Osteotoxicity after chronic dietary administration of 13-cis-retinoic acid, retinyl palmitate or selenium in mice exposed to tumor initiation and promotion

In view of the clinical trials of retinoids as therapeutic agents for premalignant skin lesions, a radiographic study was undertaken to measure skeletal toxicities after chronic dietary administration of retinoids in mice exposed to tumor initiation and promotion. CD-1 mice were initiated with 0.15 moles of 7,12-dimethylbenz[a]anthracene and promoted twice daily with 8 nmoles of 12-0-tetradecanoylphorbol-13-acetate for 23 weeks. Diets were supplemented with 60 IU, 200 IU, or 700 IU of retinyl palmitate (RP) per g diet. After 5 weeks, the 700 IU of RP /g diet was lowered to 350 IU/g diet. Administration of these diets to mice during the 23 weeks of tumor promotion resulted in a 0-fold, 2-fold, or 10-fold increase in bone fractures, respectively. Osteoporotic bone lesions identified on radiographs rose 0-fold, 0-fold, and 10-fold at the respective doses, whereas metaphyseal flares increased 0-fold, 1.4-fold, and 3.6-fold. Bone deformities were augmented 0-fold, 1.8-fold and 2.9-fold at the respective doses. Addition of selenium (2 ppm in the drinking water) did not alter the bone toxicity of RP. 13-cis-retinoic acid (CRA) was less toxic at 700 IU/g diet than was RP at that dose, as evidenced by the death of 12 of 70 mice by the 6th week of dietary RP and no deaths in the 35 mice fed 700 IU CRA/g diet for 23 weeks. CRA at 700 IU/g diet resulted in 3/4 as many osteoporotic bones, 1/3 as many bone fractures, 4/5 as many metaphyseal flares, and a similar number of bone deformities as mice fed 700/350 IU/g diet. At the dose of 200 IU/g food, osteotoxicities were similar in the mice fed diets supplemented with RP and CRA. Thus, the light dose of CRA (700 IU/g diet) was less toxic than the high dose of, RP but at a lower dose (200 IU/g), CRA was as osteotoxic as was RP. Bone fractures in mice exposed to prolonged dietary administration of retinoids was a more sensitive index of retinoid toxicity than was body weight. We have detected osteotoxicity in mice at a total dose of CRA which was about twice the total dose used clinically.     

Associations between the rates of elongation of the short bones of the hand

Data from serial radiographs of 40 children were used to study the rates of total, diaphyseal and epiphyseal elongation of the bones of the hand from 3–13 years. Communality indices were calculated from complete correlation matrices for each sex. These indices tended to be lower for boys than girls. Communality indices for distal phalanges were lower than for the other bones and those for epiphyseal elongation were lower than those for total or diaphyseal elongation. Correlations between groups of bones in their rates of elongation were higher in rows of bones than in rays. There were statistically significant neighborhood effects and a slight tendency to marginal effects in the correlations between rates of elongation in rows of bones; neither of these effects was present in rays.     

Impact of vitamin E supplement in standard laboratory animal diet on microvascular manifestation of ischemia/reperfusion injury

Aimed at improving animal fertility and health, diets for farm and laboratory animals have over the last few years been supplemented with increasing amounts of the antioxidant vitamin E. We now demonstrate by intravital microscopy that feeding hamsters with a vitamin E-supplemented “standard” rodent diet (60 ppm vitamin E) significantly reduces the microvascular manifestations of ischemia/reperfusion injury when compared to animals fed a nonsupplemented diet. Postischemic leukocyte adhesion to venular endothelium was reduced from 770 ± 204 cells/mm² at 24 h after reperfusion in control animals on the nonsupplemented diet to 403 ± 105 cells/mm² in animals on the “standard” rodent diet (means ± SD, N = 7 animals per group, p < 0.01). Animals on the nonsupplemented diet showed a dramatic loss of capillary perfusion density until 7 days after reperfusion (to 21 ± 13% of preischemic baseline values), whereas this loss was significantly attenuated (to 71 ± 12% of preischemic values, p < 0.01) in animals on the “standard” rodent diet. No difference in the extent of reperfusion injury was seen between animals on the “standard” rodent diet and animals on diets with substantially higher vitamin E supplements (300 ppm–30.000 ppm). Besides underscoring the benefit of vitamin E in reducing the extent of ischemia/reperfusion injury, this study raises the concern that vitamin E supplements in “standard” laboratory animal diets may have a far-reaching impact on biomedical research by jeopardizing established animal models of disease.     

