Abstract

Data from the National Lung Screening Trial suggested that annual computed tomography (CT) screening of at-risk patients decreases lung cancer mortality by 20%. We assessed the effects of low-dose CT radiation in mice exposed to 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) to mimic the effects of annual CT screening in heavy smokers and ex-smokers. A/J mice were treated at 8 weeks with NNK followed 1 week later by 4 weekly doses of 0, 10, 30 or 50 mGy of whole-body CT and euthanized 8 months later. Irradiated mice exhibited significant 1.8- to 2-fold increases in tumor multiplicity in males (16.1±0.8 versus 9.1±1.5 tumors per mouse; P < 0.0001) and females (21.6±0.8 versus 10.5±1.4 tumors per mouse; P < 0.0001), respectively, compared with unirradiated mice with no dose effect observed; female mice exhibited higher sensitivity to radiation exposure than did males ( P < 0.0001). Similar results were obtained when tumor area was determined. To assess if the deleterious effects of radiation could be prevented by antioxidants, female mice were fed a diet containing 0.7% N -acetylcysteine (NAC) starting 3 days prior to the first CT exposure and continuing for a total of 5 weeks. NAC prevented CT induced increases in tumor multiplicity (10.5±1.2 versus 20.7±1.5 tumors per mouse; P < 0.0001) back to levels seen in NNK/unirradiated mice (10.5±1.2). Our data suggest that exposure of sensitive populations to CT radiation increases the risk of tumorigenesis, and that antioxidants may prevent the long-term carcinogenic effects of low-dose radiation exposure. This would allow annual screening with CT while preventing the potential long-term toxicity of radiation exposure.

Introduction

Lung cancer is the leading cause of cancer-related deaths in the USA with <15% of patients surviving to 5 years following diagnosis ( 1 ). The poor survival statistics are due in part to the presentation of patients with late stage of disease, which often shows invasion and metastases at the time of diagnosis with poor chances for curative therapies. Unlike breast, prostate and colon cancer, no early detection tests have been developed that could identify lung lesions at Stage I, when 5 year survival increases to nearly 60% ( 2 ).

Recent studies ( 3–7 ) have suggested that use of annual, low-dose X-ray computed tomography (CT) screening of asymptomatic individuals at high risk for the development of lung cancer—namely heavy current and former smokers with ≥30 pack years of smoking history—could identify early stage lesions and decrease the high mortality rates in patients diagnosed with lung cancer. The largest of these trials, the National Lung Screening Trial (NLST) ( 8 ), enrolled >53 400 patients with 26 700 patients each in the CT and standard chest X-ray arms of the trial. This trial reported a 20% reduction in the mortality rate in patients enrolled in the CT screening arm, providing provocative data suggesting that an early detection test for lung cancer using CT should be implemented.

However, there are concerns that low-dose CT screening protocols may not result in overall decreases in mortality from lung cancer ( 9 ). A high rate of false-positive findings, which exceeded 95% in the NLST study, was observed, which subjected patients to further testing. Another concern is the lack of information on the long-term toxicity of exposure to low-dose radiation. The use of CT has increased >20-fold during the last 30 years, and it has been reported that approximately 29 000 excess cancers are caused by exposure to CT radiation in the USA each year ( 10–12 ). This has led to considerable concern because of the potential carcinogenic risk from these CT exposures and an estimated increase in procedure-related cancers of 0.5–5% ( 13–15 ).

As a consequence of prolonged exposure to environmental carcinogens, a “field cancerization” effect occurs in the lung ( 16 , 17) resulting in an extended region of initiated but morphologically normal cells in the damaged epithelium of current and former smokers. Continued exposure of the lung parenchyma to environmental toxicants results in further damage to the initiated cells; it is from this population of damaged cells that lung tumors are thought to be derived. Thus, annual CT scans of older current and former heavy smokers carries the risk of either initiating normal cells to become tumor cells or promoting the growth of already initiated cells into tumors. This conundrum highlights the need to better define the carcinogenic risk associated with annual CT screening of current and former smokers.

