Supplemental vitamin A prevents the tumor-induced defect in wound healing.

To test our hypothesis that supplemental vitamin A would mitigate the impaired healing that occurs in tumor-bearing animals, six groups of C3H mice, eight per group, eating a standard commercial mouse chow ad libitum that supports normal growth, reproduction, and longevity were innoculated with 200,000 C3HBA cells. When tumors measured approximately 6 mm in diameter, the mice were anesthesized and wounded (dorsal skin incisions and subcutaneous polyvinyl alcohol sponges). Twenty-four hours later, two groups (one continued on the chow and the other started on the chow supplemented with 150,000 IU vitamin A/kg chow) underwent local tumor irradiation; two groups, one ingesting the chow, the other the vitamin A supplemented chow, were started on cyclophosphamide therapy; two groups, one ingesting the chow, the other the vitamin A supplemented chow, received neither local tumor irradiation nor cyclophosphamide therapy. An additional two groups ingesting the chow, one group neither innoculated with tumor nor wounded, the other wounded by not innoculated, served as controls. Wound breaking strength and sponge reparative collagen accumulation (assessed by hydroxyproline proline measurement) were used as indicators of wound healing. The mice were killed 12 days after wounding. Tumor presence decreased wound breaking strength and sponge hydroxyproline content; these effects were largely negated by supplemental vitamin A. Local tumor irradiation diminished the adverse effect of tumor on sponge reparative collagen content but to a lesser extent than the supplemental vitamin A. Supplemental vitamin A added to the irradiation effect on healing but irradiation did not add to the vitamin A effect. Cyclophosphamide, a systemic radiomimetic anti-tumor agent, did not alter the impaired wound healing of the tumor-bearing mice. Supplemental vitamin A mitigated the impaired wound healing in the cyclophosphamide-treated tumor-bearing mice. Supplemental vitamin A also moderated the effects of wounding, tumor, and tumor therapies (local irradiation and cyclophosphamide) on the increase in adrenal size, leukopenia, thrombocytopenia, and thymic involution (except the last was not moderated in the cyclophosphamide-treated tumor-bearing rats). The splenic enlargement in the untreated tumor-bearing wounded rats and in those treated with cyclophosphamide was lessened by supplemental vitamin A. We hypothesize that these anti-stress effects of vitamin A underlie, in part, its action in mitigating the impaired wound healing of tumor-bearing mice, including those treated by local irradiation or cyclophosphamide. These findings have implications for the care of patients with malignant tumors.

Morbidity and mortality reduction by supplemental vitamin A or beta-carotene in CBA mice given total-body gamma-radiation.

Male CBA mice received graded doses (450-750 rad) of total-body gamma-radiation (TBR) from a dual-beam 137Cs irradiator. Commencing directly after TBR, 2 days later, or 6 days later, groups of mice received supplemental vitamin A (Vit A) or beta-carotene (beta-Car), compounds previously found to reduce radiation disease in mice subjected to partial-body X-irradiation. Given directly after TBR, supplemental Vit A decreased mortality, evidenced by increases in the radiation dose required to kill 50% of the mice within 30 days (LD50/30). In one experiment, Vit A increased the LD50/30 from 555 to 620 rad; in another experiment, Vit A increased the dose from 505 to 630 rad. Similarly, in a third experiment, supplemental beta-Car increased the LD50/30 from 510 to 645 rad. Additionally, each compound increased the survival times, even of those mice that died within 30 days. In addition to reduction of mortality and prolongation of survival time, supplemental Vit A moderated weight loss, adrenal gland hyperemia, thymus involution, and lymphopenia--all signs of radiation toxicity. Delaying the supplementation for 2 days after irradiation did not greatly reduce the efficacy of Vit A; however, delaying supplementation for 6 days decreased its effect almost completely.

Supplemental vitamin A prevents the acute radiation-induced defect in wound healing.

