Dose responses for adaption to low doses of (60)Co gamma rays and (3)H beta particles in normal human fibroblasts

The dose response for adaption to radiation at low doses was compared in normal human fibroblasts (AG1522) exposed to either (60)Co gamma rays or (3)H beta particles. Cells were grown in culture to confluence and exposed at either 37 degrees C or 0 degrees C to (3)H beta-particle or (60)Co gamma-ray adapting doses ranging from 0.1 mGy to 500 mGy. These cells, and unexposed control cells, were allowed to adapt during a fixed 3-h, 37 degrees C incubation prior to a 4-Gy challenge dose of (60)Co gamma rays. Adaption was assessed by measuring micronucleus frequency in cytokinesis-blocked, binucleate cells. No adaption was detected in cells exposed to (60)Co gamma radiation at 37 degrees C after a dose of 0.1 mGy given at a low dose rate or to 500 mGy given at a high dose rate. However, low-dose-rate exposure (1-3 mGy/min) to any dose between 1 and 500 mGy from either radiation, delivered at either temperature, caused cells to adapt and reduced the micronucleus frequency that resulted from the subsequent 4-Gy exposure. Within this dose range, the magnitude of the reduction was the same, regardless of the dose or radiation type. These results demonstrate that doses as low as (on average) about one track per cell (1 mGy) produce the same maximum adaptive response as do doses that deposit many tracks per cell, and that the two radiations were not different in this regard. Exposure at a temperature where metabolic processes, including DNA repair, were inactive (0 degrees C) did not alter the result, indicating that the adaptive response is not sensitive to changes in the accumulation of DNA damage within this range. The results also show that the RBE for low doses of tritium beta-particle radiation is 1, using adaption as the end point.     

Adaptive response for chromosomal inversions in pKZ1 mouse prostate induced by low doses of X radiation delivered after a high dose

Adaptive responses are induced by stress such as X radiation and result in a lower than expected biological response. Two-dose adaptive response experiments typically involve a low priming dose followed by a subsequent high radiation dose. Here, we used a sensitive in vivo chromosomal inversion assay to demonstrate for the first time an adaptive response when a low dose (0.01-1 mGy) was given several hours after a high 1000-mGy radiation dose. The adaptive responses in this study were of similar magnitude to the two-dose adaptive responses previously observed in this test system when the low dose was given first. A chromosomal inversion adaptive response was also induced by two 1000-mGy doses and when a 1-mGy dose was preceded or followed by a dose of 0.01 mGy, but not by two 4000-mGy doses. This is also the first example of an adaptive response when both doses are low. Our data agree with previous reports of an on-off mechanism of adaptive response. The induction of an adaptive response by a low dose after a high damaging dose provides evidence that the mechanisms underlying radiation adaptive responses are not due to prevention of damage induced by the high dose but to modulation of the cellular response to this damage.     

Radiation-induced bystander effects and adaptive responses--the Yin and Yang of low dose radiobiology?

Our current knowledge of the mechanisms underlying the induction of bystander effects by low doses of high or low LET ionizing radiation is reviewed. The question of what actually constitutes a protective effect is discussed in the context of adaptive (often referred to as hormetic or protective) responses. Finally the review considers critically, how bystander effects may be related to observed adaptive responses or other seemingly protective effects of low doses exposures. Bystander effects induce responses at the tissue level, which are similar to generalized stress responses. Most of the work involving low LET radiation exposure discussed in the existing literature measures a death response. Since many cell populations carry damaged cells without being exposed to radiation (so-called "background damage"), it is possible that low doses exposures cause removal of cells carrying potentially problematic lesions, prior to exposure to radiation. This mechanism could lead to the production of "U-shaped" or hormetic dose-response curves. The level of adverse, adaptive or apparently beneficial response will be related to the background damage carried by the original cell population, the level of organization at which damage or harm are scored and the precise definition of "harm". This model may be important when attempting to predict the consequences of mixed exposures involving low doses of radiation and other environmental stressors.     

