Abstract
Low-dose (≤ 0.1 Gy) radiation could reduce high-dose induced damage including tumorigenesis. However, it remains unclear whether multi-exposure to low-dose radiation at a high dose rate has any risk for increasing tumorigenesis, and whether Fhit plays any role in the process. The purpose of this study is to investigate the effects of multi-exposure to low-dose radiation at a high dose rate on tumorigenesis, and the role of Fhit in it. We irradiated Fhit+/+ and Fhit-/- mice with 1 Gy/1 or 0.1 Gy × 10 exposures at a dose rate of 1 Gy/min, sacrificed the mice at 1.5 years after radiation and observed multi-organ tumorigenesis. The results showed that although the spontaneous tumorigenesis in these mice was relatively high, 1 Gy/1-exposure dramatically increased the tumorigenesis including lung and liver tumor. Fhit-/- mice showed more tumorigenesis than Fhit+/+ mice after 1 Gy/1-exposure. However, 0.1 Gy × 10 exposures did not increase tumorigenesis, and there was no statistical difference in tumorigenesis between Fhit+/+ mice and Fhit-/- mice following 0.1 Gy × 10 exposures. Our results suggest that 0.1 Gy, even after multiple exposures, does not increase tumorigenesis, and Fhit could prevent high-dose radiation-induced tumors but has no effect in a low-dose environment.
Keywords: Fhit, low dose, ionizing radiation, tumorigenesis
Introduction
It has been known for decades that ionizing radiation increases carcinogenesis frequency. Recently, more and more data indicate that low-dose radiation (≤ 0.1 Gy) might not increase the risk for carcinogenesis. Low doses of ionizing radiation (IR) at ≤ 0.1Gy exposure could induce cell resistance to the high-dose IR-induced damage [1]. The adaptive response was then found to reduce gene mutation rates in irradiated cells [2–10] and prevented the mice from the following high dose IR-induced tumors [11, 12]. These results suggest that low dose (≤ 0.1 Gy) radiation might not be harmful to the human body. However, it remains unclear whether multi-exposure to low dose radiation (≤ 0.1 Gy) at a high dose rate could promote carcinogenesis and whether Fhit plays any role in the process.
Fhit is a gene related to human tumor progression [13], and plays a protective role in preventing mice from carcinogen induced tumors [14]. We previously reported that Fhit could prevent human cells from high-doses of UV and ionizing radiation-induced HPRT mutation [15, 16], suggesting that Fhit prevents DNA damage-induced carcinogenesis. However, it remains unclear whether Fhit could prevent high-dose radiation induced carcinogenesis and whether it plays any role in a low-dose environment. To examine whether multi-exposure of low dose radiation (≤ 0.1 Gy) at a high dose rate promotes carcinogenesis and whether Fhit plays a protective role in the process, we designed this study. The results suggest that 0.1 Gy at 1 Gy/min, even multiple exposures, does not increase tumorigenesis and Fhit could prevent high-dose radiation-induced tumors but has no effect in a low-dose environment.
Materials and methods
Mice
C57B16/J strain, wild type (Fhit+/+) and Fhit knockout (Fhit-/-) mice, 4-6 weeks old, 240 in 6 groups were used in this experiment (Table 1). The mice were sacrificed at 1.5 years after exposure to ionizing radiation. The multi-organs including lungs, liver, bones, kidneys, intestines and multi-glands were taken for histological slide preparation. The samples were fixed in buffered formalin and sent to the University's pathology facility for tissue slicing, and hematoxylin & eosin (H&E) staining. Because most tumors observed in different organs were as nests and invisible by eye directly, the size and frequency of the tumors were observed with a microscope (Nikon, Tokyo, Japan). The number of mice that generated tumors, tumor number and the tumor size in the liver or lung organ per group were analyzed and scored by using Zeiss software.
Table 1.
Mice groups
| Radiation | Fhit+/+ | Fhit-/- | ||
|---|---|---|---|---|
| Male | Female | Male | Female | |
| 0 Gy | 20 | 20 | 20 | 20 |
| 0.1 Gy ×10 | 20 | 20 | 20 | 20 |
| 1 Gy | 20 | 20 | 20 | 20 |
Irradiation
The mice (whole body) were exposed to cesium source (γ-ray) and irradiated with 1 Gy either one exposure or fractionated into 10 exposures (0.1 Gy × 10), in 10 days at the dose rate of 1 Gy/min.
Statistical analysis
Differences of tumorigenesis between the treatment groups were calculated by chi-square test. Grading data were calculated by ridit analysis. P values < 0.05 were regarded as significant.
