One of the main conclusions of the debate on the low-dose dilemma was the need to continue the epidemiological and radiobiological investigations (Köteles, 1998, 1999). The latter are expected not only to reveal the probability and mechanisms of malignant cell transformations, but also to analyze the cellular responses either in the direction of sensitization or to that of protection. Among these the genomic instability, the bystander effect, the adaptive responses have been in the limelight of research (Fry et al., 1998). Further studies are also needed on the responses of tissues, organs, and organisms to establish the up-to-date philosophy of radiological protection.
The present paper concentrates on phenomena which might modulate the biological responses though they are far from justifying the modification of the present philosophy of radiological protection including dose limitation.
ELEMENTS OF RISK ASSESSMENT
For the proper assessment of health risks the biological-pathological effects, the dose range provoking the effects, and the dose-response relationship have to be known. Radiological protection usually deals with the risks of carcinogenic effects.
The linear no threshold (LNT) model was taken as a proper approach (Koblinger, 2000), e.g. for the bronchial cancers in underground uranium miners and the survivors of atomic bombings (Chomentowski et al., 2000; Little and Muirhead, 2000). The uncertainties, however, of the epidemiological statistical analysis and conclusions below app. 100–200 mGy, or in case of radon exposure below 400 Bq ·
m–3 in air made the validity of LNT in this range questionable (Lubin and Boice, 1997; Clarke, 2000), i.e., in the dose range most relevant for radiological protection.
Accordingly, the most important element of risk assessment, i.e., the dose-response model has become the subject of criticism. In radiation biology like evaluation of radiation-induced chromosome aberrations all the three main types of relationships (linear, linear-quadratic, and curvilinear) can usually characterize the yield that might especially influence the relationships in the low dose range.
RESPONSE MODIFYING FACTORS
In radiation biology, the response modifying factors have always been taken into consideration. The physical factors are like LET values, the dose rate and mode of irradiation (acute, chronic, or fractionated). ICRP has introduced also a factor, the dose-dose-rate-effectiveness factor (DDREF) in the assessment of stochastic risks (ICRP, 1991). The chemical factors involve the endogenous and exogenous antioxidants as well as the radioprotective and radiosensitizing compounds. Collection of data on the antioxidants and individual antioxidant capacity has become of public health interest (Bognár et al., 1997; Chevion and Chevion, 2000). Among the biological factors, a few phenomena of cell biology have been known like the differences between mitotic and non-mitotic cells, and cell cycle phase-dependency. Recently, studies on gene activation and inactivation, the checking points in the regulation of cell cycle, the cell-to-cell communication have brought new results which might modify the radiation response (Somosy, 2000; Köteles and Somosy, 2000). However, doses, studied in these contexts are rather high, in the order of magnitude of grays. Therefore, it would be important to know the dose ranges of these relations and to clarify whether they appear in response to a threshold dose. The setting of the latter, however, depends also on the sensitivity of detection techniques. Hardly any data are available on the type of dose-response relations of these radiation-induced phenomena. For example no dose-dependency was found in provoking genetic instability between 2 and 12 Gy (Little, 1998). Does it mean that even lower doses can produce the effect? It would be important to know for its stochastic risk assessment.
A few cell biological phenomena that might modify radiation responses are shown in Fig. 1. Among these, the risks might be increased by the development of genom instability and bystander effects, and the risks might be decreased by the adaptive responses and hormesis.
Fig. 1. New biological phenomena as potential risk modifying factors.
GENOM INSTABILITY
Several observations indicate that ionizing radiation induces genom instability, a feature that can be transmitted in cell cultures over generations. Increased number of mutations, mitotic capability, chromatic aberrations, decreased life-span, increased frequency of malignant transformations could be observed in later generations (Little, 1998). This status of cells might be the basic condition for carcinogenesis (Ullrich and Ponnaiya, 1998). Rather high doses, 4–12 Gy were applied both with low and high LET radiation. As app. 10–20% of cell cultures were altered and the genom mutation rate for such doses is in the order of 10–5 to 10–8, the conclusion could be drawn that the target is the whole genom. This genom-wide feature might serve as a basis for later exposures aggravating the genotoxic effects. In the development of this hipermutability even epigenetic factors might contribute like the inhibition of apoptosis through TP53 gene mutation or cellular membrane lipids.
Biochemically the gene instability can be characterized by the increases of intracellular oxidants – superoxid anion radicals, partial deletions of gene segments, and increased oxygen radical attacks against the membranes (Wright, 1998).
BYSTANDER EFFECT
Cells neighboring the irradiated cells can be damaged; sister chromatid exchanges, gene activation like that of TP53 or cell cycle genes, increased frequency of HPRT mutations could be observed (Nagasawa and Little, 1999; Seymour and Mothersill, 2000). Linear dose-response relationships could be fitted between 5 cGy and 1,2 Gy. at low doses even higher mutation rate was found than expected from extrapolations.
The mechanism of the effect might be cell-to-cell communication through metabolites, cytokines and diffusion of free radicals. Investigations demonstrated that the cytokine release from irradiated cultured cells really increased upon relatively low dose irradiation (Fig. 2; Köteles et. al., 1994 a, b, 1995).