Effect of low-dose radiation on repair of DNA and chromosome damage

In this report results of studies on the effect of different doses of low LET (linear energy transfer) radiations on the unscheduled DNA synthesis (UDS) and DNA polymerase activity as well as the induction of adaptive response in bone marrow cells (BMC) by low dose radiation were presented. It was found that whole-body irradiation (WBI) with X-ray doses above 0.5 Gy caused a dose-dependent depression of both UD5 and DNA polymerase activity, while low dose radiation below 250 mGy could stimulate the DNA repair synthesis and the enzyme activity. WBI of mice with low doses of X-rays in the range of 2-100 mGy at a dose rate of 57.3 mGy per minute induced an adaptive response in the BMC expressed as a reduction of chromosome aberrations following a second exposure to a larger dose (0.65 mGy). It was demonstrated that the magnitude of the adaptive response seemed to be inversely related to the induction dose. The possibility of induction of adaptive response in GO phase of the cell cycle and the possibility of a second induction of the adaptive response were discussed.     

Adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture.

This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose-response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture. When the single-cell microbeam delivered from 1 to 12 alpha particles through the nuclei of 10% of C3H 10T(1/2) cells, more cells were inactivated than were actually traversed by alpha particles. The magnitude of this bystander effect increased with the number of particles per cell. An adaptive dose of 2 cGy of gamma rays, delivered 6 h beforehand, canceled out about half of the bystander effect produced by the alpha particles.     

Protection from radiation-induced apoptosis by the radioprotector amifostine (WR-2721) is radiation dose dependent

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure. We chose to study a relatively low dose which is the maximum radiation dose for radiation emergency workers (0.25 Gy) and a high dose relevant to radiotherapy exposures (6 Gy). Mice were administered 400 mg/kg amifostine 30 min before, or 3 h after, whole-body irradiation with 0.25 or 6 Gy X-rays and apoptosis was analysed 3 or 7 h later in spleen and bone marrow. We observed a significant increase in radiation-induced apoptosis in the spleen of mice when amifostine was administered before or after 0.25 Gy X-rays. In contrast, when a high dose of radiation was used (6 Gy), amifostine caused a reduction in radiation-induced apoptosis 3 h post-irradiation in spleen and bone marrow similar to previously published studies. This is the first study to investigate the effect of amifostine on radiation-induced apoptosis at a relatively low radiation dose and the first to demonstrate that while amifostine can reduce apoptosis from high doses of radiation, it does not mediate the same effect in response to low-dose exposures. These results suggest that there may be a dose threshold at which amifostine protects from radiation-induced apoptosis and highlight the importance of examining a range of radiation doses and timepoints.     

Radioresponsiveness at low doses: hyper-radiosensitivity and increased radioresistance in mammalian cells

The rationale for and importance of research on effects after radiation at "low doses" are outlined. Such basic radiobiological studies on induction of repair enzymes, protective mechanisms, priming, and hypersensitivity are certainly all relevant to treatment of cancer (see Section 1, Studies at low doses - relevance to cancer treatment). Included are examples from many groups, using various endpoints to address the possibility of an induced resistance, which has been compared to the adaptive response [M.C. Joiner, P. Lambin, E.P. Malaise, T. Robson, J.E. Arrand, K.A. Skov, B. Marples, Hypersensitivity to very low single radiation doses: its relationship to the adaptive response and induced radioresistance, Mutat. Res. 358 (1996) 171-183.]. This is not intended to be an exhaustive review--rather a re-introduction of concepts such as priming and a short survey of molecular approaches to understanding induced resistance. New data on the response of HT29 cells after treatment (priming) with co-cultured activated neutrophils are included, with protection against X-rays (S1). Analysis of previously published results in various cells lines in terms of increased radioresistance (IRR)/intrinsic sensitivity are presented which complement a study on human tumour lines [P. Lambin, E.P. Malaise, M.C. Joiner, Might intrinsic radioresistance of human tumour cells be induced by radiation?, Int. Radiat. Biol. 69 (1996) 279-290].It is not feasible to extrapolate to low doses from studies at high doses. The biological responses probably vary with dose, LET, and have variable time frames. The above approaches may lead to new types of treatment, or additional means to assess radioresponsiveness of tumours. Studies in many areas of biology would benefit from considerations of different dose regions, as the biological responses vary with dose. There may also be some implications in the fields of radiation protection and carcinogenesis, and the extensions of concepts of hyper-radiosensitivity (HRS)/IRR extended to radiation exposure are considered in Section 2, Possible relevance of IRR concepts to radiation exposure (space). More knowledge on inducible responses could open new approaches for protection and means to assess genetic predisposition. Many endpoints are used currently--clonogenic survival, mutagenesis, chromosome aberrations and more direct--proteins/genes/functions/repair/signals, as well as different biological systems. Because of scant knowledge of the relevant aspects at low doses, such as inducible/protective mechanisms, threshold, priming, dose-rate effects, LET within one system, it is still too early to draw conclusions in the area of radiation exposure. Technological advances may permit much needed studies at low doses in the areas of both treatment and protection.     