Results
Whole body exposure to 0.1 Gy X 10 did not increase the frequencies of multi-organ tumorgenesis
To investigate the effects of multi-exposure to low dose (≤ 0.1 Gy) radiation on tumorigenesis, we compared tumorigenesis among six groups of C57B16/J strain mouse (Fhit+/+ and Fhit-/- as indicated in table 1) including: the mice without radiation, the mice exposed to 0.1 Gy × 10 exposures (0.1 Gy/exposure, 1 exposure/day), and the mice exposed to 1 Gy (1 exposure). We sacrificed the mice at 1.5 years following radiation and observed the number of mice with tumors, tumor number and tumor size in multi-organs including lungs, liver, bones, kidneys, intestines, ovaries, testis and salivary glands. The results showed that although the Fhit+/+ mice had a high spontaneous carcinogenesis in multi-organs, especially in the liver and lung (total tumorigenesis frequency ∼30% and some mice showed multi-organ tumors) (Figure 1) (Table 2), Fhit-/- mice didn't show increased tumorigenesis without radiation. Multi-exposure of 0.1 Gy (0.1 Gy × 10) did not increase the tumorigenesis frequency in Fhit+/+ or Fhit-/- mice. Sex glands including ovary and testis tumorigenesis also did not increase frequencies in 0.1 Gy × 10 irradiated mice and there was no difference in the sex gland tumorigenesis between Fhit+/+ and Fhit-/- mice after exposure to 0.1 Gy × 10. However, 1 Gy with 1 exposure dramatically increased the tumorigenesis frequency in Fhit+/+ and Fhit-/- mice when compared with that in non-irradiated control mice (P<0.01) (Table 2). These results suggest that ≤0.1 Gy is a safe dose for tumorigenesis even at a high dose rate with multiple exposures. Although tumorigenesis frequency is higher in Fhit-/- mice (75%) than in Fhit+/+ mice (60%) following 1 Gy/1 exposure, there is no statistical difference between these two groups, which might be due to the small number of animals in each group (40 mice). We then did the following analysis.
Figure 1.
Different type tumors were observed in the mice. The mice were sacrificed as described in Materials and Methods. The multi-organs including lung, liver, kidney, intestine and multi-glands including ovary, testis, parotid and sublingual gland were taken for histological slide preparation. The samples were fixed in buffered formalin and sent to the University' pathology facility for tissue slicing and hematoxylin & eosin (H&E) staining. The magnification is 100 X.
Table 2.
Total Tumorigenesis Frequency
| Group | Radiation | Tumorigenesis Frequency |
| Fhit+/+ | 0 Gy | (12/40) 30% |
| Fhit+/+ | 0.1 Gy × 10 | (14/40) 35% |
| Fhit+/+ | 1 Gy | (24/40) 60%* |
| Fhit-/- | 0 Gy | (14/40) 35% |
| Fhit-/- | 0.1 Gy × 10 | (12/40) 30 % |
| Fhit-/- | 1 Gy | (30/40) 75%** |
p<0.05
p<0.01 (Comparison between irradiated group and non-irradiated group)
Fhit prevents high-dose radiation promoted tumorigenesis
To further investigate the effects of Fhit on high-dose radiation promoted tumorigenesis, we analyzed the tumor size in the lung and liver organs from Fhit+/+ and Fhit-/- mice following radiation (0.1 Gy × 10 or 1 Gy × 1). We divided the tumor size into four grades by observing the sections under microscopy: +: tumor diameter < 0.1 mm; ++: tumor diameter 0.1-0.5 mm; +++: tumor diameter 0.5-1 mm; ++++: tumor diameter > 1 mm (Figure 2). We calculated the tumor diameter by combining tumors in one mouse if the mouse had more tumors in liver or lung. We scored the total tumor index by using the number of mice bearing tumors in each category to time grade: for example, 2 mice with tumor + equaling tumor index 2 (derived from 2 × 1) and 2 mice with tumor ++ equaling tumor index 4 (derived from 2 × 2). In this way, we compared the index for liver and lung tumors among different groups. The results showed that both 1 Gy/1 exposure irradiated Fhit+/+ and Fhit-/- mice dramatically increased both indexes of liver and lung tumors when compared with non-irradiated control groups and 0.1 Gy × 10 exposures irradiated groups (Table 3 and 4) (P< 0.01). Interestingly, although there is no statistical difference of total liver or lung tumor frequency between Fhit+/+ and Fhit-/- mice following 1 Gy/1 exposure, there are significant differences in the indexes for both liver and lung tumors between the two groups: Fhit+/+ group showed lower indexes of liver and lung tumors than in Fhit-/- group (Table 3 and 4) (P< 0.05). These results further confirm that low-dose (0.1 Gy) at high dose rate, even with multi-exposure, could not increase tumorigenesis frequency compared to non-irradiated controls. In addition, these results indicate that Fhit could prevent high-dose induced tumorigenesis.
Figure 2.
