster V79-4 cells irradiated as a monolayer with characteristic carbon K-shell (CK) (0.28 keV), aluminum K-shell (AlK) (1.49 keV), and titanium K-shell (TiK) (4.55 keV) ultrasoft X rays under aerobic and anaerobic conditions. Relative to (60)Co gamma rays, the relative biological effectiveness (RBE) for cell inactivation at 10% survival and for induction of DSBs increases as the photon energy of the ultrasoft X rays decreases. The RBE values for cell inactivation and for induction of DSBs by CK ultrasoft X rays are 2.8 +/- 0.3 and 2.7 +/- 0.3, respectively, and by TiK ultrasoft X rays are 1.5 +/- 0.1 and 1.4 +/- 0.1, respectively. Oxygen enhancement ratios (OERs) of approximately 2 for cell inactivation and induction of DSBs by ultrasoft X rays are independent of the photon energy. The time scale for rejoining of DNA DSBs is similar for both ultrasoft X rays and 60Co gamma rays. From the size distribution of small DNA fragments down to 0.48 kbp, we concluded that DSBs are induced randomly by CK and AlK ultrasoft X rays. Therefore, ultrasoft X rays are more efficient per unit dose than gamma radiation at inducing DNA DSBs, the yield of which increases with decreasing photon energy.

Induction and rejoining of DNA double-strand breaks in V79-4 mammalian cells following gamma- and alpha-irradiation.

The induction and rejoining of DNA double-strand breaks (dsbs) in V79-4 mammalian cells following irradiation by 60Co gamma-rays and 238Pu alpha-particles (average LET 120 keV microns-1) under aerobic conditions have been determined using both the sucrose sedimentation and filter elution techniques under non-denaturing conditions. Cellular inactivation was also determined. The dependence of the initial yield of dsbs at 277 K on dose under aerobic conditions is linear with a relative biological effectiveness (RBE) for alpha-particles of 0.85 +/- 0.14 (sedimentation) and 0.68 +/- 0.12 (elution) compared with 60Co gamma-rays. The ability of the cells to rejoin dsbs at 310K is significantly reduced for alpha-irradiations with only 30-50% rejoined for a 3-h incubation period. With low LET radiation, > 90% of the dsbs are rejoined within 3 h at a dose of 20 Gy. The RBE for cellular inactivation was determined to be 4.0 at the 1% survival level. From the cellular dimensions and the D0-value for cellular inactivation by alpha-particles, it is determined that, on average, 4.7 tracks traverse the cell nucleus per lethal lesion. Under hypoxic conditions, the RBE values for induction of dsbs and cellular inactivation (10% level) by alpha-particles are approximately 3.0 and approximately 11.8 respectively. From these findings, it is suggested that the residual DNA damage and not the initial damage is reflected in the cellular inactivation. It is inferred that the difference in repair of the various lesions is a reflection of the differences in the complexity of the clustered damage produced by these radiations.

Direct comparison of biological effectiveness of protons and alpha-particles of the same LET. III. Initial yield of DNA double-strand breaks in V79 cells.

The results reported form part of a series of experiments to substantiate and extend the findings by Belli et al. (1989) that protons are more biologically effective at cell killing than alpha-particles of the same LET. The irradiations were carried out using the Variable Energy Cyclotron (VEC) at the Harwell Laboratories. V79-4 Chinese hamster cells were exposed to alpha-particles and protons with LETs of 20 and 23 keV microns-1 in the dose range 40-150 Gy. X-rays were also used for comparison. Two methods were used for measurement of initial DNA double-strand breaks: sedimentation and DNA precipitation assays. The dose-response relationships were found to be well fitted by straight lines in all cases. With the sedimentation assay a slightly lower yield of dsb was found from protons than from alpha-particles of the same LET. The yield from X-rays was not significantly different from either. The precipitation assay showed similar yields of DNA damage from both particle types but significantly higher yields from X-rays. This may reflect a difference in the type of lesions scored by the two methods. Since the initial amount of dsb does not account for the observed differences in cellular response to radiations of different qualities, it is likely that these are related to the nature of the dsb (affecting reparability) or to the occurrence of other types of molecular damage.

