Osteogenic Disorder Shionogi Research Articles

3] Expression of recombinant l-gulono-γ-lactone oxidase

This chapter analyzes the sequence of cDNA encoding rat liver L -gulono-y-lactone oxidase (GLO). It also discusses the reality of the etiology of the GLO deficiency of the osteogenic disorder Shionogi (ODS) rat by constructing minigenes for wild-type and mutant GLOs and introducing them into COS-1 cells. The enzyme was found to be expressed in the microsomal fraction of the cell, as is the case for rat liver cells. Because the efficiency of expression was not high, owing to low efficiency of transfection, a baculovirus vector was utilized for the expression. The specific activity of GLO expressed in silkworm cells was comparable to the high level found in rat liver microsomes. Using the baculovirus expression system, the apoenzyme of GLO was produced by culturing virus-infected cells in a riboflavin-deficient medium. Several methods utilized for the expression of the recombinant GLO and for detection of the expressed enzyme are described in the chapter.

Growth and bone remodelling in a scorbutic rat model

This study used the scorbutic Osteogenic Disorder Shionogi rat model and a specific diet to reliably induce a state of sub-scurvy scorbutus. Under these conditions overall somatic growth was assessed, as well as that of the caudal vertebrae, as an example of scorbutic bone growth. Tail loops were then used to mechanically stress mature caudal vertebrae under scorbutic conditions, and the vertebrae's adaptation to these applied forces was assessed, using measurements of bend deformation and histologic analysis of osteogenesis. Scorbutic animals exhibited significant somatic growth retardation (p < 0.05), and abnormal reductions in osteogenesis and periosteal responsiveness to growth. Scorbutic vertebrae also showed greater bend angles of deformation (p < 0.05), and a marked reduction in cortical osseous remodelling and periosteal differentiation. It appeared that the sub-scurvy scorbutic bones were smaller, weaker and less able to adapt to physical stresses: this pattern was reflected at the histologic level.

Induction by 4‐nitroquinoline‐l‐oxide of oral epithelial dysplasia and neoplasia in scurvy‐prone osteogenic disorder shionogi (ODS) rats

Carcinogenesis by 4-nitroquinoline-1-oxide (NQO) in the oral mucosa is a reliable method of obtaining oral mucosal squamous cell carcinoma (OMSCC) and allows examination of various stages of oral cancer development. In vivo and in vitro studies have indicated that L-ascorbic acid (AA) may have a role in cancer prevention. The Wistar "scurvy-prone" osteogenic disorder Shionogi (ODS) rat of the od/od substrain is unable to synthesize AA and requires supplementation for its survival. This study examined the effects of NQO on the oral mucosa of ODS and outbred Wistar rats. NQO (0.5%) was applied topically to the palatal mucosa of 72 male ODS and 36 outbred Wistar rats three times weekly for 4, 8, 12, 16, 20, and 24 wks. The ODS rats were divided so that 36 rats were given 2.5 g/l AA in the drinking water and 36 rats were given 0.33 g/l AA. Vehicle-treated and untreated control animals were included. The rats were killed two weeks after the final NQO application, and the tissues were examined. Epithelial dysplasia was assessed using a modified Smith and Pindborg (1969) index. The ordered categorical scores were analyzed appropriately. Plasma AA levels were checked in ODS and outbred rats at the start and end of the experiment. The results indicated that the oral mucosa of the ODS and outbred rats were susceptible to NQO but that the rate of dysplasia and OMSCC development differed between them, with more rapid changes being found in the ODS rats (p < or = 0.05). No significant difference was found in the dysplasia scores and in the rate of OMSCC development between ODS rats given 2.5 g/l of AA and ODS rats given 0.33 g/l of AA (p > 0.05). No epithelial changes were observed in the palatal mucosa of vehicle-treated and untreated controls. The plasma AA level mean (+/- SEM) was 56 +/- 6 microM for the outbred rats, 8 +/- 1 microM for the ODS rats given 0.33 g/l AA supplementation, and 29 +/- 2 microM for the ODS rats given 2.5 g/l AA. It was concluded that the chronic AA-deficient state in ODS rats played an insignificant role in oral carcinogenesis and that other factors, for example, genetic differences in susceptibility to NQO, contributed to the present findings.

