The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1661-1664

Vitamin A Inhibits Radiation-Induced Pneumonitis in Rats^1,2

Carrie A. Redlich^{*, , 3}, Sara Rockwell^**, Joyce S. Chung, Andrew G. Sikora, Marianne Kelley^**, and Susan T. Mayne

^* Section of Pulmonary and Critical Care Medicine,  Occupational and Environmental Medicine Program, Department of Internal Medicine, the Departments of  Epidemiology and Public Health and ^** Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Radiation-induced lung injury frequently limits the total dose of thoracic radiotherapy that can be delivered, and the determinants of host susceptibility are poorly understood. To test the hypothesis that vitamin A status may be an important, modifiable host determinant of radiation-induced lung injury, we determined the effect of altered vitamin A status on radiation-induced lung inflammation in rats. WAG-Rij Y rats were fed a diet deficient in or supplemented with vitamin A (0 units/kg or 80,000 units/kg diet). After 5 wk of consuming the prescribed diet, rats were irradiated with 15 Gy of 250 kV X-rays to the whole thorax. At 4-5 wk post-irradiation, there were significantly fewer neutrophils on bronchoalveolar lavage in rats fed the vitamin A-supplemented diet (8.8 ± 1.2% neutrophils) compared with those fed the vitamin A-deficient diet (20.8 ± 3.4% neutrophils, P < 0.01). At the termination of the experiment, 4-5 wk postradiation, lung retinol levels of the vitamin A-supplemented group were 19.6 ± 1.8 nmol/g, whereas those in the vitamin A-deficient group were significantly lower, 1.7 ± 0.5 nmol/g (P < 0.01). These findings suggest that supplemental vitamin A may reduce lung inflammation after thoracic radiation and be an important modifiable radioprotective agent in the lung.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Ionizing radiation is commonly used to treat many thoracic and chest wall malignancies, including primary carcinoma of the lung and breast, Hodgkin's disease and metastases to the lung (Movsas et al. 1997, Rockwell and Roberts 1998). Radiation-induced pneumonitis and subsequent pulmonary fibrosis can result in significant morbidity and mortality; this potentially life-threatening toxicity limits the dose of radiotherapy that can be administered to cancer patients and can limit the possibility of curative treatment (McDonald et al. 1995). Marked differences in host susceptibility clearly exist; clinical radiation pneumonitis is frequently unpredictable and sporadic (Rockwell and Roberts 1998). However, the determinants of these differences are poorly understood.

The most important risk factors for developing radiation pneumonitis are the dose of radiation and the volume of lung irradiated (Movsas et al. 1997, Rockwell and Roberts 1998). Chemotherapy, intercurrent infections and injury, and withdrawal of steroids may also increase the risk of radiation pneumonitis (Movsas et al. 1997). However, there remains substantial unexplained variability in individual responses to radiation. A potentially important determinant of risk may be vitamin A nutritional status.

Vitamin A (retinol) and its analogs, the retinoids, are potent regulators of epithelial proliferation and differentiation (Jetten et al. 1992, Lotan 1997). In rodents, vitamin A deficiency has been shown to increase susceptibility to lung infection (Chytil 1992) and lung cancer (Sankaranarayanan and Mathew 1996), and to increase lung toxicity from agents such as ozone (Paquette et al. 1996). Human studies have also found an association between low vitamin A intake and/or reduced serum retinol levels and increased risk of lung infection (Chytil 1992, Ross and Hammerling 1994), and reduced lung function (Paiva et al. 1996). It has been estimated that 20-40% of the U.S. population may have suboptimal vitamin A nutritional status based on below normal liver retinol stores (Raica et al. 1972, Underwood et al. 1970); our own studies have found surprisingly low levels of vitamin A in human lung tissues obtained from patients undergoing thoracotomy and lung resection (Redlich et al. 1996). This suggests that patients, especially those with lung cancer, who may undergo thoracic irradiation, may indeed have reduced lung retinol levels and may be at an increased risk of radiation-induced lung injury.

We hypothesized that altered vitamin A status could modify radiation-induced lung inflammation. To test this hypothesis, studies were undertaken to examine the effects of altered vitamin A status on radiation pneumonitis in rats. These studies demonstrate that, in this model system, a diet high in vitamin A reduces radiation pneumonitis after thoracic irradiation.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  Male WAG-Rij Y rats were bred and maintained at Yale as specific pathogen-free animals and were housed in a moderate security barrier facility throughout the experiments (Rockwell and Kelley 1989). Rats were treated with radiation in a small laboratory within this barrier facility. This species was chosen on the basis of earlier studies that established the radiation-pneumonitis model (Rockwell et al. 1995, Rosiello et al. 1993).