In order to assess the long-term effects of radiation exposure from clinically relevant low-dose CT, we initiated studies in which two different murine models of lung cancer were subjected to multiple whole-body CT doses approximating the NLST screening protocol. Utilizing a transgenic model in which the mutant human Ki- rasG12C gene is expressed in the lung from a doxycycline-inducible promoter, we previously demonstrated that mice expressing the mutant RAS transgene exhibited a 43% increase in lung tumor multiplicity when exposed to low-dose CT radiation ( 18 ), an effect not seen in control irradiated mice, which did not express the mutant gene. This suggests that individuals previously sensitized to tumor formation as a result of prior exposure to the carcinogens contained in cigarette smoke could be at greater risk for radiation-induced tumor formation. In this study, we utilized a chemical model of lung carcinogenesis in which A/J mice were treated with the tobacco-specific carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and exposed to a similar CT screening protocol as were the transgenic mice. In addition, as radiation has been shown to cause oxidative stress and the production of reactive oxygen species (ROS) ( 19 , 20) , we also determined the effects of concurrent treatment with an antioxidant to prevent radiation-induced cancer formation. In this study, we report that mice treated with NNK and exposed to low doses of CT radiation exhibited 1.8- to 2-fold increases in tumor multiplicity and tumor area compared with unirradiated mice with no dose-response effect observed. Female mice were more sensitive to the tumor enhancing effects of low-dose CT radiation than were male mice. When female mice were treated concurrently with the antioxidant N -acetylcysteine (NAC), the radiation-mediated increase in tumor multiplicity was prevented, suggesting that pre- and/or co-treatment with an antioxidant may prevent the long-term carcinogenic effects of low-dose radiation exposure. This will allow for annual screening of high-risk patients with CT while preventing the potential long-term toxicity of radiation exposure.

Materials and methods

Animal treatment and histological analysis of lungs

All animal studies were conducted under a protocol approved by the institutional animal care and use committee. Seven-week-old female A/J mice were obtained from The Jackson Laboratory and allowed to acclimate to the animal facility for 1 week. The mice were divided into four groups consisting of 20 mice per group for each sex. At 8 weeks of age, the mice were treated intraperitoneally with a single dose of 100mg/kg of NNK dissolved in saline using the protocol described by Lu et al. ( 21 ), which results in a 100% tumor incidence. One week after treatment with NNK, mice were irradiated using a clinical multi-row detector (eight-slice) helical CT scanner with 4 weekly doses of 0, 10, 30 or 50 mGy of whole-body 100 kVp radiation for total radiation doses of 0, 40, 120 and 200 mGy. An additional two groups of female mice comprising the radioprotective arm of the study were co-treated for 5 weeks with NAC at 0.7% (wt/wt) in the AIN-76A diet beginning 3 days prior to CT irradiation and continuing for a total of 5 weeks throughout the CT exposure period ( Figure 1 ). Dosimetric parameters measurements were determined as described previously ( 18 ). These mice were treated with either 0 or 200 mGy total dose of CT radiation as described above. The NAC containing AIN-76A diet was formulated by the Wake Forest University Diet Core Laboratory.

Fig. 1.

Experimental timeline. At 8 weeks of age, mice were treated with 100mg/kg of NNK. One week after treatment with NNK, mice were anesthetized and either sham irradiated or irradiated once per week for 4 weeks with 10, 30 or 50 mGy of 100 kVp X-rays from a clinical helical CT scanner. Mice in the chemopreventive arm of the experiment were co-treated with 0.7% (wt/wt) of NAC in the diet starting 3 days before the first fraction and continuing for 5 weeks. Mice were euthanized at 8 months following the last fraction of radiation, at which time the lung tissue was removed and tumor multiplicity and area determined.

Fig. 1.

