Shu-Zheng Liu

The shape of the dose-effect curve of biological parameters depends on a number of factors among which the radiosensitivity of the target under study, the dose spacing and dose rate are important ones. When one goes down to the lower dose range, especially with doses below 0.2 Gy, changes of many biological parameters in the opposite direction to those observed with doses higher than 0.5 Gy are often documented, thus resulting in a U- or J-shaped or an inverted U- or J-shaped curve. In the present paper such a phenomenon is reviewed by giving data from the author’s laboratory demonstrating the difference in nature of effects induced by low versus high dose X-rays at molecular and cellular levels as well as their implications in the intact organism. A hypothetical dose-effect model is suggested to summarize the observations.

Key words: radiation dose-effect model; genes; cell proliferation; cell cycle control; signal transduction; cytokines

[Ed. note: See Figure captions at the bottom of the text - to be revised.]

One of the most important features of radiobiology research is the establishment of dose-effect relationship of the biological parameters under study. With the diversity of biological phenomena it is not easy to establish a universal dose-effect model. In the literature a dose-effect curve has often been formulated by extrapolation from data obtained with doses above 0.5 or 1 Gy. The matter may be simplified by doing so, but the seemingly accurate formulation may actually deviate from reality. The reason is very clear since there have been in recent years radiobiological data obtained with doses within 0.1 or 0.2 Gy demonstrating deviations from the suggested "ideal" dose-effect curve. When one goes down to the lower dose range changes of many biological parameters in the opposite direction are often observed, thus resulting in a U-shaped (J-shaped) or an inverted U- or J-shaped curve. In the present paper the dose-effect relationship of biological parameters at cellular and molecular level as well as their association with events in the whole organism after exposure with low LET radiation will be analyzed.

One of the most prominent effects of ionizing radiation is its suppressive action on cell survival and proliferation. This occurs with doses above 0.5 Gy [1]. But with doses below 0.5 Gy, there often appears a stimulation of cell proliferation as demonstrated in splenocytes after whole-body irradiation (WBI) [2] and also in EL-4 cells after in vitro irradiation, thus an inverted J-shaped dose-effect curve could be formulated (Fig 1). The distinct effect of low versus high dose radiation on cell proliferation has its molecular basis as shown in panels A and B of figure 2 in which the mRNA transcription and protein expression of molecules related to cell survival and growth after 0.075 and 2 Gy are illustrated, respectively (Fig. 2). In this figure the level of transcription 2h after WBI and the level of protein expression 24h after WBI relative to sham-irradiated control are shown since in most of the cases these are the times for transcription and expression reaching the peak (or going down to the nadir), respectively. However, there are cases in which the peak or the nadir occurs before or after the above specified times. These were given in a previous report [3]. Panel A of figure 2 shows the transcription level of 5 genes in the thymus (odd numbers) and spleen (even numbers) related to cell survival, namely, c-fos, c-myc, bcl-2, p53 and ICE. It is clear that the direction of change is distinctly different for the low (0.075 Gy) and high (2 Gy) doses though the amplitude of the changes varies. The transcription of the survival gene bcl-2 is suppressed by more than 50% after 2 Gy and up-regulated after 0.075 Gy and the transcription of the death gene ICE takes an opposite direction in the thymus with a nearly doubled increase of its transcription after 2 Gy. It should be pointed out that the transcription of c-fos gene is up-regulated by 75% 2h after WBI with 0.075 Gy, but it reacts to WBI promptly with an increase of about 4 fold 30 min after this dose as shown in [3]. Panel B demonstrates the expression of proteins related to cell survival in the thymus (except columns 10 and 14 which stand for Peyer’s patch). The change of the expression of these proteins occurs in opposite directions except that of c-Myc and even in this case the high dose causes marked increase of its expression and the low dose shows little effect (column 3). The most marked changes are seen in expression of Bad (an increase of more than 300% after 2 Gy and a decrease by 60% after 0.075 Gy, column 8), and the Bcl-X_L/Bad ratio (an increase of more than 200% after 0.075 Gy and a decrease by more than 90% after 2 Gy, column 9). These composite changes at the molecular level give support to the different cellular response to low versus high dose radiation.

Cellular functions may be depressed by high dose X-rays, but are usually stimulated by low dose radiation (LDR). This can be well illustrated by examples in the immune system, such as NK (natural killer) and ADCC (antibody-dependent cell-mediated cytotoxicity) activity [4]. Another important immune function is the synthesis and secretion of cytokines. The transcription level of IL-2, IL-4, IL-6, IL-10, IL-12p35, TNF-b (lymphotoxin) and IFN-g in the thymus (odd numbers) and spleen (even numbers) is shown in panel D of figure 2 that demonstrates opposite directions of deviation from control for most of the cytokines examined. As for changes 2h after WBI shown in this panel, more marked deviations from control are seen for IFN-g (interferon-gamma) in the thymus (column 5), IL-6 in the spleen (column 10) and IL-12p35 (column 13). When the time course of the transcription level is considered a striking difference in that of IL-12p35 and IL-10 transcription after low versus high dose radiation is seen as shown in figure 3 with opposite changes of the transcription level of these two cytokines in the spleen, i.e., LDR (0.075 Gy) caused up-regulation of the transcription level of IL-12p35 and down-regulation of that of IL-10, and high dose radiation (2 Gy) exerted opposite effects (adopted from [3]). These changes could well explain the stimulatory effect of LDR on cell-mediated immunity as verified in previous studies [5]. Another example of the distinct effect of low versus high dose radiation on cellular function is the secretion of IFN-g the dose-effect curve of which is shown in figure 4.