Alpha and fission fragment autoradiography with superimposed tissue images in CR-39 plastic.

A technique is described by which the distributions of both fissionable and alpha-emitting radionuclides in histological sections of lung are visualised in the nuclear track recording plastic CR-39. Fission fragments and alpha-particles from radionuclides contained within the section register as tracks in CR-39 and an image of the tissue structure is induced, superimposed upon the track distribution, by means of low-energy (less than 1 MeV) alpha-particles from an external 238Pu source. Thus accurate apposition of the track distribution and tissue structure is achieved. The efficiencies of this system for alpha-particle and fission fragment detection are 0.51 +/- 0.09 (SD) and 0.48 +/- 0.08 (SD) respectively.

Further experiments to study whether localised fission fragment irradiation of rat lung causes tumours.

Male albino rats inhaled an aerosol of 235UO2 (mass median aerodynamic diameter = 2.8 micrometers and geometric standard deviation = 1.6). Approximately 20 h or 7 d post-inhalation the rats were exposed briefly to 10(12) slow neutrons cm-2 in a nuclear reactor, causing the retained 235UO2 particles of approximate mass 40 or 400 micrograms to emit fission fragments which irradiated the lungs. The mean absorbed doses from the fission fragments were either 80 ot 800 cGy approximately and in addition the lungs were exposed to a background of alpha-rays throughout the rats' life-time from the retained 235UO2 which gave mean doses of about half that from the fission fragments. The animals were kept for their life-time and killed when they became moribund. Malignant tumours were found in the lungs (squamous cell carcinoma and adenocarcinoma) which were probably induced by the alpha-rays rather than the fission fragments. Because of insufficient numbers of animals in the experimental groups, however, some statistical uncertainty exists as to whether the fission fragments were in fact less effective than the alpha-rays per unit absorbed dose in causing malignant tumours of the lung.

Influence of macrophages on microdistribution of inhaled UO2 aerosol in rat lung.

Following the inhalation of an aerosol of UO2 (mass median aerodynamic diameter = 3 microns and geometric standard deviation = 1.6) the lungs of male albino rats were populated by foci containing UO2 particles. A method of neutron-induced autoradiography on Lexan plastic was used to reveal these foci in thin sections cut from the lung. For masses of UO2 in the lung that differed by more than a factor of 10 (39 to 450 micrograms) the number of foci per g of lung increased, but not in proportion to the mass of UO2 deposited. This limited increase in the number of foci was considered to result from physiological limitations on the number of alveolar macrophages available for engulfing the UO2 particles.

Do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla cells?

Androgens regulate many aspects of human hair growth in both sexes. After puberty they transform tiny vellus follicles in many areas, e.g. the face, to terminal ones producing long, thick, pigmented hairs. In genetically predisposed individuals, androgens also cause the reverse transformation of terminal scalp follicles into vellus ones, causing balding. In the current hypothesis for androgen action, androgens control most follicular cells indirectly acting via the mesenchyme-derived dermal papilla which regulates many aspects of follicular activity. In this model androgens binding to androgen receptors in dermal papilla cells alter their production of regulatory molecules which influence other follicular components; these molecules may be soluble paracrine factors and/or extracellular matrix proteins. This hypothesis is supported by immunohistochemical localisation of androgen receptors in dermal papilla cell nuclei and the demonstrations that androgen receptor content and testosterone metabolism patterns of cultured dermal papilla cells from various body sites reflect hair growth in androgen-insensitivity syndromes. The next question is whether androgens alter the paracrine factors secreted by dermal papilla cells. Cultured dermal papilla cells do release soluble, proteinaceous factors into their media which stimulate the growth of keratinocytes and other dermal papilla cells. This mitogenic potential can cross species from humans to rodents. Importantly, testosterone in vitro stimulates the mitogenic potential of beard cells, but in contrast inhibits production by balding scalp cells reflecting their in vivo androgenic responses. Since androgens in vitro do alter the secretion of paracrine factors the current focus lies in identifying specific factors produced, e.g. IGF-I and stem cell factor (SCF), using ELISA and RT-PCR, and comparing their expression in cells from follicles with varying responses to androgens in vivo or under androgen stimulation in vitro. This should lead to greater understanding of androgen action and enable the development of better treatment for androgen-potentiated disorders.