Experimental timeline. At 8 weeks of age, mice were treated with 100mg/kg of NNK. One week after treatment with NNK, mice were anesthetized and either sham irradiated or irradiated once per week for 4 weeks with 10, 30 or 50 mGy of 100 kVp X-rays from a clinical helical CT scanner. Mice in the chemopreventive arm of the experiment were co-treated with 0.7% (wt/wt) of NAC in the diet starting 3 days before the first fraction and continuing for 5 weeks. Mice were euthanized at 8 months following the last fraction of radiation, at which time the lung tissue was removed and tumor multiplicity and area determined.

Mice were euthanized 8 months following the last fraction of radiation by CO 2 asphyxiation and either cervical dislocation or thoracotomy. The lungs were removed, carefully examined for pulmonary masses and all macroscopic pulmonary lesions were recorded and size determined with electronic calipers. Tumor multiplicity was determined from these macroscopically visible surface lesions. The lung tissue was processed for histopathology by fixation in methacarn (methanol/chloroform/acetic acid, 6:3:1) for 48h, embedded in paraffin and prepared for routine microtomy. Tissue slices cut 4 microns thick were stained with hematoxylin and eosin and used to determine the stage of microscopic lesions throughout the lung (hyperplasia, adenoma or adenocarcinoma). Lesion stage was determined from at least five slides taken from individual mice (one slide per mouse) within each group and sex and was conducted with the pathologist blinded to the identity of the slides being examined. Proliferative lesions were classified with respect to standard murine pulmonary tumor characteristics ( 22 ).

Statistical analysis

Lung lesions presented as individual (non-coalesced) or multiple tumors consisting of at least two tumors that grew together (coalesced). Our initial analyses utilized non-coalesced tumors as the coalesced tumors accounted for only 13, 10, 9 and 10% of the lesions from mice exposed to 0, 40, 120 and 200 mGy, respectively, and 20% of the lesions in both groups of mice treated with NAC.

The analysis of non-coalesced tumors was done by evaluating the three outcomes of tumor multiplicity, total tumor area and tumor size. Tumor multiplicity is defined as the number of observed tumors within a mouse. Total tumor area (mm 2 ) was calculated by multiplying the length and width (length and width are the same for non-coalesced tumors) of each tumor and summing all tumors within each mouse. Tumor size (mm) describes the diameter of a tumor. Descriptive statistics were initially used to describe tumor multiplicity and total tumor area by treatment group. Two-sided t -tests were used to make comparisons, which involved the NAC only and 200 mGy/NAC groups and were restricted to female mice only. To make comparisons between treatment groups of different radiation doses and genders, a linear model was used. Model adjusted means were used to describe the effects seen in the different treatment arms.

To determine the effects of treatment on tumor size for non-coalesced lesions, a generalized estimating equations longitudinal model (to account for multiple tumors within a mouse) with a multinomial distribution was used to model the effects of the different treatment groups on the ordinal tumor size outcome. For tumor stage, a Fisher’s exact test comparing all groups and all stages was used.

In secondary analyses, coalesced tumors were included in the area, and tumor multiplicity estimations and the same analysis methods were used. For the secondary analysis of tumor multiplicity, each coalesced tumor was counted as two tumors because at least two smaller tumors grew together to form the larger tumor. As similar results were obtained with and without including coalesced tumors in the analyses, results are reported for non-coalesced tumors only unless otherwise specified.

All values are expressed as either the least squares mean (when modeling interactions for gender and radiation dose) or raw mean ± standard error of the mean. Analyses were done with SAS 9.2 (SAS Institute, Cary, NC).