Acute radiation injury leads to thymic involution, adrenal enlargement, leukopenia, thrombocytopenia, gastrointestinal ulceration, and impaired wound healing. The authors hypothesized that supplemental vitamin A would mitigate these adverse effects in rats exposed to acute whole-body radiation. This hypothesis was based on previous experiments in their laboratory that showed that supplemental vitamin A is thymotropic for normal rodents and lessens the thymic involution, lymphopenia, and adrenal enlargement that follows stress, trauma, and neoplasia, largely obviates the impaired wound healing induced by the radiomimetic drugs streptozotocin and cyclophosphamide, lessens the systemic response (thymic involution, adrenal enlargement, leukopenia, lymphocytopenia) to local radiation, and shifts the median lethal dose (LD50/30) following whole-body radiation to the right. To test their hypothesis, dorsal skin incisions and subcutaneous implantation of polyvinyl alcohol sponges were performed in anesthetized Sprague-Dawley rats at varying times following sham radiation or varying doses of whole-body radiation (175-850 rad). In each experiment, the control diet [which contains about 18,000 IU vit. A/kg chow (3 X the NRC RDA for normal rats)] was supplemented with 150,000 IU vit. A/kg diet beginning at, before, or after sham radiation and wounding or radiation and wounding. The supplemental vitamin A prevented the impaired wound healing and lessened the weight loss, leukopenia, thrombocytopenia, thymic involution, adrenal enlargement, decrease in splenic weight, and gastric ulceration of the radiated (750-850 rad) wounded rats. This was true whether the supplemental vitamin A was begun before (2 or 4 days) or after (1-2 hours to 4 days) radiation and wounding; the supplemental vitamin A was more effective when started before or up to 2 days after radiation and wounding. The authors believe that prevention of the impaired wound healing following radiation by supplemental vitamin A is due to its enhancing the early inflammatory reaction to wounding, including increasing the number of monocytes and macrophages at the wound site; possible effect on modulating collagenase activity; effect on epithelial cell (and possible mesenchymal cell) differentiation; stimulation of immune responsiveness; and lessening of the adverse effects of radiation.

Effect of supplemental vitamin A on the healing of colon anastomosis.

The effect of dietary supplementation with vitamin A on the healing of colon anastomoses was studied. Fifty adult male Sprague-Dawley rats were divided into two groups: (1) rats fed a standard chow which contains the equivalent of about 15 IU vitamin A/g diet; (2) rats fed the chow supplemented with an additional 150 IU vitamin A/g diet. Rats were prefed for 5 days; on Day 6 under ether anesthesia the colon was divided 1-in. distal to the ileocecal junction and then reanastomosed. The rats were maintained on the above diets for 5 days and killed on the sixth postoperative day with ether and the segment of colon containing the anastomosis was resected. In 15 rats of each group, the breaking strength of the anastomosis was measured. In the remaining 10 rats of each group, the bursting strength of the anastomotic site and a segment of normal distal colon was measured. Samples of colon from the anastomotic site and the normal segment were analyzed for hydroxyproline. There was a significant decrease in hydroxyproline content at the anastomotic site when compared to the normal distal colon segment in each group of rats (P less than 0.01). The hydroxyproline content of both normal colon and the anastomotic site was significantly higher in the vitamin A-supplemented rats than in the control diet rats (P less than 0.01). There was also a significant increase in bursting strength in the vitamin A-supplemented rats both of the anastomotic site (P less than 0.01) and of the normal colon segment (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of vitamin A and beta carotene on intra-abdominal sepsis.

Vitamin A may play a role systemically and locally in controlling intra-abdominal sepsis. Adult male rats were divided into three groups. Group 1 ate a standard rat laboratory chow (not vitamin A deficient), group 2 ate the same chow supplemented with vitamin A, and group 3 ate the chow supplemented with beta carotene. All animals underwent cecal ligation, and the cecum was perforated either with a 27-gauge or an 18-gauge needle. Vitamin A dietary supplementation had a significant protective effect, which was manifested by improved survival in the animals whose cecum was perforated with an 18-gauge needle, prevention of postoperative hypothermia, maintenance of peripheral WBC counts at normal or above-normal values, and better localization of the intra-abdominal inflammatory process. Dietary supplementation with beta carotene had a lesser protective effect.