Fig. 2. Effect of various doses of X-irradiation upon the IFN-gamma and
TNF-alpha secretion of human leukocytes during one hour of incubation.
ADAPTIVE RESPONSE
The adaptation is manifested in higher resistance of cells against high doses provided a low dose was given in a few hours before. This adaptive response was observed in various types of human cells, normal and tumors, in G1 lymphocytes and fibroblasts (Raaphorst and Boyden, 1999). Lymphocytes in G0 phase also reacted with adaptation (Fig. 3; Köteles et al., 1997). In certain cultured cells – C3H10T1/2 – a few mGy resulted in decreased rate of spontaneous malignant transformation (Azzam et al., 1996).
It is interesting to note that the adaptive response appears in various cells only in a certain dose window, between 5 and 200 mGy. Its mechanism probably involves more efficient detoxification of free radicals, induced synthesis of repair enzymes, more efficient elimination of damaged cells from the organism through apoptosis (Fig. 4; Köteles et al., 1997).
In cultured cells or living tissues the conditions for adaptive response could lead to the appearance of a threshold dose below which neither any cell biological phenomena like clastogenic effects nor malignant transformations appear.
Fig. 3. Adaptive response of G0 human lymphocytes X-irradiated in whole blood, in vitro.
Fig. 4. Reduction of radiation-induced micronucleus-carrying CB cells
upon treatment of rabbits in vivo with mistletoe lectins.
GENE REGULATION
The relevant literature on radiation-induced gene mutation and their regulation is very rich. The processes might lead to simultaneous gene induction with various kinetics and are dependent on radiation types. The alterations may occur within a few minutes following even 10 mGy. Data are available on TNF-a
activation through proteinkinase C, TP53 gene alteration following alpha irradiation, activation of kappa nuclear factor through oxygen radical signaling (Fry et.al, 1998; Schmidt-Ullrich et al., 2000). The inducing signal might originate from DNA damage, or through epigenetic mechanisms from cytoplasm or membrane damages. An example for the latter is the apoptosis inhibition by membrane damage derived ceramide formation leading to genomic instability as well as by the loss of TP53 gene.
The widely discussed hormesis, the stimulatory or beneficial effect of low doses also falls into the category of adaptation.
INDIVIDUAL SUSCEPTIBILITY AND SENSITIVITY
In the new trend of radiation protection to alter the dose limit for the population the knowledge on the individual sensitivity gains more importance. In human population the mutation rate varies by a factor of app. 10. DNA repair mechanisms and their deficiencies are a well-known phenomenon in certain diseases like Bloom’s syndrome, Fanconi anemia, Werner’s syndrome, ataxia teleangiectasia, etc. Recently, even following 100 mGy the increase of HPRT mutations has been demonstrated in a population of Chernobyl clean-up workers (Moore et al., 1997). Therefore, it is a question to be answered in the future whether “mutator phenotype” persons exist in the population with higher sensitivity against ionizing radiation or against any combined clastogenic or mutagenic effects.
Individual sensitivity is influenced by endogenous protective mechanisms like the antioxidant system. It can also be supported by the supply of exogenous antioxidants. The antioxidant capacity in sera of various individuals varies by a factor of 2 and it has also been demonstrated that the uptake of exogenous compounds can be measured and controlled. It is of practical importance to note that at a higher antioxidant status a lower clastogenic effect is produced by irradiation (Köteles et al., 1999).
LEVELS OF REACTIONS
The various levels of modification of radiation effects include nuclear level in the chromatin structure, cytoplasmic sites, tissue, organ, and organism level up to individual sensitivity (Fig. 5). The various injuries of DNA from point mutations up to “multiple damaged sites” can be prevented or corrected by radical scavengers, antioxidants, and enzymatic repair mechanisms. Pathological cell processes induced involve nuclear or cytoplasmic structures. The epigenetic changes provide a feedback to the nuclear centers. The radiosensitivity of tissues and organs depend on their cell compositions. Reaching the organism level, it is obvious that individual susceptibility is influenced by many factors. This has to be considered especially when combined environmental effects occur. Therefore, in research activities with a purpose to increase radioresistance, to treat injuries, to develop diagnostic assays, to assess the complex risks of several environmental interactions with the cells or organisms, these phenomena have to be considered.
Fig. 5. Levels of reactions against ionizing radiation.
CONCLUSIONS
The effects of low doses are influenced by many biological factors and the reactions themselves do not necessarily mean harm. The lower the dose the more decisive the protective mechanisms are. Harms are caused by doses breaking through various levels of protection.
The research and development on properly sensitive biological assays to detect cellular alterations have great importance as our knowledge on biological effects depends on our capability to detect. This is especially important when combined environmental effects and risks have to be seen and assessed. The biological indicators should point to acute effects exerted in the present or the past time, through a retrospective analysis to point also to combined effects which might increase future risks.
The low dose dilemma is rather complex, the carcinogenic risk – if there is any at a certain level of doses – has to be studied by epidemiology, the process of malignant transformation has to be studied by radiation biology including response modifying cellular phenomena.
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