Results

NNK-treated mice represent the population of heavy ex-smokers who are asymptomatic but contain occult lesions that could develop into overt lung tumors. In order to determine the effects of exposure to clinically relevant low-dose CT radiation on this sensitized population, mice were treated with a single 100mg/kg dose of NNK followed by 4 weekly CT scans and euthanized 8 months after the last fraction of radiation. Unirradiated control mice and irradiated/NNK-naïve mice were not included in the study as A/J mice exhibit a very low tumor incidence in the absence of chemical treatment ( 21 , 23 , 24) . Our prior studies with a transgenic mouse model found no effect of radiation in irradiated mice that did not express the mutant Ki- ras transgene ( 18 ). In addition, a comprehensive review of radiation exposure studies in animal models ( 25 ) describes the lack of effect of radiation on murine lung cancer formation at dose below 2 Gy (see Table 32 in reference 25).

As the tumor incidence of NNK-treated mice in this study was 100%, we determined the effects of different doses of radiation on the tumor multiplicity, tumor area and tumor size of the macroscopically visible surface tumors. In our initial analysis, we combined the data from male and female mice. As shown in Figure 2 , mice treated with NNK and subsequently exposed to radiation exhibited approximately 2-fold increases in tumor multiplicity ( Figure 2A ; P < 0.001) and tumor area ( Figure 2B ; P < 0.005) with no dose-response effect observed. As an overwhelming number of lesions was <2mm in size (<10% of lesions overall and in each individual group were >2mm), we divided our assessment of tumor size into three categories: (i) <0.5mm, (ii) 0.5 to <1mm and (iii) ≥1mm ( Table I ). Tumors in the radiation-treated groups were significantly smaller than those from unirradiated mice ( P ≤0.0001); however, there were no radiation-mediated differences in the morphology or relative stage of the lesions as determined from microscopic slides of tumor sections ( Table II ).

Table I.

Distribution of tumor sizes in irradiated and non-irradiated mice (%)

 No radiation No radiation + NAC  40 mGy a  120 mGy b  200 mGy b  200 mGy + NAC c 
<0.5 mm 18.6 14.3 33.2 37.0 41.8 10.1 
0.5 to <1 mm 47.3 38.8 43.9 40.9 34.4 38.0 
≥1 mm 34.1 46.9 22.9 22.0 23.8 52.0 
 No radiation No radiation + NAC  40 mGy a  120 mGy b  200 mGy b  200 mGy + NAC c 
<0.5 mm 18.6 14.3 33.2 37.0 41.8 10.1 
0.5 to <1 mm 47.3 38.8 43.9 40.9 34.4 38.0 
≥1 mm 34.1 46.9 22.9 22.0 23.8 52.0 

All mice were treated with 100mg/kg of NNK. Lesion size was determined by measuring the diameter of visible surface tumors at their longest length with electronic calipers. Data shown are combined for males and females as they exhibited similar tumor distributions. Groups treated with NAC consisted only of female mice. There were no gender-related differences in tumor diameter. Radiation dose is expressed in total milligray (mGy) radiation received from the four fractions.

a Significantly different from 0 mGy control, P = 0.0001.

b Significantly different from 0 mGy control, P < 0.0001.

c Significantly different from 0 mGy control, P < 0.003.

Table II.

Histological stage of the mouse lung lesions

 No radiation No radiation + NAC 40 mGy 120 mGy 200 mGy 200 mGy + NAC 
Hyperplasias (%) 85 94 96 91 82 79 
Adenomas (%) 15  6  4  6 15 21 
Adenocarcinomas (%)     3  3  
 No radiation No radiation + NAC 40 mGy 120 mGy 200 mGy 200 mGy + NAC 
Hyperplasias (%) 85 94 96 91 82 79 
Adenomas (%) 15  6  4  6 15 21 
Adenocarcinomas (%)     3  3  

All mice were treated with 100mg/kg of NNK. Lesion stage was determined from a total of at least five slides from each group and sex, with each slide obtained from an individual mouse. Data for male and female mice from each group were combined as they exhibited similar tumor distributions. Radiation dose is expressed in total mGy radiation received from the four fractions. A Fisher’s exact test comparing all groups and all stages found no significant differences between groups ( P = 0.29).