All studies were prereviewed and approved by the Yale Animal Care and Use Committee and were performed in full compliance with all institutional and governmental animal welfare standards.

Diets.  Animals had free access to food and hyperchlorinated water. At the time of weaning (3 wk) the rats were randomly separated into two groups. One group was fed an autoclavable vitamin A-deficient diet (ICN Biomedicals, Costa Mesa, CA); the other group received an identical diet supplemented with 80,000 unit/kg vitamin A (retinyl acetate).⁴ This dose of vitamin A was chosen because it was approximately the same amount of vitamin A found in the diet routinely used in the Yale animal facility (Prolab R-M-H 3500, Agway, Syracuse NY). Before weaning all rats had received the autoclavable Prolab R-M-H 3500 diet. All food, caging, bedding, and supplies used in the specific pathogen-free animal facility must be autoclaved to ensure the microbiological status of the animals. Autoclaving reduces the vitamin A content of the food by ~50%. Rats were weighed weekly throughout the experiments.

Radiation and experimental design.  At 8 wk of age, groups of WAG-Rij Y rats that had been fed either the vitamin A-supplemented or the vitamin A-deficient diet for 5 wk were randomly assigned to receive either sham irradiation (unirradiated) or thoracic irradiation. For irradiation, rats were anesthetized with a single intraperitoneal injection of sodium pentobarbital and positioned on their backs in a Lucite positioning jig. The thorax was irradiated with 15 Gy of 250 kV X-rays (15 mA, 2 mm A1 filtration), delivered at a dose rate of 1.28 Gy/min using a Siemens Stabilipan (Siemens AG, Erlangen, Germany) located within the barrier facility as described previously (Rosiello et al. 1993). The head and neck and the remainder of the body, including the long bones, were shielded with lead and received a dose <5% of that delivered to the lungs. Sham-irradiated rats were anesthetized and positioned as if for irradiation for a time similar to the duration of the irradiation. There were a total of 20 rats in the irradiated groups (10 fed the vitamin A-supplemented diet and 10 fed the vitamin A-deficient diet) and 15 rats in the unirradiated groups (7 fed the vitamin A-supplemented diet and 8 fed the vitamin A-deficient diet).

Assessment of radiation reactions.  Bronchoalveolar lavage (BAL) was performed as described previously (Rosiello et al. 1993) 4-5 wk after irradiation (i.e., after 9-10 wk of consuming the experimental diets). These times were chosen as a result of our more detailed studies examining radiation pneumonitis in this same experimental system (Rockwell et al 1995, Rosiello et al. 1993), which showed maximal changes in the cells recovered in the BAL fluid during this period after irradiation. Rats were given a lethal dose of sodium pentobarbital intraperitoneally (100-150 mg/rat); then a cervical incision was made and blunt dissection was performed, exposing the trachea. A 14-gauge angiocatheter was inserted into the trachea and sutured in place. Seven individual 7-mL aliquots of physiologic saline were instilled and removed. Effluent was recovered by gravity drainage, and all aliquots from a single rat were pooled. The BAL fluid was centrifuged at 400 × g for 15 min. The cell pellet was suspended in 1 mL of Ca⁺⁺/Mg⁺⁺-free Hanks' balanced salt solution (GIBCO, Grand Island, NY) and cells were counted with a hemocytometer. Differential cell counts were performed by scoring 200 cells on slides prepared by cytocentrifugation (Shandon Southern Instruments, Sewickley, PA) and stained by the Diff Quick method (Scientific Products, McGaw Park, IL). The numbers of macrophages, neutrophils, lymphocytes and eosinophils recovered are expressed as a differential percentage of total cells recovered. The absolute numbers of the different BAL cells were calculated by multiplying the total number of nucleated cells recovered by the percentages of the different cell types. Total protein in the BAL supernatant was determined by a colorimetric assay, using Coomassie Blue as the dye and bovine serum albumin as the standard (Rylatt and Parish 1982).

Nutrient level determinations.  Lung samples and a subset of all liver and serum samples were obtained. Retinol and -tocopherol levels for lung, liver and serum were determined by HPLC according to modifications of established methods (Mayne and Parker 1986). All procedures were performed under dimmed lights. Serum samples were thawed and an aliquot was removed and transferred into a screw-capped test tube. Tocol (Kodak, Rochester, NY) in 100% ethanol was added as an internal standard, and the sample was extracted twice using hexane. The hexane phase was collected, evaporated under nitrogen gas and the residue reconstituted in the mobile phase for HPLC injection.