Vitamin C deficiency in captive nonhuman primates fed commercial primate diet

Scurvy was diagnosed in 19 rhesus monkeys (Macaca mulatta) and four squirrel monkeys (Saimiri sciureus) from a colony of nonhuman primates maintained on a commercial diet. Signs of weakness, reluctance to move, gingival hemorrhage, bruising, proximal and distal metaphyseal fractures, weight loss and anemia appeared in juvenile and young adult rhesus monkeys over a 2 week period. Clinical signs subsided after 5 days of vitamin C therapy. At the same time, cephalohematomas and weakness developed in squirrel monkeys, which failed to respond to treatment. These cases were associated with manufacturer's admitted error in preparation of the commercially prepared monkey diet.     

Dietary alpha-tocopherol affects differential gene expression in rat testes

Gene-chip technology was employed to study the effect of dietary vitamin E (VE) on gene expression in rat testes. Male albino rats were fed with either a diet deficient in VE or a standard diet containing VE. Differential gene expression was monitored at five individual time-points over a period of 14 months with all animals individually profiled. Low VE intake resulted in the consistent up-regulation of 7-dehydrocholesterol reductase and GATA binding protein 4, both involved in testosterone synthesis. Cyclin D3, important in cell cycle progression and Wilms tumor 1, related to cancer development, were also up-regulated in the vitamin E deficient animals. This study demonstrates that low dietary VE intake has long-term effects on gene expression in the testes. Our data provides insights into the possible molecular mechanisms underlying the beneficial effects of vitamin E on the male reproductive organ.     

The effect of TCDD on acyl CoA:retinol acyltransferase activity and vitamin A accumulation in the kidney of male Sprague-Dawley rats

Previous studies have shown that rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show signs of toxicity that are similar to the responses of animals to a vitamin A-deficient diet. These include hypophagia, loss of body weight, loss of hepatic vitamin A, and accumulation of renal retinoids. Male Sprague-Dawley rats treated with 10, 30, or 100 nmol/kg of TCDD accumulated renal vitamin A, with retinyl palmitate concentrations reaching 8 times those of control animals, similar to that of male rats fed a vitamin A-free diet for 26 days. Acyl CoA:retinol acyltransferase (ACARAT) activities in both TCDD-treated rats and rats fed a vitamin A-free diet for 26 days were similarly elevated, and were strongly and positively correlated with the renal retinyl palmitate concentrations. Retinol concentrations in the kidneys of rats treated with TCDD or fed a vitamin A-free diet were only slightly elevated when compared to control rats. We suggest that accumulation of retinyl esters in the kidneys of rats treated with TCDD or fed a vitamin A-free diet occurs as a result of increased rates of retinol esterification.     

Generation of prostacyclin-like substance and lipid peroxidation in vitamin E-deficient rats

Endogenous generation of prostacyclin (PGI₂)-like substance and lipid peroxidation were studied in the aorta of rats fed on vitamin E-deficient diet and/or vitamin E-supplemented one for 4 to 10 months after they were weaned at 4 weeks. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and was estimated by comparison of its antiaggregatory activity with that produced by known amounts of synthetic PGI₂. Thiobarbituric acid-reacting substance (TBARS) was determined as an indicator of lipid peroxidation. The generation of PGI₂-like substance was significantly reduced in rats fed on vitamin E-deficient diet for 8 and 10 months as compared with that in the animals fed on vitamin E-supplemented one for the same period (p<0.001). Mean concentration of TBARS in the aortae of rats fed on vitamin E-deficient diet for 10 months was significantly higher than that of the animals fed on vitamin E-supplemented diet for the same feeding period (p<0.001). These alterations in the aortae of rats fed on vitamin E-deficient diet were corrected by feeding them on vitamin E-supplemented diet for subsequent 2 months.     