Fig. 2.

Low-dose CT radiation increases lung tumor development in NNK-treated mice. ( A ) Least squares mean number (± standard error of the mean) of tumors per mouse for each total dose level for male and female mice combined. Radiation exposure caused a significant increase in tumor multiplicity ( P < 0.001) independent of dose. ( B ) Least squares mean (± standard error of the mean) of tumor area (mm 2 ) for each total dose level for male and female mice combined. Radiation exposure caused a significant increase in tumor area ( P < 0.005) independent of dose.

Fig. 2.

Low-dose CT radiation increases lung tumor development in NNK-treated mice. ( A ) Least squares mean number (± standard error of the mean) of tumors per mouse for each total dose level for male and female mice combined. Radiation exposure caused a significant increase in tumor multiplicity ( P < 0.001) independent of dose. ( B ) Least squares mean (± standard error of the mean) of tumor area (mm 2 ) for each total dose level for male and female mice combined. Radiation exposure caused a significant increase in tumor area ( P < 0.005) independent of dose.

Because there was no dose-response effect seen with different doses of radiation, we combined all the dose groups and compared the effects of low-dose CT radiation on male and female mice. We did not detect any gender difference in either tumor multiplicity (10.5±1.4 tumors per mouse in females versus 9.1±1.5 tumors per mouse in males) or tumor area (9.4±1.3mm 2 in females versus 7.0±1.4mm 2 in males) between male and female unirradiated mice of the macroscopically visible surface tumors. However, upon exposure to CT radiation, female mice exhibited significantly higher values for both tumor multiplicity (21.6±0.8 versus 16.1±0.8 tumors per mice; P < 0.0001) and tumor area (13.6±0.8 versus 10.7±0.8mm 2 ; P = 0.01) relative to male mice ( Figure 3 ). Although data shown in Tables I and II combine both male and female mice, separate analyses found that gender had no effect on the tumor size of visible surface lesions, lesion morphology, and distribution of lesions by stage as determined from microscopic slides.

Fig. 3.

Higher sensitivity of female mice to low-dose radiation-mediated carcinogenesis. Least squares mean number (± standard error of the mean) of tumors per mouse in irradiated versus unirradiated mice for both female and male mice. Radiation exposure caused a greater increase in ( A ) tumor multiplicity ( P < 0.0001) and ( B ) tumor area ( P = 0.01) in female mice. Bars with different letters are significantly different from one another.

Fig. 3.

Higher sensitivity of female mice to low-dose radiation-mediated carcinogenesis. Least squares mean number (± standard error of the mean) of tumors per mouse in irradiated versus unirradiated mice for both female and male mice. Radiation exposure caused a greater increase in ( A ) tumor multiplicity ( P < 0.0001) and ( B ) tumor area ( P = 0.01) in female mice. Bars with different letters are significantly different from one another.