Regression of C3HBA mouse tumor due to X-ray therapy combined with supplemental beta-carotene or vitamin A.

Male CBA/J mice, ingesting a vitamin A- and beta-carotene-sufficient laboratory chow, were inoculated in a hind limb with 2 X 10(5) C3HBA adenocarcinoma cells. When the mean tumor size was 6.2 mm, the mice were divided randomly into groups; some groups received supplemental vitamin A or beta-carotene, some received 3,000 rad local radiation to the tumor, and others received both radiation and one of the supplements. All mice that received only radiation or one of the dietary supplements died within 3 months. When local irradiation and supplemental vitamin A or beta-carotene were coupled, "complete" tumor regression occurred in every case (12/12), and tumor regrowth in and death of the mice occurred in only 1 of 12 in each of these groups during the succeeding 12 months. One year after irradiation and dietary supplementation, half the surviving mice were switched back to the control chow. During the next year, none of the mice remaining on the vitamin A or beta-carotene supplements developed tumors; however, of 6 mice switched from vitamin A, 5 had tumors that reappeared. In contrast, tumors recurred in only 2 of 6 mice after they were switched from beta-carotene. A second experiment yielded similar results. These results show that both vitamin A and beta-carotene supplementation added remarkably to the antitumor effect of local irradiation. beta-Carotene supplementation produced a greater residual antitumor action than vitamin A supplementation after the supplements were discontinued, which may have been due to greater tissue storage of beta-carotene.

Wound healing and thymotropic effects of arginine: a pituitary mechanism of action.

Supplemental dietary arginine HCl (ARG-HCl) minimizes immediate post-wounding weight loss, accelerates wound healing, and is thymotropic for uninjured and wounded rats. The present experiments were to determine if arginine-pituitary interactions underlie these effects because arginine is a growth hormone secretagogue. Effects of 1% dietary ARG-HCl supplements (0.5% added to a regular commercial rat diet containing 1.8% ARG, 0.5% in drinking water) were studied in (a) hypophysectomized (hypox) rats supplemented with ACTH, L-thyroxine, testosterone propionate, (b) such hypox rats additionally supplemented with bovine growth (hypox + bGH) hormone, (c) intact rats (Int), and (d) intact rats supplemented with growth hormone (Int. bGH). Group (a) hypox rats healed their wounds as rapidly as intact rats (dorsal skin incision breaking strength, accumulation of reparative collagen in sc polyvinyl alcohol sponges). Group (b) hypox, bGH rats showed increased wound breaking strength and accumulation of reparative collagen in the sc sponges to levels significantly greater than those of intact controls; bGH given to intact controls did not affect these indices of wound healing. Supplemental ARG-HCl given intact rats significantly minimized immediate postoperative weight loss, increased wound breaking strength and sponge reparative collagen accumulation, and increased thymic weight. None of these effects of supplemental ARG-HCl were observed in group (a) hypox rats or group (b) hypox + bGH rats. We conclude that an intact hypothalamic-pituitary axis is necessary for these beneficial effects of supplemental ARG-HCl given wounded rats.

Prophylactic and therapeutic actions of supplemental beta-carotene in mice inoculated with C3HBA adenocarcinoma cells: lack of therapeutic action of supplemental ascorbic acid.

Decreased tumor incidence, increased latent period, and increased survival time were observed in C3H/HeJ mice fed supplemental beta-carotene for 3 days and then inoculated with 10(4) C3HBA (syngeneic) tumor cells. In addition, C3H/HeJ, C3H/He, and CBA/J mice, fed supplemental beta-carotene beginning immediately after they were inoculated with 2 X 10(5) C3HBA tumor cells, showed decreased tumor growth and increased survival time. When beta-carotene was fed to mice in which palpable tumors were already present, it similarly slowed tumor growth and extended animal survival time. Ascorbic acid supplementation (5 g/kg diet), introduced into the experiment as a possible synergist for beta-carotene's antitumor action, was without therapeutic action when tested in the presence or absence of beta-carotene supplements. The basal diet, a standard commercial mouse chow, contains more vitamin A than the National Research Council's recommended dietary allowance for normal rodents and supports normal growth, reproduction, and longevity of normal mice. The work reported here is the first demonstration of the antitumor action of beta-carotene in animals with a transplanted tumor.