Free Radical-Induced Liver Injury. I. Effects of Dietary Vitamin E Deficiency on Triacylglycerol Level and its Fatty Acid Profile in Rat Liver

Effects of dietary vitamin E deficiency on the fatty acid compositions of total lipids and phospholipids were studied in several tissues of rats fed a vitamin E-deficient diet for 4, 6, and 9 months. No significant differences were observed between the vitamin E deficiency and controls except in the fatty acid profiles of liver total lipids. Triacylglycerol (TAG) accumulation was found in the liver of rats fed a vitamin E-deficient diet. The levels of TAG-palmitate and -oleate increased particularly in the liver from such animals. The fatty acid compositions of hepatic phospholipids were not affected by the diet. Increased TAG observed in the liver of rats fed a vitamin E-deficient diet was restored to normal when the diet was supplemented with 20 mg α-tocopheryl acetate/kg diet. These findings indicate that dietary vitamin E deficiency causes TAG accumulation in the liver and that the antioxidant, vitamin E, is capable of preventing free radical-induced liver injury.     

Vitamin K deficiency of germfree mice caused by feeding standard purified diet sterilized by gamma-irradiation.

Germfree mice died when they were fed a purified diet of AIN-76 formula sterilized by gamma-irradiation. Vitamin K deficiency was suspected and this study was performed to confirm the cause of the death. Germfree mice were fed purified diets of AIN-76 or AIN-93M formula, which were pelleted and sterilized by gamma-irradiation at a dose of 50 kGy. One half of the mice fed the AIN-76 diet died within two weeks and the surviving animals were also in poor health, while 91% of mice fed the AIN-93M diet survived. No hemorrhage was observed grossly in any organs of the surviving animals. Histologically, degeneration with inflammatory cell infiltration was observed as well as hemorrhage and fibrosis in the heart muscles of mice fed the AIN-76 diet. No microscopic lesions were observed in the other organs. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were extremely prolonged when mice were fed the AIN-76 diet. The animals totally recovered when they were intragastrically administered 1 microg/day of vitamin K(3) from the third day of feeding of the AIN-76 diet, except for PT and APTT which were still slightly longer than in mice fed the AIN-93M diet. The concentration of vitamin K(3) supplied in the AIN-76 diet decreased to an undetectable level after gamma-irradiation, while the AIN-93M diet contained 240 microg/kg of vitamin K(1). These results indicate that the deaths of the germfree mice fed the gamma-irradiated AIN-76 diet were caused by vitamin K deficiency. Vitamin K deficiency may cause fatal degeneration of cardiac muscle cells.     

The effect of TCDD on Acyl CoA: Retinol acyltransferase activity and vitamin a accumulation in the kidney of male sprague-dawley rats

Previous studies have shown that rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show signs of toxicity that are similar to the responses of animals to a vitamin A-deficient diet. These include hypophagia, loss of body weight, loss of hepatic vitamin A, and accumulation of renal retinoids. Male Sprague-Dawley rats treated with 10, 30, or 100 nmol/kg of TCDD accumulated renal vitamin A, with retinyl palmitate concentrations reaching 8 times those of control animals, similar to that of male rats fed a vitamin A-free diet for 26 days. Acyl CoA:retinol acyltransferase (ACARAT) activities in both TCDD-treated rats and rats fed a vitamin A-free diet for 26 days were similarly elevated, and were strongly and positively correlated with the renal retinyl palmitate concentrations. Retinol concentrations in the kidneys of rats treated with TCDD or fed a vitamin A-free diet were only slightly elevated when compared to control rats. We suggest that accumulation of retinyl esters in the kidneys of rats treated with TCDD or fed a vitamin A-free diet occurs as a result of increased rates of retinol esterification.     

Metacarpophalangeal length changes in humans during adulthood: a longitudinal study.