Because radiation can cause oxidative stress and enhance the formation of ROS, we next determined whether the low-dose radiation-mediated increases in tumor formation could be prevented by concurrent treatment with an antioxidant. We chose NAC as this agent has been shown to inhibit lung tumor formation in mice ( 26–29 ) and to attenuate increases in ROS and subsequent DNA damage by a variety of environmental stressors ( 30 , 31) . In addition, NAC is a relatively inexpensive nutriceutical that is readily available over the counter. Because female mice were the most sensitive to the effects of low-dose CT radiation, we treated two additional groups, one unirradiated and one exposed to 200 mGy of total CT radiation, with 0.7% (wt/wt) NAC in the diet starting 3 days prior to the first radiation fraction and continuing for 5 weeks (approximately 5 days after the last fraction of radiation). Results shown in Figure 4 demonstrate that NAC significantly and completely inhibited the increase in visible surface tumor formation in irradiated mice (20.7±1.5 versus 10.5±1.2 tumors per mouse in irradiated versus NAC/irradiated mice; P < 0.0001) to tumor multiplicities observed in the unirradiated control groups (10.5±1.1 and 9.8±0.6 tumors per mouse in NNK and NNK/NAC groups, respectively). The decrease in non-coalesced tumor area, however, did not reach statistical significance (14.0±0.9mm 2 in irradiated mice versus 12.6±1.6mm 2 in irradiated/NAC mice; P = 0.46) as shown in Table III . In addition, there was no significant difference in tumor area between the irradiated/NAC mice and the unirradiated 0 mGy controls. Similar results were obtained when coalesced tumors were included in the analysis (24.5±1.9 versus 22.6±2.2mm 2 in irradiated versus irradiated/NAC mice; P = 0.52). No difference in tumor area was noted when comparing NNK treatment to NNK/NAC-treated mice in the unirradiated controls, although tumor area in the irradiated/NAC mice remained significantly greater relative to the 0 mGy controls without NAC ( P < 0.05; Table III ). The lack of a significant effect of NAC on the tumor area is due to a small but significant increase in tumor size in the 200 mGy/NAC-treated group, which contained significantly more lesions ≥1mm relative to the 0 mGy control group ( Table I ). NAC co-treatment had no effect on the morphology or histological staging of the lesions ( Table II ).

Table III.

Tumor area (mm 2 ) in female mice treated or untreated with NAC

 0 mGy 0 mGy/NAC 200 mGy 200 mGy/NAC 
Non-coalesced  9.4±1.0 10.4±1.4  14.0±0.9 a,b 12.6±1.6 
Non-coalesced and coalesced 15.8±2.0 18.2±2.1  24.5±1.9 a,c  22.6±2.2 d 
 0 mGy 0 mGy/NAC 200 mGy 200 mGy/NAC 
Non-coalesced  9.4±1.0 10.4±1.4  14.0±0.9 a,b 12.6±1.6 
Non-coalesced and coalesced 15.8±2.0 18.2±2.1  24.5±1.9 a,c  22.6±2.2 d 

All mice were treated with 100mg/kg of NNK. Total surface tumor area for non-coalesced lesions only and combined non-coalesced and coalesced lesions was calculated by multiplying the length and width of each tumor and summing all tumors within each mouse.

aP < 0.005 when compared with 0 mGy control group.

bP < 0.05 when compared with 0 mGy/NAC group.

cP = 0.054 when compared with 0 mGy/NAC control group.

dP < 0.05 when compared with 0 mGy control group.

Fig. 4.

Protective effects of NAC on low-dose radiation-mediated carcinogenesis. Mean number (± standard error of the mean) of tumors per mouse in female mice ± 200 mGy of total radiation and ± treatment with 0.7% NAC in the diet. NAC caused a significant decrease in tumor multiplicity in the irradiated mice back to levels observed in unirradiated mice (0 mGy, P < 0.005; 0 mGy/NAC, P < 0.05).

Fig. 4.

Protective effects of NAC on low-dose radiation-mediated carcinogenesis. Mean number (± standard error of the mean) of tumors per mouse in female mice ± 200 mGy of total radiation and ± treatment with 0.7% NAC in the diet. NAC caused a significant decrease in tumor multiplicity in the irradiated mice back to levels observed in unirradiated mice (0 mGy, P < 0.005; 0 mGy/NAC, P < 0.05).

Discussion

Estimates of the carcinogenic potential of low-dose radiation (<100 mGy) in the range emitted from medical devices was addressed by the National Academy’s Seventh Committee on the Biological Effects of Ionizing Radiation ( 19 ). This report extrapolated risk estimates based on the Japanese atomic bomb survivors and assumed that the effects of ionizing radiation followed a Linear No-Threshold (LNT) model, with radiation acting as an initiator of cancer as a result of stochastic effects, such as DNA damage. However, the committee also pointed out that genetic susceptibility to radiation-mediated damage could alter the risk for cancer induction. This suggests that, in susceptible populations, low doses of radiation could act as a tumor promoter and once promotion has occurred, additional increases in the radiation dose may not increase the risk further ( 32–35 ). It is thus possible that, in this scenario, the LNT model would not be able to accurately predict the risk of cancer from low-dose exposures ( 15 , 36 , 37) . Our results suggest that the LNT model may not be applicable to susceptible populations exposed to the low-dose, low-energy radiation emitted from CT scanners.