Moloney murine sarcoma virus tumors in CBA/J mice: chemopreventive and chemotherapeutic actions of supplemental beta-carotene.

Decreased tumor frequency, increased latent period, and increased rate of tumor regression were observed in male inbred CBA/J mice fed supplemental beta-carotene before and/or after they were inoculated with the Moloney sarcoma virus. When beta-carotene feeding was begun after tumors were already present, it markedly increased the rate of tumor regression. beta-Carotene minimized the virus-induced thymus gland involution that accompanies tumor growth, and this action on the thymus gland was believed to underlie part of beta-carotene's antitumor activity. The basal diet, a standard commercial mouse chow containing more vitamin A than the National Research Council recommends as a daily allowance for rodents, supported normal growth, reproduction, and longevity of normal mice. The work reported here is the first demonstration of the antitumor action of beta-carotene in mice inoculated with an oncogenic virus.

Decreased resistance of C3H/HeHa mice to C3HBA tumor transplants; increased resistance due to supplemental vitamin A.

Groups of inbred C3H/HeHa and C3H/HeJ mice were inoculated with either a low or a high number of C3HBA tumor cells, C3H/HeHa mice were less resistant to tumor development and growth than C3H/HeJ mice as judged by tumor incidence, latent period, tumor size (growth rate), and survival time. Resistance to decrease and death following inoculation with tumor cells was related to thymus status in the following way: Thymic involution was associated with decreased resistance of the mice to tumor development. When C3H/HeHa mice were fed supplemental vitamin A, and treatment that increases their thymus size and numbers of thymic small lymphocytes, their resistance to the C3HBA tumor was markedly increased.

Vitamin A inhibits some aspects of systemic disease due to local x-radiation.

We have previously reported that supplemental vitamin A ameliorates the stress response to a wide variety of noxious agents. The present study was carried out to determine how supplemental vitamin A influences the course of radiation sickness in C3H female mice subjected to 3000 R irradiation of one lower hind limb. All mice ingested a chow diet containing about 13,000 units of vitamin A/kg diet (about half as preformed vitamin A and half as beta-carotene) which supports normal growth, development, and reproduction of normal mice. One hundred fifty thousand units of vitamin A/kg chow was added for the vitamin A supplemented mice. All mice ate and drank ad libitum. The supplemental vitamin A feeding was begun either 3 days before radiation or immediately after radiation. There were no significant differences in the effects of these two regimens. The supplemental vitamin A prevented the weight loss, moderated the adrenal hypertrophy, prevented the thymic involution, and lessened the lymphopenia due to radiation. We conclude that supplemental vitamin A has both prophylactic and therapeutic benefits in radiation-induced disease.

Impaired wound healing in streptozotocin diabetes. Prevention by supplemental vitamin A.