Total lengths of the 19 diaphyseal hand bones were measured from standardized radiographs of healthy American whites as young adults (ca. 21 years) and again at ca. 55 years of age. The four hand-bone rows exhibit distinctive length changes: Distal and middle phalanges continue to increase significantly in length, proximal phalanges constitute a transition zone of little change, and metacarpals uniformly decrease in length. Clear-cut sex differences are noteworthy: Males change more (lose more in some bone rows, gain more in others) than females. Progressive elongation was greatest in the distal phalanges where apposition around the distal aspect ("tufting") is not constrained by a joint or epiphysis. Loss of bone length in the metacarpals by subchondral resorption is consistent with documented reductions in activity levels and grip strength with age, as well as diminished joint spaces which alter loading of the joints.     

Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster

Abstract. Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well-established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A-supplemented and vitamin A-deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1 -9 μg/g liver, respectively, at 28 d. In contrast, in animals on the vitamin A-deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0–32 and 0–09 μg/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d; their tracheas were depleted of retinol at 28 d. No vitamin A-associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A-deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild-to-severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A-deficiency when the tissue levels of the vitamin are depleted.     

Evidence that sodium deprivation influences vitamin D dependent rat renal calcium binding protein

In order to provide some insight concerning the role of renal calcium binding protein (CaBP) in the functioning of the mammalian kidney, the response of renal CaBP to dietary alterations was examined. Three week old rats were fed diets deficient in calcium, phosphorous or sodium supplemented with vitamin D for a four week period. The specific activity of renal CaBP (as measured by the chelex resin assay; Ca²⁺ bound protein/Ca²⁺ bound resin per mg protein) in the 28,000 M_r region was found to increase four fold in rats fed the low phosphorus diet and two fold rats fed the low calcium diet when compared to rats fed the control diet. Renal CaBP/mg protein from rats fed the low sodium diet decreased 50% from the control values. Changes in renal CaBP were confirmed by polyacrylamide gel analysis of the 28,000 M_r fraction by densitometric tracing using a purified CaBP marker. The greater response to dietary phosphorus restriction suggests that renal CaBP may be regulated by a mechanism different from that of intestinal CaBP. The decrease in renal CaBP in rats fed the low sodium diet suggests for the first time that sodium is required for vitamin D dependent distal tubular calcium transport processes.     

Inhibitory effect of vitamin E on proinflammatory cytokines-and endotoxin-induced nitric oxide release in alveolar macrophages

Nitric oxide is thought to be an important modulator of various functions in normal and inflamed airways. In the present study, we evaluated the effects of high vitamin E (250 mg and 1250 mg alpha-tocopheryl acetate (TA)/kg diet/10 days) on nitric oxide (NO(.)) release by alveolar macrophages (AMs) in response to lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha). LPS and IL-1beta treatment (1-10 microg/ml) enhanced NO(.) release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration dependent manner. However, this enhancement of NO(.) was attenuated in the AMs of animals fed with 250 mg or 1250 mg vitamin E/kg diet. TNF-alpha had no effect in eliciting the release of NO(.) in AMs obtained either from control or from hyper vitamin E fed animals. Further, LPS (1-10 microg/ml) enhanced the inducible nitric oxide synthase (iNOS) activity of AMs of control group and TA-fed animals almost to equal extent. Similarly, LPS-induced formation of N-nitrosamine (N-nitroso-L-[(14)C]-proline) in AMs of control and TA-supplemented animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 microM) in AMs of control animals, when triggered with 10 microg LPS/ml, caused a significant decrease in N-nitroso-L-[(14)C]-proline formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and proinflammatory cytokines-induced NO(.)-mediated damage by AMs.     

Guidelines for selective radiological assessment of inversion ankle injuries.

A prospective study was performed to establish definitive guidelines for selective use of radiography in the assessment of inversion ankle injuries. Five hundred patients were included, representing 3.2% of the workload of the department during the study period. There were 379 soft tissue injuries, 56 malleolar fractures, 40 avulsion fractures, 21 fractures at the base of the fifth metatarsal, and four calcaneal fractures. Multiple logistic regression identified distal fibular tenderness, age, and ability to bear weight as the most important clinical variables in predicting important fractures (p less than 0.001). A policy of requesting x ray examination of only those patients with distal fibular tenderness or inability to bear weight or aged over 60, with a further proviso that no foot radiographs should be obtained, would produce a 60% reduction in ankle radiography in this centre without detriment to patient care.     