Although mice and humans differ in their relative sensitivity to ionizing radiation, research conducted over several decades has clearly shown that rodent models have generated radiobiological data of direct relevance to humans. Indeed, mice demonstrate similar responses to radiation in terms of types of morphological lesions and genetic damage induced following exposure to low- and high-dose radiation. Studies of rodent models have generated mechanistic insights that continue to be translated to the human population in terms of normal tissue response ( 38 , 39) , effects of low-dose radiation ( 20 , 40) and carcinogenesis ( 41 ).

Because it is not possible to obtain this type of data from human studies without first placing individuals on a CT screening protocol and assessing tumor incidence 20 years later, we utilized a well-characterized mouse model ( 21 , 42) for lung cancer in which mice were treated with the tobacco-specific carcinogen NNK and were placed on a CT screening protocol that approximated the NLST screening trial. Using 4 weekly radiation doses of 10, 30 and 50 mGy, we found that exposure to radiation (i) increased the tumor multiplicity and tumor area by 1.5- to 2-fold in irradiated versus unirradiated mice ( Figure 2 ), (ii) exhibited no dose-response effect, (iii) had no effect on tumor morphology or tumor stage ( Table II ) and (iv) caused significantly greater increases in tumor formation in female as compared with male mice ( Figure 3 ).

It was noted that lesions from irradiated mice were significantly smaller compared with those in non-irradiated mice. This suggests that low-dose CT may be promoting the growth of a distinct set of lesions that would not otherwise have developed into tumors in the absence of the radiological stimulus. These lesions may have a slight growth disadvantage relative to those initially induced by NNK. For example, low-dose radiation might not only induce new lung lesions but also exert growth inhibitory effects by temporarily inhibiting cell cycle progression. This could occur as a result of the up-regulation of tumor suppressor genes following radiation-induced damage ( 35 , 43 , 44) . These radiation-induced tumors would thus tend to be smaller than those resulting from treatment with NNK, resulting in the shift of lesion size to smaller diameter tumors. Overall, these results suggest that the high-risk group of current and former heavy smokers, who would most probably be entered into annual CT screening protocols, may be at greater risk for the long-term carcinogenic effects of radiation exposure than the general population even at these low clinical doses of radiation.

Previous studies from our laboratory found similar results using a transgenic mouse model in which the mutant human Ki- rasG12C was conditionally expressed in the lung from a tetracycline-inducible promoter ( 18 ). Exposure to low-dose CT radiation at total doses ranging from 80–160 mGy resulted in a 43% increase in tumor multiplicity in irradiated mice with no dose-response effects. No effects were seen on morphology, and there were significantly higher radiation-mediated increases in tumorigenesis in the female mice. In that study, we also examined the effect of radiation on mice that were not treated with doxycycline and hence did not express the mutant RAS gene. Low-dose CT radiation appeared to have little or no effect in this population, providing further support for the hypothesis that these levels of clinically relevant radiation from diagnostic medical devices may cause tumor formation in individuals who are highly susceptible as a result of genetic polymorphisms or prior environmental exposures ( 35 , 41 , 44–47 ). In addition, studies by Grudzenski et al. ( 48 ) have shown that irradiation of murine fibroblasts with 10 mGy of 90kV X-rays failed to stimulate repair of DNA double-stand breaks , which did occur at doses ≥100 mGy, raising the possibility that lack of adequate DNA repair, especially in sensitive individuals, could lower the threshold for tumor formation.