Goodson and Hunt showed that wound healing is impaired in streptozotocin (Sz) diabetic rats; we speculated that this impairment results from defective early inflammatory responses to wounding. Because we had shown that supplemental vitamin A stimulates the early inflammatory response to wounding in nondiabetic rats, we studied the effect of supplemental vitamin A on wound healing in rats with Sz-induced diabetes. Male Sprague-Dawley rats were fed a commercial rat chow containing twice the amount of vitamin A recommended by the NRC for healthy rats. The rats ate and drank (tap water) ad libitum. Two-thirds of the rats were injected (intravenously) with Sz 60 mg/kg body weight. All of these rats became diabetic (hyperglycemia greater than 350 mg/dl, hyperphagic, polydipsic, polyuric, glycosuric greater than 2%). Seven days later, half of the Sz-injected rats were continued on the chow (Group 2) while the other half (Group 3) were switched to the chow supplemented with 150,000 units of vitamin A/kg chow. The next day, all were wounded (7 cm skin incisions and s.c. polyvinyl alcohol sponge implants). Similarly wounded saline injected nondiabetic rats ingesting the unsupplemented chow served as controls (Group 1). The wounds of Group 2 rats healed poorly compared to Group 1 (breaking strength of skin incisions, 308 +/- 19 g vs 584 +/- 23 g, p less than 0.001; hydroxyproline of the sponge reparative tissue, 0.87 mg vs 2.40 mg/100 mg sponge p less than 0.001). Supplemental vitamin A (Group 3) did not affect the hyperglycemia, hyperphagia, polydipsia or glycosuria, but increased the breaking strengths of the incisions of the diabetic rats (468 +/- 40 g, p less than 0.001), and the sponge hydroxyproline (2.38 mg/100 mg sponge, p less than 0.001). In another experiment, in which the wounding and start of supplemental vitamin A were delayed until 28 days after streptozotocin administration (50 mg/kg body weight), similar results were obtained. Streptozotocin diabetes also caused a decrease in the cross-linking of reparative collagen as judged by the ratio of breaking strengths of skin incisions before and after formalin fixation. Supplemental vitamin A did not influence this defect. Sz also caused peripheral lymphocytopenia, adrenal hypertrophy and thymic involution which responded to the supplemental vitamin A. Based upon experimental data and theoretical considerations we conclude Sz diabetes causes two defects in wound healing: a) quantitatively (reduction in reparative collagen accumulation) and b) qualitative reduction in the degree of cross-linking of reparative wound collagen. The action of supplemental vitamin A in correcting the impaired wound healing, adrenal enlargement, thymic involution and lymphocytopenia of Sz-diabetic rats is independent of an effect on their disturbed carbohydrate metabolism.

Thymic stimulatory actions of arginine.

Various arginine HCl supplements (0.5-3%), half added to a basal commercial rodent chow (1.8% arginine) and half to the drinking water, were given to 8- to 9-week-old male CBA/J mice for 6 days. Control animals were fed the basal chow and drank tap water. All mice ate and drank ad libitum. Weight gain and food intake were similar in all groups. All arginine supplements increased significantly: thymic weight (average 22%), thymic lymphocyte content (average 45%), and the in vitro reactivity of thymic lymphocytes judged by the incorporation of 3H-leucine into the TCA-precipitable protein fraction in response to stimulation by phytohemagglutinin and concanavalin A. All these thymic effects resulted from the 0.5% arginine hydrochloride supplement; further increases in arginine supplementation did not increase these effects. These data suggest that supplemental arginine may improve host defence mechanisms and thereby may play an important role in the care of severely injured or ill patients, since it is well established that their defense mechanisms are reduced.

Supplemental arginine increases thymic cellularity in normal and murine sarcoma virus-inoculated mice and increases the resistance to murine sarcoma virus tumor.

Arginine supplements were given to 6 week old CBA mice beginning 3 days prior to inoculation with a murine sarcoma virus, the Moloney Sarcoma Virus (MSV). Although the basal diet contained 1.8% arginine and was therefore not arginine-deficient, supplementation of the diet and the drinking water with 0.5% arginine HCl reduced tumor incidence, lengthened the latency period, decreased tumor size, and hastened tumor regression. Arginine also increased thymic weight and cellularity in normal and in MSV-inoculated mice. The antitumor action of arginine may be related to its effect on the thymus.

Arginine: an essential amino acid for injured rats.