The cell cycle progression is controlled by a number of checkpoints that allow the transition of cells through the different phases. Under such transition ‘stations’ there lay accurate molecular machineries. Ionizing radiation perturbs the normal transition of cell cycle progression when the dose is high enough. These are often designated as G₁ arrest, S delay and G₂ arrest, the molecular regulation of the latter being most fully studied after irradiation [6]. Figure 5 shows the dose-effect relationship of G₂ arrest of the thymocytes after WBI as assessed by flow cytometry. In this figure the percentage of cells in G₂/M phase was significantly increased 24h after WBI with doses above 1 Gy (G₂ block), but with lower doses the number of cells in G₂/M phase was even found to be less than control, thus giving rise to a J-shaped curve [7]. It is known that the transition of cells from G₂ to M phase is governed by MPF (meiosis promoting factor) that is formed by two subunits, i.e., cyclin B₁ and p34^cdc2 [6]. We examined both the transcription level of these two molecules by Northern blotting and their protein expression by immunohistochemistry in the thymus after WBI with 0.075 and 2 Gy and found significant suppression of the two molecules both at mRNA transcription and protein expression levels after 2 Gy and their slight up-regulation after 0.075 Gy [3]. This provides the molecular basis of the cellular changes occurring after WBI with low versus high doses.

Signal transduction in cells forms the basis of the molecular mechanisms of the changes in cellular functions in response to external and internal stimuli. Many of the genetic changes in the thymus after exposure to LDR are closely related to shifts in the signal molecules in the transduction pathways. It has previously been disclosed that at least two important signal transduction pathways are involved in the activation of thymocytes in response to low dose radiation [3,8,9]. The first of these is the Ca²⁺- PKC (protein kinase C) pathway and the other is the cAMP (cyclic adenosine monophosphate) pathway. It was found that after WBI with low dose X-rays the Ca²⁺-PKC pathway was stimulated and the cAMP pathway was down-regulated. Panel C of figure 2 shows the changes in the molecular cascades of these two pathways after WBI with low versus high dose X-rays. These are shown in the first through the 9^th columns. Another factor to be considered for signal transduction pathways in the lymphocytes is the prostaglandin (PG) system. It is known that PGE antagonizes the action of IL-2 and suppresses immune reactions via stimulation of cAMP production. PLA (Phospholipase A) is one of the signal molecules that induce PGs [10]. As seen in column 10 of panel C, the activity of PLA₂ was down-regulated after 0.075 Gy and stimulated after 2 Gy. Thus three signal pathways are interlaced resulting in facilitation of nuclear translocation of NF-kB, which would cause induction of a series of genes related to cellular activation, such as IFN-g and IL-2. The actual situation may be more complicated with many other signal molecules involved. The dose-effect relationship of the signal molecules 24h after WBI is shown in figure 6 (the PKCs are not included in this figure). The nuclear translocation of NF-kB (nuclear factor kappaB) may be one of the common pathways in the realization of cell survival and activation following LDR. It is known that the Rel/NF-kB family consists of different dimers among which the p65/p50 heterodimer exerts an activating effect on transcription while the p50/p50 homodimer causes transcription suppression, thus the ratio of p65/p50 to p50/p50 in the nucleus may be an important factor determining the outcome of the cellular effects [11]. The level of nuclear expression of CREB (cAMP response element binding protein) is a transcription factor that has a suppressive effect on transcription activation in immune cells. It was found that the dose-response curve of the ratio of p65/p50 to p50/50 and that of the DNA binding activity of CREB took an opposite direction (He et al, in press) (Fig. 7). Therefore, in the context of cell signaling different or opposite changes are also observed after low versus high dose radiation.

The changes at cellular and molecular levels after exposure to different doses of ionizing radiation are also reflected on the intact whole organism. Carcinogenesis is the most concerned effect of ionizing radiation, especially when low-level radiation is considered. It is well known that medium to high doses of radiation would lead to an increase in cancer risk in the human population. However, it remains to be clarified whether the risk of cancer induction by LDR could be extrapolated from data obtained from higher doses as suggested by the linear no-threshold hypothesis. This was discussed in papers concerning epidemiological studies in human populations presented in this conference. In animal studies opposite effects of low versus high doses have also been observed in this aspect. In some strains of mice fractionated high dose radiation would lead to significant increase of thymic lymphoma, e.g., when C57BL/6J mice are subjected to 1.75 Gy X-rays once a week for 4 consecutive weeks, thymic lymphoma will appear in about half of the animals in 6 months [12]. If each dose of 1.75 Gy is preceded by a low dose (0.025, 0.075 and 0.1 Gy) X-rays with an interval of 6 to 24h, the incidence of thymic lymphoma could be reduced to one third or one half of the high dose exposed controls [12]. The authors considered the lowered incidence of cancer with the intervention of a preceding low dose to be related to the enhancing effect of LDR on immunity [13].