Finally, because radiation can increase the production of free radicals that can damage DNA, we tested the ability of NAC as a potential inhibitor of the radiation-mediated increases in lung tumor formation. Our results demonstrate that pre- and concurrent treatment with NAC completely inhibited tumor multiplicity back to levels seen in unirradiated mice ( Figure 4 ). However, NAC did not cause a statistically significant decrease in tumor burden ( Table III ). The significance of this finding is not clear at this time. We had anticipated that the short 5 week period of NAC treatment would have prevented the radiation-mediated increase in tumor formation but not the effects on tumor growth, as mice co-treated with 200 mGy of radiation and NAC exhibited a small but significant increase in tumor size ( Table I ). The mechanism by which the presence of NAC during the early phases of tumorigenesis helps to promote increased growth of some tumors remains to be determined.

One possibility is that NAC may provide a protective effect, whereby scavenging of radiation-induced ROS that may form during early tumor development allows some tumors to expand more rapidly over the 5 week treatment period. Once NAC is withdrawn, this enhanced tumor growth may once again be restrained by normal cellular mechanisms, thus accounting for the limited increase in tumor size and minimal effects on tumor area. Recent studies have shown, for example, that irradiation of breast cancer cells increases manganese superoxide dismutase activity ( 49 ), which has been shown to inhibit cell proliferation in various cancer cell lines ( 49–51 ). The lack of effect of irradiation and/or NAC treatment on tumor stage ( Table II ) suggests that all the lesions are fairly uniform in terms of their differentiation status and thus probably equivalent in terms of their malignant potential. A definitive assessment requires further genetic testing of the individual tumors. As reviewed in De Flora et al. ( 30 ), several studies have shown that the ability of NAC to scavenge ROS is due to the molecule’s intrinsic chemistry and its ability to stimulate the synthesis of glutathione. It has been well established in a variety of in vivo and cell culture systems that NAC can directly scavenge ROS ( 52 , 53) and increase the levels of glutathione ( 54–62 ) with a concomitant increase in the levels of cysteine ( 56 , 63 , 64) and manganese superoxide dismutase (MnSOD) ( 65–70 ). Secondary to these antioxidant and ROS scavenging effects, NAC treatment results in alterations of gene expression for a variety of signaling pathways, which influence proliferative, apoptotic, DNA repair, invasive and metastatic pathways ( 30 ). Which of these functions of NAC can account for its observed effects in this study remain to be determined.

In summary, our results demonstrate, in a murine chemical lung cancer model, that exposure to low doses of CT radiation that approximate those of the NLST trial increase tumor formation in the sensitized population of mice previously exposed to a tobacco-specific carcinogen. Pre- and concurrent treatment with an antioxidant prevents the radiation-mediated increase in tumor multiplicity. These data also support our previous suggestion that the LNT model of radiation-induced carcinogenesis may not apply to low levels of radiation exposure in cancer-prone populations. With the importance of CT screening in diagnostic procedures, the protective effects observed with NAC in our study suggest that susceptible populations may be screened with CT without the risk of future radiation-induced damage.

Funding

National Cancer Institute (grant R01-CA136910 to M.T.M.); the Wake Forest University Cancer Center Support Grant (P30-CA12197) and a partner grant from the Comprehensive Cancer Center and the Department of Radiation Oncology at the Wake Forest University School of Medicine.

Conflict of Interest Statement: None declared.

Acknowledgements

A preliminary report of this work was presented at the 51st annual meeting of the Society of Toxicology, 11–15 March 2012, San Francisco, CA ( Toxicological Sciences, Supplement (Toxicologist) 126(1-S): 78, 2012).

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Abbreviations:

    Abbreviations:
  • CT

    computed tomography

  • LNT

    Linear No-Threshold model

  • NAC

    N -acetylcysteine

  • NNK

    4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone

  • NLST

    National Lung Screening Trial

  • ROS

    reactive oxygen species.