Liver and lung samples underwent extraction and saponification as described previously (Mayne and Parker 1988). Thawed samples were minced, ground with hexane/isopropanol (3:2, v/v, containing butylated hydroxyanisole, butylated hydroxytoluene and pyrogallol as antioxidants), then filtered. Extracts were washed with sodium sulfate, then dried under nitrogen gas and mixed with ethanol containing antioxidants for saponification. Potassium hydroxide was added and samples were saponified by heating at 50°C for 8 min. The postsaponification solution was re-extracted with hexane, evaporated under nitrogen gas and the residue reconstituted in the mobile phase for HPLC injection.

The HPLC system consisted of two Rainin Rabbit-HP pumps with a Rainin Dynamax Dual Chamber mixer (Emeryville,CA), a Gilson Model 401 dilutor, a Gilson Model 231 autosampler (Middleton, WI), a Rheodyne model 7010 injection valve with a 20 µL loop (Cotati, CA), a Rainin column temperature controller, a Hewlett Packard model 1050 multiple wavelength detector (Palo Alto, CA), a Beckman DABS Ultrasphere 250 × 4.6 mm, 5µm column (Fullerton, CA) and the Rainin Dynamax HPLC Method Manager 1.2 interfaced with a dedicated Macintosh SE Computer.

The HPLC assay was performed via a gradient system beginning with 100% methanol and changing to methanol/acetonitrile/tetrahydrofuran (60:22:18, v/v/v, all HPLC grade) over a period of 25 min. The flow rate was 1.3 mL/min, and the column was thermostatically controlled at 30°C. Retinol and -tocopherol were monitored on one channel at 300 nm. Peaks were identified by comparison of retention times with those of external standards. The laboratory that performed the nutrient assays is a participating member of the National Institute of Standards and Technology Micronutrient Measurement Proficiency Testing Program.

Statistical methods.  Values are expressed as the arithmetic means ± SEM, unless otherwise noted. Statistical comparisons were made between the rats fed the supplemented and deficient vitamin A diets within radiation treatment groups. Nutrient level comparisons were also made between rats that underwent sham irradiation and irradiation within dietary groups. Nutrient levels and BAL data were assessed for significance by Student's t tests. Data were analyzed using Data Desk 5.0 (Data Description, Ithaca, NY).

	RESULTS

Abstract Introduction Methods Results Discussion References

Altered vitamin A status.  WAG-Rij Y rats with altered vitamin A status were obtained by using diets deficient in or supplemented with vitamin A. As shown in Table 1, lung retinol concentrations of rats fed the vitamin A-supplemented diet were significantly greater than those of rats fed the vitamin A-deficient diet. Liver retinol concentrations were also markedly different in the two groups, with reduced but not absent liver stores in the irradiated rats fed the vitamin A-deficient diet compared with those rats fed the vitamin A-supplemented diet. Serum retinol levels in rats fed the vitamin A-deficient diet were slightly but not significantly lower than those in the vitamin A-supplemented group, consistent with a reduced but not deficient vitamin A status. Serum -tocopherol levels did not differ between the diet groups (Table 1). Irradiation of the rats had little effect on any of the nutrient levels; nutrient levels in the unirradiated and irradiated groups fed the same diet were similar, except for elevated lung -tocopherol levels in the irradiated rats (Table 1).

View this table: [in this window] [in a new window]   Table 1. Lung, liver, and serum retinol and -tocopherol concentrations in rats fed vitamin A-deficient and -supplemented diets1

Effect of dietary change on radiation induced pneumonitis.  To determine whether the dietary modifications altered the inflammatory response in the lung after chest irradiation, rats fed the two diets were killed 4-5 weeks postradiation. As shown in Table 2, there was a significant increase in both lymphocytes and neutrophils after irradiation, as well as an increase in total BAL fluid protein. Dietary modification alone, without irradiation, had no effect on these variables (Table 2). In irradiated rats, however, there were 58% fewer BAL neutrophils in rats fed the vitamin A-supplemented diet compared with those fed the vitamin A-deficient diet. A significant reduction in lymphocytes was also seen in the irradiated rats fed the supplemented vitamin A diet compared with those fed the vitamin A-deficient diet.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The data presented here demonstrate for the first time that vitamin A supplementation may reduce the severity of radiation pneumonitis. The radiation reactions in the lungs of the rats irradiated during these experiments were similar to those we described in detail previously in WAG-Rij rats receiving radiation alone (Rosiello et al. 1993). The changes include a small initial protein leak at 24 h, followed 4-5 wk later by an inflammatory cell recruitment or alveolitis and a larger protein leak. Our earlier studies demonstrated that BAL fluid changes were more sensitive than histopathology as an indicator of pneumonitis. The lavage changes seen in rats receiving whole-thorax irradiation are similar to those described in human patients who exhibit an analogous delayed inflammatory response (Movsas et al. 1997, Rockwell and Roberts 1998.) Rats fed the diet supplemented with vitamin A showed a >50% reduction in the lung inflammatory response after chest irradiation, with a decline in inflammatory cells from 20.8 to 8.8% neutrophils.