In conclusion, the dose-effect relationships of many biological parameters may not follow a simple linear or linear quadratic pattern as expected. Such a pattern can only be observed when the dose-effect curve is constructed with data from doses above 0.5 Gy. If the dose spacing is shifted to the lower range in the experimental design, e.g., when the changes after doses of 0.025 to 0.2 Gy are carefully observed, one can often find shifts in the opposite direction of those with doses above 0.5 Gy. This pattern of dose-response curves has been illustrated in the present paper both at cellular and molecular level. A hypothetical dose-effect model is suggested in figure 8. Such a formulation may have important implications for the whole organism as well as the population if more studies along this direction are made.

Acknowledgment: This work is supported by grants from NSFC.

1. Liu S-Z. Radiation Hormesis with low-level exposures. Beijing: Science Publishers, 1996.

2. Li X-Y, Lu Z, Zhang YC, et al. Enhancing effect of low dose radiation on the proliferative activity of thymocytes in mice. J Radiat Res Radiat Proc 1994; 12, 124-126.

3. Liu S-Z, Bai O, Chen D, et al. Genes and protein molecules involved in the cellular activation induced by low dose radiation. J Radiat Res Radiat Proc 2000; 18: 175-186.

4. Liu S-Z. Biological defense and adaptation induced by low dose radiation. Human Ecol Risk Assess (HERA) 1998; 4:1217-1254.

5. Liu S-Z. Cellular and molecular basis of the stimulatory effect of low dose radiation on immunity. In Wei, L-X, Sugahars, T. and Tao, Z-F. (eds.), High Levels of Natural Radiation. Amsterdam: Elsevier Science BV, 1997; 341-353.

6. McKenna WG, Maity A, Muschel RJ. Radiation and the molecular biology of the cell cycle. Radiation Research 1895-1995, Proc 10^th ICRR, Wurzburg, Germany, Aug. 27-Sept. 1, 1995; 629-634.

7. Ye F, Liu S-Z. Effects of whole body irradiation with X-rays on cell cycle progression of thymocytes in mice. Radiat Prot 2000; 20:189-192.

8. Liu S-Z, Xie F. Involvement of the Ca²⁺-protein kinase C and adenylate cyclase signal pathways in the activation of thymocytes in response to whole-body irradiation with low dose X-rays. Chin Med Sci J 2000; 15:1-7.

9. Liu S-Z, Su X, Zhang YC, et al. Signal transduction in lymphocytes after low dose radiation. Int J Occup Med Toxicol 1994; 3: 107-117.

10. Danet-Desnoyers G, Meyer MD, Gross TS, et al. Regulation of endometrial prostaglandin synthesis during early pregnancy in cattle: effects of phospholipases and calcium in vitro. Prostaglandins 1995; 50: 313-330.

11. Ghosh S, May MJ, Kopp EB. NF-kappaB and Rel proteins: evolutionarily conserved mediators of immune response. Annu Rev Immunol 1998; 16: 225-260.

12. Li X-Y?13. Li X-J?14. Zhang Y?15. et al. Influence of low dose ionizing radiation on thymic lymphoma induced by carcinogenic dose radiation in C57BL/6J mice. Acad. Period Abst Chin 1998; 4: 1406-1407.

16. Li X-J, Yang Y, Li X-Y, et al. Immunologic mechanism of the suppressive effect of low dose radiation on thymic lymphoma induced by radiation. J Radiat Res Radiat Proc 1999; 17: 125-128.

Figure 1 Proliferation of EL-4 cells after irradiation

Figure 2 Molecular changes after whole-body irradiation with low versus high doses

A. Cell survival related genes—mRNA transcription (odd numbers for thymus and even numbers for spleen);
B. Cell survival related genes—protein expression (in thymus except columns 10 and 14 which stand for Peyer’s patch);
C. Signal trasnduction molecules of thymus;
D. Interleukin genes—mRNA (odd numbers for thymus and even numbers for spleen)

Figure 3 Transcription of IL-12p35 and IL-10 in the spleen after whole-body irradiation with low versus high dose X-rays

Figure 4 Dose-response curve of interferon-gamma secretion by splenocytes after whole-body irradiation

Figure 5 Changes in number of G₂ phase cells in the thymus after whole-body X-irradiation

Figure 6 Dose-response curves of signal molecules in the thymus after whole-body X-irradiation

Figure 7 Dose-effect relationship of thymocyte NF-kB/Rel and CREB 12h after whole-body X-irradiation

Figure 8 Hypothetical dose-effect model in radiobiological studies

MH Radiobiology Research Unit

Norman Bethune University of Medical Sciences

Changchun 130021, China

Email:drliusz@yahoo.com