Vitamin E deficiency accentuates adriamycin-induced cardiomyopathy and cell surface changes

Free radicals have been suggested to play a role in adriamycin-induced cardiomyopathy. Adriamycin-induced myocardial effects were examined in rats maintained on a vitamin E deficient diet. Animals were divided into four groups: I, control; II, adriamycin-treated; III, vitamin E deficient diet; IV, vitamin E deficient diet plus adriamycin treatment. Adriamycin-treated animals (groups II and IV) were given six injections (i.p.) over two weeks for producing a cumulative dose of 15 mg/kg. Animals in groups III and IV were placed on vitamin E deficient diet starting two weeks prior to the first injection of adriamycin or vehicle. Myocardial tissue analysis were performed on animals sacrificed 1 week after the last injection. Mortality was significantly higher in group IV which also showed doubling of myocardial malondialdehyde content relative to the non-adriamycin-treated vitamin E deficient group (III). Myocardial cell damage in group IV was characterized by separation of the external lamina, subsarcolemmal changes, mitochondrial swelling and myofibril dropout. Group II hearts showed only a mild dilation of the sarcotubules and swelling of the mitochondria. Total sialic acid content of the sarcolemma in groups II, III and IV was 55, 90 and 24% of the control values in group I. These data show a characteristic sarcolemmal injury produced by adriamycin in hearts of animals with reduced antioxidant capacity which is probably mediated by increased free radical activity as well as lipid peroxidation.     

Effect of Trisetum flavescens P.B. on calcium and phosphorus metabolism in rats. Experimental evidence for the principle actively affecting phospho-calcium homeostasis.

After reviewing the literature on the vitamin D3-like activity of certain plants [Solanum malacoxylon, Cestrum diurnum and Trisetum flavescens], the authors present results of experiments in which dried Trisetum flavescens was administered perorally to rats. The addition of 2.5% dried Trisetum flavescens to the standard laboratory diet caused a drop in blood phosphate levels and a simultaneous marked increase in the calcium and phosphorus content of bones of the experimental animals. The mechanism of the action of Trisetum flavescens on phosphocalcium metabolism is discussed.     

Moderate zinc deficiency negatively affects biomechanical properties of rat tibiae independently of body composition

To guide development of novel nutritional strategies aimed at reducing the incidence of stress fractures, we observed the effects of manipulating dietary zinc (Zn) content on bone integrity in Sprague–Dawley rats fed either a severely Zn-deficient (ZnD; 1 ppm), a moderately Zn-deficient (MZnD; 5 ppm) or a Zn-adequate (ZnAD; 30 ppm) diet for 6 weeks. At the completion of the diet period, body composition, bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) were determined in vivo by using dual-energy X-ray absorptiometry. Following euthanasia, long bones were collected for determination of Zn content and biomechanical strength testing. Despite significant positive correlations between dietary Zn and both body weight (BW) and bone Zn content for the entire cohort (r=.77 and r=.83, respectively), rats fed MZnD or ZnAD diets did not differ in feed intakes, body composition, BMC, BA, BMD or BW. Tibial bones, but not femur bones, appear to be more responsive to dietary Zn manipulation, as all bone biomechanical strength indices in the ZnAD-fed rats were significantly greater than in rats fed the ZnD diets. Rats fed either MZnD or ZnAD diets had stronger tibiae (129% increase in maximum load and stress at maximum load, P<.01) compared with those fed ZnD diets. The load at breakage for the tibial bones of rats fed MZnD diets was not different from the ZnD rats, but lower (P<.05) than that of the ZnAD rats. These results suggest that since feed intakes, body composition, BMC, BA, BMD and BW were not significantly different between the MZnD- and ZnAD-fed animals, the reduced bone integrity observed in the MZnD-fed rats resulted from dietary Zn inadequacy, and not as a result of the reduced growth that is typically associated with Zn deficiency.     

Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster

Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well-established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A-supplemented and vitamin A-deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1.9 micrograms g liver, respectively, at 28 d. In contrast, in animals on the vitamin A-deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0.32 and 0.09 micrograms/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d: their tracheas were depleted of retinol at 28 d. No vitamin A-associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A-deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild-to-severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A-deficiency when the tissue levels of the vitamin are depleted.