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.

Vitamin A and retinoic acid: induced fibroblast differentiation in vitro.

The role of vitamin A in wound healing and fibroplasia has been studied extensively in vivo but the mechanism(s) of its action has not been established. In this study the effect of vitamin A and retinoic acid on fibroblast growth and collagen accumulation in vitro was examined. Vitamin A and retinoic acid added to Balb 3T3 mouse fibroblasts in tissue culture resulted in induction of cell differentiation as manifested by a decrease in cell growth rate, enhanced collagen accumulation, and morphologic differentiation. The results of this in vitro study suggest that the stimulatory in vivo effect of vitamin A and retinoic acid on collagen accumulation and fibroplasia in healing wounds is due in a major way to fibroblast differentiation and enhanced collagen synthesis.

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.

Carnitine biosynthesis from gamma-butyrobetaine and from exogenous protein-bound 6-N-trimethyl-L-lysine by the perfused guinea pig liver. Effect of ascorbate deficiency on the in situ activity of gamma-butyrobetaine hydroxylase.

The production of carnitine from peptide-bound 6-N-trimethyl-L-lysine (Lys(Me3)) or 4-N-trimethyl-aminobutyrate(gamma-butyrobetaine) perfused through isolated guinea pig livers was investigated. [Methyl-3H] Lys(Me3)-labeled agalacto-orosomucoid (AGOR) and asialofetuin were rapidly taken up and degraded by the perfused liver. Most of the free Lys(Me3) derived from Lys(Me3)-AGOR was released unmodified into the perfusion medium. However, Lys(Me3), arising from Lys(Me3)-asialofetuin was converted mostly to gamma-butyrobetaine and carnitine. gamma-Butyrobetaine added to the perfusion medium was hydroxylated to carnitine by the liver at a rate of 2.3 mumol/h. Guinea pigs maintained on an ascorbate-free diet for 17-60 days showed lowered ascorbate contents in all tissues measured and, coincidentally, a sharp reduction in carnitine levels in kidney, liver, and cardiac, and skeletal muscle. Carnitine production from [1,2,3,4-14C]gamma-butyrobetaine and [methyl-3H]Lys(Me3)-asialofetuin was reduced in perfused livers obtained from ascorbate-deficient guinea pigs. Although hydroxylation of gamma-butyrobetaine to carnitine was effectively depressed in the perfused isolated livers from ascorbate-deficient animals, hydroxylation of [methyl-3H]Lys(Me3) (derived from asialofetuin) to [methyl-3H]3-hydroxy-6-N-trimethyl-L-lysine was unaffected. Prior administration of ascorbate to the medium perfusing the isolated livers caused carnitine biosynthesis from all precursors examined to return to control values.

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.

Cyclosporine A impairs wound healing in rats.

Cellular immune responses may play an important role in the early inflammatory and cellular phases of wound healing. Cyclosporine A (CSA), a new immunosuppressive agent, impairs cellular immunity and T-cell-dependent humoral immunity. Therefore, the effect of CSA-induced immunosuppression in a rat wound-healing model was studied. Sprague-Dawley rats underwent a standardized skin incision and subcutaneous implantation of sterile polyvinyl alcohol sponges. CSA was dissolved in olive oil and given by gavage to one group of animals at a total dose of 125 mg/kg/10 days. The control group received an equivalent volume of olive oil. Ten-day-old wounds were weaker in CSA-treated animals, both in the fresh state (282 +/- 19 g vs 380 +/- 27 g, P less than 0.01), and after formalin fixation (1111 +/- 74 g vs 1419 +/- 57 g, P less than 0.01). In addition, CSA-treated rats accumulated significantly less hydroxyproline in the wound sponge granuloma, an index of reparative collagen deposition. The impairment in wound healing occurred without differences in body weight gain or organ weights. There was a profound immunosuppression in the animals receiving CSA as determined by thymic lymphocyte blastogenesis in response to Con A and PHA. These findings suggest that immunosuppression in otherwise healthy animals impairs wound healing.

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.

Increased survival due to radioactive estradiol in mice with C3HBA or BW 10232 tumors.

The influence of progesterone and estradiol labeled with tritium was studied in mice inoculated with transplantable mammary adenocarcinomas C3HBA or BW 10232. Tumor size, tumor growth rate, and host survival were measured. Radioactive [3H]estradiol administration increased survival time and inhibited tumor growth in mice inoculated with these tumor lines. Tumor growth retardation depended on the amount of radioactivity injected and nonradioactive estradiol was without any salutary effect on tumor size or host survival. Neither survival times nor tumor growth rate were altered by radioactive [3H]progesterone. The underlying mechanism(s) is (are) referable to ionizing radiation by the specific carrier estradiol or to an isotope effect of [3H]estradiol.

Granulopoiesis associated with the C3HBA tumor in mice.

Female C3H/HeJ mice were inoculated with syngeneic breast adenocarcinoma cells (C3HBA). A progressive neutrophilia developed as the tumors grew. A linear relationship was demonstrated between the tumor diameter and the extent of the neutrophilia. Local tumor excision caused a rapid fall (3 days) in the neutrophil count. Media conditioned with tumor cells, normal mouse kidney, and bone marrow of normal or tumor-bearing mice were prepared. Tumor cell-conditioned medium was found to have marked stimulating activity for granulocytic colony formation of mouse bone marrow cells. Sera from tumor-bearing mice also had colony-stimulating activity. This finding strongly suggested that the neutrophilia was caused by the release of a neutrophil-stimulating factor from the tumor.

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.

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.

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.