Variation of the radiosensitizing efficiency of RSU-1069 with pre-irradiation contact times: a rapid mix study.

Using a cellular fast-mixing technique, the time course of radiation sensitization of hypoxic, V79 cells by various concentrations of RSU-1069 (0.25-2.5 mmol dm-3) and misonidazole (2.5-50 mmol dm-3) have been studied to distinguish between fast chemical processes and the much slower biochemical responses to ionizing radiation and the monofunctional alkylating action of RSU-1069. Under conditions of equi-concentration, misonidazole and RSU-1069 show similar radiosensitizing efficiencies for pre-irradiation contact times up to 1 s. The values of the sensitizer enhancement ratio of approximately 1.5 for both 2-nitroimidazoles (2.5 mmol dm-3) is considerably less than that of 1.9-2.8 determined with misonidazole for a pre-irradiation contact time of 1 h under hypoxia. It is proposed that the enhanced radiosensitizing efficiency of RSU-1069 compared to that of misonidazole after long contact times involves, in part, the formation of 'sub-toxic' damage probably involving monofunctional and/or bifunctional action of RSU-1069 prior to irradiation.

The carcinogenic effect of localized fission fragment irradiation of rat lung.

In a preliminary investigation of 'hot particle' carcinogenesis uranium oxide particles were introduced into the lungs of rats either by intubation of a liquid suspension of the particles or by inhalation of an aerosol. Subsequently the animals were briefly exposed to slow neutrons in a nuclear reactor, resulting in localized irradiation of the lung by fission fragments emitted from 235U atoms in the oxide particles. The uranium used in the intubation experiments was either enriched or depleted in 235U. Squamous cell carcinomas developed at the site of deposition of the enriched uranium oxide in many cases but no lung tumours occurred in the rats with the depleted uranium oxide, in which the lung tissue was exposed to very few fission fragments. Only enriched uranium oxide was used in the inhalation experiments. Pulmonary squamous cell carcinomas occurred after the fission fragment irradiation but were fewer than in the intubation experiments. Adenocarcinomas of the lung were seen in rats exposed to uranium oxide without subsequent irradiation by neutrons in the reactor and in rats irradiated with neutrons but not previously exposed to uranium oxide. It is concluded that (i) fission fragments were possibly implicated in the genesis of the squamous cell carcinomas, which only developed in those animals exposed to enriched uranium oxide and neutrons and (ii) the adenocarcinomas in the rats inhaling enriched uranium oxide only were likely to have been caused by protracted irradiation of the lung with alpha-rays emitted from the enriched uranium.

The hair follicle: a paradoxical androgen target organ.

Androgens are the main regulator of normal human hair growth. After puberty, they promote transformation of vellus follicles, producing tiny, unpigmented hairs, to terminal ones, forming larger pigmented hairs, in many areas, e.g. the axilla. However, they have no apparent effect on the eyelashes, but can cause the opposite transformation on the scalp leading to the replacement of terminal hairs by vellus ones and the gradual onset of androgenetic alopecia. This paradox appears to be an unique hormonal effect. Hair follicles are mainly epithelial tissues, continuous with the epidermis, which project into the dermis. A mesenchyme-derived dermal papilla enclosed within the hair bulb at the base controls many aspects of follicle function. In the current hypothesis for androgen regulation, the dermal papilla is also considered the main site of androgen action with androgens from the blood binding to receptors in dermal papilla cells of androgen-sensitive follicles and causing an alteration of their production of paracrine factors for target cells e.g. keratinocytes. Studies of cultured dermal papilla cells from sites with different responses to androgens in vivo have confirmed the paradoxical responses. All dermal papilla cells from androgen-sensitive sites contain low capacity, high affinity androgen receptors. However, only some cells formed 5alpha-dihydrotestosterone, e.g. beard but not axillary cells, in line with hair growth in 5alpha-reductase deficiency. Incubation with androgens also stimulated the mitogenic capacity of beard cell media, but inhibited that produced by scalp cells. This suggests that the paradoxical differences are due to differential gene expression within hair follicles, presumably caused during embryogenesis.