The influence of arginine supplements on growth and healing of skin incisional wounds was studied in rats fed either a chemically defined diet lacking arginine or a laboratory chow containing 1.8% arginine. Rats fed the arginine-free diet grew more poorly than did arginine-supplemented rats (1.8 vs. 7.0 gm/day) in the preoperative period. After operation arginine-deficient animals grew very poorly (1 gm/day), while arginine-supplemented rats gained 4.3 gm/day. Arginine-deficient animals showed impaired wound healing, as judged by the breaking strengths of their incisions 10 days after wounding (228 vs. 293 gm for the arginine-supplemented rats). Arginine-deficient rats also showed decreased collagen deposition in a specific wound site, as indicated by the decreased content in hydroxyproline in sponge granulomas (2.5 vs. 4.2 mg/100 mg. of sponge for the arginine-supplemented rats). In rats fed commercial chow, 1% arginine decreased the postoperative weight loss associated with injury (0.7 vs. 5.2 gm) in one experiment and improved wound strength in two experiments (312 vs. 188 gm in one experiment and 309 vs. 246 gm in another). Arginine also increased hydroxyproline deposition in a specific wound area (5.5 vs. 4.1 mg in one experiment and 3.1 vs. 1.9 mg. in another). It is concluded that arginine has two roles in wounded animals. It is essential for the synthesis of the increased amounts of reparative collagen required for wound healing, and it decreases some of the negative aspects of the metabolic responses to injury. These are thought to be associated with an arginine-induced growth hormone release.

White cell involvement in the inflammatory, wound healing, and immune actions of vitamin A.

Stress or injury-induced phenomena, such as impaired wound healing and immune depression, may be related to impaired function of certain leukocyte populations. Since vitamin A prevents some aspects of stress, we studied its effect on various white cell populations in normal and injured rats. Supplemental vitamin A (150,000 IU/kg chow) to normal rats resulted in marked increases in thymic weight and lymphocytes without any effct on adrenal weight. The basal chow contains 13,700 IU vitamin A per kg. In rats subjected to moderately severe injury (dorsal wounding or unilateral femoral fracture), supplemental vitamin A greatly diminished the thymic involution observed in chow-fed controls and delayed or minimized the accompanying adrenal hypertrophy. In uninjured rats, supplemental vitamin A induced in three to four days a temporary circulatory leukocytosis characterized by lymhocytosis, monocytosis, and a relative neutropenia. These changes in the blood picture persisted one day after femoral fracture. On the second and third day postfracture the lymphocyte and neutrophil values returned to normal while the monocytosis persisted. Polyvinyl alcohol sponges implanted next to the fracture site demonstrated that supplemental vitamin A consistently increased the number of white blood cells migrating into the wound area and showed significantly larger numbers of monocytes/macrophages. These data suggest that vitamin A influences the numbers and nature of white cells involved in immune, inflammatory, and wound healing processes. In addition to the known antiglucocorticoid activity of vitamin A, these effects may represent a direct beneficial action of dietary vitamin A supplements for stressed and injured animals.

Antipyretic and antiviral action of vitamin A in Moloney sarcoma virus- and poxvirus-inoculated mice.

Six week-old male CBA/J mice fed a commercial powdered laboratory chow or the same chow supplemented with vitamin A palmitate (150,000 U/kg) were inoculated with either the Moloney strain of murine sarcoma virus (M-MuSV) or poxvirus. Central body temperature was measured daily. Both viruses elicited fevers, but the fevers were less pronounced and of shorter duration in the mice ingesting the vitamin A-supplemented diet. Palpable M-MuSV-induced tumors appeared later, were less frequent, grew more slowly, and were resorbed sooner in the mice fed the vitamin A supplement. Similarly, in these mice the appearance of pox lesions was delayed, their numbers reduced, and their disappearance hastened.

Influence of vitamin A on wound healing in rats with femoral fracture.

Groups of healthy wounded rats with and without comminuted femoral fractures, and maintained on nutritionally complete commercial rat chow with and without supplemental vitamin A, were studied. The test wounds were standard dorsal skin incisions and s.c. polyvinyl alcohol sponge implants. In some experiments the rats were pair-fed; the rats with femoral fracture not receiving supplemental vitamin A were the lead group for determining food allowanced. In other experiments, the rats were allowed food ad libitum. We found that wound healing of rats with femoral fracture was increased when supplemental vitamin A was given, but the supplemental vitamin A did not completely obviate the adverse effects of fracture. The ratio of the breaking strengths of the skin incisions after formalin fixation to the breaking strengths of the incisions in the fresh state was higher in the unsupplemented rats, supporting the results of our earlier experiments that vitamin A increases the rate of collagen cross-linking.