These data are consistent with other rodent studies that have demonstrated a protective effect of vitamin A or retinoid supplementation, e.g., protection against the development of lung injury after exposure to ozone (Paquette et al. 1996) and bleomycin (Habib et al. 1993). Retinoids have also been found to protect against injury from whole-body irradiation (Seifter et al. 1984), radiation injury to the gastrointestinal tract (Mason and Tofilon 1994) and radiation injury to cells in culture (Kennedy and Krinsky 1994). The findings are also consistent with dermatologic studies demonstrating beneficial effects of retinoids in the treatment of several inflammatory and fibrotic dermatologic conditions, including psoriasis, acne, keloids and folliculitis (Futoryan and Gilchrest 1994).

Of concern is whether vitamin A supplementation might enhance tumor development or interfere with the efficacy of radiotherapy in treating the tumor. Vitamin A and its analogs have been shown to suppress lung carcinogenesis in animal models and to prevent upper aerodigestive tract cancers, including secondary lung cancers, in some human chemopreventive trials (Lotan 1996). However, two recent large controlled clinical chemoprevention trials found an increased risk of lung cancer in subjects supplemented with -carotene alone (The Alpha-Tocopherol, -Carotene Cancer Prevention Study Group 1994) or in combination with vitamin A (Omenn et al. 1996). This question therefore remains unresolved.

Irradiation itself had no effect on lung, liver or serum retinol levels. Higher lung -tocopherol levels were seen in irradiated compared with unirradiated rats, with no significant differences in serum or liver -tocopherol levels. This effect was not related to vitamin A status because lung -tocopherol levels did not differ between the low and high vitamin A groups. The reason for this finding is unclear. It has been shown that after ozone and nitrogen dioxide exposure, the vitamin E content in the lung increases, possibly due to increased mobilization of vitamin E to the lung in response to oxidant exposures (Elsayed 1993). Thoracic irradiation may result in a similar increased -tocopherol uptake by lung tissue.

Despite the great interest in the role of dietary factors such as vitamin A as determinants of host responses, surprisingly little is known about the storage and metabolism of this and other nutrients in nonhepatic tissues, including the lung (Blaner and Olson 1994). Because plasma vitamin A levels are maintained within a fairly narrow range despite large fluctuations in dietary vitamin A intake and tissue stores, serum vitamin A levels are poor indicators of total body vitamin A status, making it difficult to evaluate vitamin A status in humans (Gerster 1997). Lung retinol levels obtained in the rats fed the two diets were compared with the limited data available on human lung levels (Redlich et al. 1996). Rats fed the vitamin A-supplemented diet had lung retinol levels that were markedly higher (18.9 ± 2.6 nmol/g lung) than those of patients undergoing thoracic surgery (0.52 ± 0.21 nmol/g lung; n = 21). Rats fed the vitamin A-deficient diet had lung retinol levels (2.7 ± 0.6 nmol/g lung) similar to, but still higher than levels measured in human lung tissue, suggesting that lung retinol stores in patients undergoing thoracic surgery may be very low, comparable to those in the rats fed the vitamin A-deficient diet. These data raise the possibility that patients undergoing chest irradiation might benefit from vitamin A supplementation before and during radiotherapy.

Whether rodent models accurately reflect human vitamin A metabolism and storage is an important question that merits further investigation. Our findings suggest that vitamin A-supplemented rodents may not be an optimal animal model with which to investigate the effects of radiation and other toxic exposures in humans. The investigation of interspecies differences in vitamin A storage and metabolism and the use of animal diets that more closely reflect the nutritional intakes of the relevant human populations should be explored.

Further studies are required to understand more fully the mechanisms by which vitamin A plays the protective role demonstrated here. The actions of retinoids, such as promoting cell differentiation, are believed to be mediated by different classes of retinoic acid nuclear receptors that are expressed in a number of tissues including the lung, and that bind retinoic acid and regulate the transcription of a number of retinoid responsive genes (Lotan 1997). Retinoids may also exert their effects through the modulation of inflammatory cytokines and growth factors. Our own studies have shown that retinoids inhibit fibroblast interleukin-1-induced interleukin-6 and interleukin-8 production (Redlich et al. 1993, Zitnik et al. 1994).

These data suggest that additional studies are required to ascertain whether vitamin A levels are important determinants of the severity of lung injury after radiotherapy and/or after other pneumotoxic exposures. Whether dietary supplementation with vitamin A can protect human cancer patients from the lung injury and perhaps other toxicities produced by radiotherapy and/or chemotherapy warrants further investigation.

	FOOTNOTES

Manuscript received 23 February 1998. Initial reviews completed 9 March 1998. Revision accepted 23 June 1998.

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