Adaptive response to gamma radiation in mammalian cells proficient and deficient in components of nucleotide excision repair

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.     

Radiomodification of glyoxalase I in the liver and spleen of mice: Adaptive response and split-dose effect

Glyoxalase system, particularly glyoxalase I (Gly I) plays an important role in regulation of cell division and is considered to be a metabolic indicator of cell proliferation. The glyoxalase system is likely to have a close link with cellular radiosensitivity. Therefore, we have examined the effect of adaptive and split-dose of -rays on the activity of Gly I in the liver and spleen of mice. For the adaptive response studies, mice pre-treated with a conditioning dose of 0.5 Gy were given a challenging dose of 4 Gy at varying time intervals. In the split-dose studies, a dose of 4 Gy was delivered into two equal fractions and spaced at different time intervals. The results show that pre-exposure to a conditioning dose or the fractionation of total dose decreased the specific activity of Gly I in the liver and spleen of mice. The decreased activity of Gly I was suggestive of protective action induced by the conditioning dose and fractionation of dose. The similar pattern of radiation response of Gly I probably supported the possibility of involvement of a common pathway in the radiation-induced adaptive and split-dose effect. From these observations a close link between the Gly I and the adaptive-response as well as the split-dose effect is speculated. Since, the glyoxalase system is vital for a variety of biological functions including cell division and repair, the present findings may have relevance in understanding the dose-fractionation as well as the biological defence induced by low doses of radiations.     

Neoplastic transformation in vitro by low doses of ionizing radiation: role of adaptive response and bystander effects

The shape of the dose-response curve for cancer induction by low doses of ionizing radiation is of critical importance to the assessment of cancer risk at such doses. Epidemiologic analyses are limited by sensitivity to doses typically greater than 50-100 mGy for low LET radiation. Laboratory studies allow for the examination of lower doses using cancer-relevant endpoints. One such endpoint is neoplastic transformation in vitro. It is known that this endpoint is responsive to both adaptive response and bystander effects. The relative balance of these processes is likely to play an important role in determining the shape of the dose-response curve at low doses. A factor that may influence this balance is cell density at time of irradiation. The findings reported in this paper indicate that the transformation suppressive effect of low doses previously seen following irradiation of sub-confluent cultures, and attributed to an adaptive response, is reduced for irradiated confluent cultures. However, even under these conditions designed to optimize the role of bystander effects the data do not fit a linear no-threshold model and are still consistent with the notion of a threshold dose for neoplastic transformation in vitro by low LET radiation.     