Grade of the size in liver or lung tumors. The size of liver or lung tumors was graded as: – (no tumor), + (tumor size < 0.1 mm), ++ (tumor size is between 0.1-0.5 mm), +++ (tumor size is between 0.5-1mm), and ++++ (tumor size > 1 mm). The indexes were calculated by combining the diameters of all liver or lung tumors in each mouse. The magnification is 100 X.
Table 3.
Liver Tumor Index
| Group | Radiation | + | ++ | +++ | ++++ | Index |
|---|---|---|---|---|---|---|
| Fhit+/+ | 0 Gy | 7 | 2 | 1 | 0 | 14 |
| Fhit+/+ | 0.1 Gy × 10 | 9 | 1 | 1 | 0 | 14 |
| Fhit+/+ | 1 Gy | 15 | 3 | 1 | 1 | 28** |
| Fhit-/- | 0 Gy | 7 | 2 | 1 | 0 | 14 |
| Fhit-/- | 0.1 Gy × 10 | 7 | 2 | 0 | 0 | 11 |
| Fhit-/- | 1 Gy | 11 | 8 | 5 | 4 | 58**/* |
p<0.05
p<0.01 (Comparison between irradiated group and non-irradiated group
Comparison between 1 Gy-irradiated Fhit+/+ and Fhit-/- groups)
Table 4.
Lung Tumor Index
| Group | Radiation | + | ++ | +++ | ++++ | Index |
|---|---|---|---|---|---|---|
| Fhit+/+ | 0 Gy | 10 | 1 | 0 | 0 | 12 |
| Fhit+/+ | 0.1 Gy × 10 | 9 | 1 | 0 | 0 | 11 |
| Fhit+/+ | 1 Gy | 17 | 5 | 0 | 0 | 27** |
| Fhit-/- | 0 Gy | 6 | 3 | 1 | 0 | 15 |
| Fhit-/- | 0.1 Gy × 10 | 8 | 3 | 0 | 0 | 14 |
| Fhit-/- | 1 Gy | 10 | 6 | 6 | 4 | 56**/* |
p<0.05
p<0.01 (Comparison between irradiated group and non-irradiated group
Comparison between 1 Gy-irradiated Fhit+/+ and Fhit-/- groups)
Discussion
Low dose radiation exposure does not increase the tumorigenesis frequency
Our data support that low dose radiation (≤ 0.1 Gy) does not increase tumorigenesis although high dose radiation promotes carcinogenesis, which is believed to be linked to low dose radiation induced adaptive response that could protect cells from high dose radiation-induced killing. Such adaptive responses must promote repairing DNA double strand breaks (DSBs) because DSBs are the severest damage for radiation induced mammal killing. Two major kinds of DSB repair exist in mammalian cells, homologous recombination repair (HRR), and non-homologous end-joining (NHEJ). The NHEJ pathway requires Ku80, Ku70, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ligase IV, XRCC4 and Artemis. The HRR pathway requires Rad51, Rad52, Rad54 as well as the Rad51 paralogs including XRCC2, XRCC3, Rad52B, Rad51C, and Rad51D. The NHEJ pathway is not only involved in DNA repair but is also involved in the immuneresponse through regulating the variable diversity joining V(D)J recombination [17–19]. The V(D)J recombination is important for generating a diverse repertoire of T cell receptor (TCR) and immunoglobulin (Ig) molecules that are necessary for the recognition of diverse antigens including dysfunctional cells such as tumor cells [20]. It is evident that low dose radiation stimulates immunal response, which plays a positive role in preventing carcinogenesis [21]. The detailed mechanism by which low dose radiation stimulates the repair pathways needs to be elucidated in future studies.
Fhit prevents high-dose radiation promoted tumorigenesis
Since Fhit was cloned from the chromosome fragile site 3p14.2 in 1996 [22], the effects of Fhit on tumor development have been widely studied [13, 23–31]. All these data support that Fhit plays a role in preventing tumor development. Our data in this study showed that Fhit could prevent high dose radiation promoted tumorigenesis. Combining these data with our previous published data that Fhit could prevent human cells from high-doses of UV and ionizing radiation-induced HPRT mutation [15, 16], we can conclude that Fhit plays an important role in preventing DNA damage promoted tumorigenesis. The mechanism related to Fhit preventing tumorigenesis might involve its role in promoting apoptosis [31], maintaining normal checkpoint response [32, 33], regulating Cyclophilin A [34], and the Akt transduction pathway [35]. However, at this moment, we still do not understand the exact mechanism by which Fhit prevents high-dose radiation induced mutation [16] and tumorigenesis, which needs more studies in the future.
Taken together, our results indicate that low-dose radiation (0.1 Gy) at high dose rate, even with multi-exposure, would not increase the tumoriginesis, and Fhit could protect high-dose radiation-induced tumorigenesis but has no effect in a low-dose environment.
Acknowledgements
We thank Dr. Kay Huebner for the Fhit-/- mice. This work was supported by DOE grant (DE-FG02-09ER64755).
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