Variability of the adaptive response to ionizing radiations in humans

Human lymphocytes exposed to low doses of ionizing radiations from incorporated tritiated thymidine ([3H]dThd) or from X-rays become less susceptible to the induction of chromatid aberrations by high doses of X-rays. This indicates that low doses of ionizing radiation can produce an effect similar to the adaptive response observed with alkylating agents in prokaryotes, animal and plant cells. To determine whether there is individual variability in the adaptive response to ionizing radiations we exposed human lymphocytes from 18 different healthy donors to 'adapting' doses of [3H]dThd (0.01 microCi/ml) or X-rays (0.01 Gy) and subsequently to a 'challenge' treatment of 0.75 Gy of X-rays delivered 2 h before fixation. Four of the 18 donors did not show an adaptive response; in some cases in these individuals a synergistic response of increased, rather than decreased, damage was found. Two of these 4 donors showed no adaptive response in 3 subsequent experiments separated by 4-month intervals. This suggests that the human population exhibits a heterogeneity in the adaptive response to ionizing radiations which might be, at least in part, genetically determined.     

Interrelationships amongst radiation-induced genomic instability, bystander effects, and the adaptive response

Over the past two decades, our understanding of radiation biology has undergone a fundamental shift in paradigms away from deterministic "hit-effect" relationships and towards complex ongoing "cellular responses". These responses include now familiar, but still poorly understood, phenomena associated with radiation exposure such as bystander effects, genomic instability, and adaptive responses. All three have been observed at very low doses, and at time points far removed from the initial radiation exposure, and are extremely relevant for linear extrapolation to low doses; the adaptive response is particularly relevant when exposure is spread over a period of time. These are precisely the circumstances that are most relevant to understanding cancer risk associated with environmental and occupational radiation exposures. This review will provide a synthesis of the known, and proposed, interrelationships amongst low-dose cellular responses to radiation. It also will examine the potential importance of non-targeted cellular responses to ionizing radiation in setting acceptable exposure limits especially to low-LET radiations.     

Characterization of the adaptive response to ionizing radiation induced by low doses of X rays to human lymphocytes

In previous studies we have shown that low doses of radiation from incorporated tritiated thymidine can make human lymphocytes less susceptible to the genetic damage manifested as chromatid breakage induced by a subsequent high dose of X rays. We have also shown that this adaptive response to ionizing radiation can be induced by very low doses of X rays (0.01 Gy; i.e., 1 rad) delivered during S phase of the cell cycle. To see if a low dose of X rays could induce this response in cells at other phases of the cell cycle, human lymphocytes were irradiated with 0.01 or 0.05 Gy before stimulation by phytohemagglutinin (G0) or with 0.01 Gy at various times after stimulation (G1), followed by 1.5 Gy (150 rad) at G2 phase. Although G0 lymphocytes failed to exhibit an adaptive response, G1 cells irradiated as early as 4 h after stimulation did show the response. Experiments were also carried out to determine how long the adaptive response induced by 0.01 Gy could persist. A 0.01-Gy dose was delivered to lymphocytes in the first S phase, followed by 1.5 Gy in the same or subsequent cell cycles. Lymphocytes receiving a 1.5-Gy dose at 40, 48, or 66 h after stimulation exhibited an adaptive response, whereas those receiving a 1.5-Gy dose at 90 or 114 h did not. Duplicate cultures containing bromodeoxyuridine showed that at 40 h all the lymphocytes were in their first cell cycle after stimulation, at 48 h half of the lymphocytes were in their first cell cycle and half in their second, and at 66 h 80% of the lymphocytes were in their third cell cycle. Thus the adaptive response persists for at least three cell cycles after it is induced by 0.01 Gy of X rays. In other experiments, the time necessary for maximal expression of the adaptive response was determined by delivering 0.01 Gy at hourly intervals 1-6 h before the 1.5-Gy dose. While a 4-h interval was enough for expression of the adaptive response, shorter intervals were not.     

The effects of increasing dosages of X-radiation on the chromosomes of the central mudminnow, Umbra limi (Kirtland) (Salmoniformes: Umbridae)

Fish subjected to 350 R, 660 R and 990 R of X-radiation showed chromosomal aberrations such as chromatid breaks and gaps, and chromatid exchanges between several chromosomes. The frequency of aberrations/metaphase increased with radiation dosage. Likewise, the percentage of aberrant cells increased with increased irradiation. The countable metaphases fish was lower for higher doses of radiation. At lower doses single chromatid breaks accounted for most of the aberrations whereas complex aberrations involving the breakage and exchange of fragments between several chromosomes were more frequent in fish subjected to 990 R. Gill tissue yielded three times as many countable metaphases as did spleen tissue.     

Mathematical analysis of genetic effects of low doses of ionizing radiation

The comparative study of effects of low doses of radiation on peripheral blood lymphocytes of persons occupationally exposed to radiation and non-exposed ones was carried out. The main attention was paid to radio-adaptive response forming under consistent exposure to low (0.05 Gy) and damaging (2 Gy) doses of gamma-irradiation. Noticeable heterogeneity in capacity for adaptive response forming in occupational group was revealed. The mathematical model adequate to experimental material was constructed using Kohonen neuronets.     

Radio-adaptive response: characterization of a cytogenetic repair induced by low-level ionizing radiation in cultured Chinese hamster cells

Pretreatment with low doses of beta-rays from incorporated tritiated thymidine ([3H]dThd) or of Co-60 gamma-rays (1 or 5 cGy) rendered actively growing Chinese hamster V79 cells more resistant to the induction of micronuclei or sister-chromatid exchanges (SCEs) by a subsequent high dose of gamma-rays (1 Gy). This adaptive response to ionizing radiation (radio-adaptive response) can be induced by an optimal range of low doses of 3H beta-rays, but not by much lower or higher adapting doses. Full expression of the adaptive response induced by the exposure to low doses of 60Co gamma-rays occurred 4 h after the adapting dose. The cells pre-exposed to low doses of gamma-rays showed cross-resistance to challenge doses of gamma-rays themselves and also of mitomycin C (MMC) and near ultraviolet light (UV-B, 313 nm), but not to those of ethyl methanesulfonate (EMS) or cis-platinum (II) diammine dichloride (cisplatin) for SCE induction. These results suggest that the radio-adaptive response mechanistically couples to the repair network which copes with chromatin lesions induced by MMC and UV-B.     

Acceleration of DNA-double strand rejoining during the adaptive response of Chlamydomonas reinhardtii

Newly developed constant-field low voltage electrophoresis (adapted for algae cells by us) was applied to quantify the induction and repair of nuclear DNA double-strand breaks, by measuring the movement of DNA out of the starting wells into the electrophoresis gel using a UV-gel scan and computer analysis of DNA-ethidium bromide fluorescense (Syngene; Gene tools). A cell-wall-less mutant strain of Chlamydomonas reinhardtii (CW15) was used; the DNA and proteins are easily accessible because of the lack of an outer cell wall. Our results showed that giving a small priming dose (50 Gy) led to a small acceleration of dsb rejoining. When the magnitude of the priming dose was progressively increased, there was a corresponding decrease in the fraction of damage remaining at 4 hours after radiation exposure (to a test dose of 500 Gy). This indicates an upregulated rejoining of dsb following exposure of cells to the priming dose, which may be related to the strong adaptive response in this organism. Protein synthesis inhibitors were found to reduce the rate of rejoining of dsb, and from earlier results are known to inhibit the adaptive response. Thus, the adaptive response is likely to be dependent on increased dsb rejoining and depends on de novo protein synthesis. The nature of these proteins has not yet been established. C. reinhardtii CW15 is an attractive model system in which to study the underlying mechanisms of the adaptive response to ionizing radiation, and its underlying link with dsb rejoining. The results are interesting both from a basic biological point of view, and as a means to further understand the response of tumour cells to radiation therapy since the adaptive response has been postulated to determine the shape of the "shoulder" region of the survival curve of cells at low doses of radiation.     

The combined action of N-methyl-N'-nitro-N-nitrosoguanidine and UV light on Bacillus subtilis

The mutagenic interaction between ultraviolet irradiation and the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine was studied in repair-competent and excision-deficient strains of Bacillus subtilis. Pre-exposure to low doses of MNNG with following treatment by low and intermediate doses of UV light increase the resistance of Bac. subtilis to UV radiation (antagonistic effect). Probably pre-exposition with MNNG leads to induction of enzymes reparation, UV damages being controlled with adaptive response genes.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.