Change in the Level of Vitamin C and Lipid Peroxidation in Tissues of the Inherently Scorbutic Rat during Ascorbate Deficiency

To investigate an accurate profile of vitamin C deficiency, ascorbate deficiency was caused in the inherently scorbutic rat [Osteogenic Disorder Shionogi (ODS)], and changes in the level of the vitamin in 12 tissues of the animals (plasma, liver, stomach, small and large intestines, lung, heart, kidney, adrenal gland, spleen, muscle, and brain) were followed based on the specific method (Kishida et al. Anal. Chem. 1992, 64, 1505-1507). The level of ascorbate in plasma decreased most rapidly, and the rate of decline of the vitamin was the slowest in the brain among the 12 tissues. Based on the kinetic profile of ascorbate decay, these tissues were classified into four groups. After 25 days of ascorbate deficiency, indicators of oxidative stress changed significantly compared with the control group. The indices included increased lipid hydroperoxide level determined by the specific method (Tokumaru et al. Anal. Chim. Acta 1995, 307, 97-102) in the brain, elevated thiobarbituric acid-reactive substances (TBARS) and glutathione peroxidase activity in the heart, and the fall of glutathione in plasma and the liver.

Ascorbic acid inhibits chemically induced uroporphyria in ascorbate-requiring rats

Ascorbate was previously shown to suppress accumulation of uroporphyrin (URO) in cultured chick embryo hepatocytes and to competitively inhibit microsomal oxidation of uroporphyrinogen catalyzed by cytochrome P4501A2. Here we used the Osteogenic Disorder Shionogi (ODS) mutant rat, which cannot synthesize ascorbic acid, to examine the in vivo effect of ascorbic acid on hepatic URO accumulation caused by treatment with 3-methylcholanthrene (MC) and 5-aminolevulinate (ALA). Female mutant rats maintained on three levels of dietary ascorbate (15,200, and 800 ppm) were treated for a total of 24 days. On the 11th and 16th days, rats were administered 3-methylcholanthrene, and 5-aminolevulinate was present continuously in the drinking water from day 14. Hepatic URO accumulated at the two lowest ascorbate levels, but not at 800 ppm ascorbate. The latter dose produced normal hepatic ascorbate levels. Plasma ascorbate levels were proportional to the hepatic values. Male rats also accumulated URO at the low dietary dose of ascorbic acid. The methylcholanthrene-induced increase in microsomal levels of CYP1A1 and CYP1A2, total cytochrome P450, and activities of uroporphyrinogen oxidation and ethoxyresorufin deethylase were not affected by the dietary level of ascorbate. Neither male nor female Fischer 344 rats accumulated URO when treated with the MC/ALA regime. Hepatic ascorbate concentrations in these rats were five-fold to seven-fold higher than they were in mutant rats that developed uroporphyria on 150 ppm dietary ascorbate. In ODS rats fed ascorbate at 90 but not 900 ppm in the diet, hexachlorobenzene caused hepatic URO accumulation, indicating that the effect of ascorbic acid is not unique to the regimen using methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbic acid inhibits chemically induced uroporphyria in ascorbate-requiring rats

Ascorbate was previously shown to suppress accumulation of uroporphyrin (URO) in cultured chick embryo hepatocytes and to competitively inhibit microsomal oxidation of uroporphyrinogen catalyzed by cytochrome P4501A2. Here we used the Osteogenic Disorder Shionogi (ODS) mutant rat, which cannot synthesize ascorbic acid, to examine the in vivo effect of ascorbic acid on hepatic URO accumulation caused by treatment with 3-methylcholanthrene (MC) and 5-aminolevulinate (ALA). Female mutant rats maintained on three levels of dietary ascorbate (15,200, and 800 ppm) were treated for a total of 24 days. On the 11th and 16th days, rats were administered 3-methylcholanthrene, and 5-amino-levulinate was present continuously in the drinking water from day 14. Hepatic URO accumulated at the two lowest ascorbate levels, but not at 800 ppm ascorbate. The latter dose produced normal hepatic ascorbate levels. Plasma ascorbate levels were proportional to the hepatic values. Male rats also accumulated URO at the low dietary dose of ascorbic acid. The methylcholanthrene-induced increase in microsomal levels of CYP1A1 and CYP1A2, total cytochrome P450, and activities of uroporphyrinogen oxidation and ethoxyresorufin deethylase were not affected by the dietary level of ascorbate. Neither male nor female Fischer 344 rats accumulated URO when treated with the MC/ALA regime. Hepatic ascorbate concentrations in these rats were five-fold to seven-fold higher than they were in mutant rats that developed uroporphyria on 150 ppm dietary ascorbate. In ODS rats fed ascorbate at 90 but not 900 ppm in the diet, hexachlorobenzene caused hepatic URO accumulation, indicating that the effect of ascorbic acid is not unique to the regimen using methylcholanthrene. These results are consistent with a role for ascorbate in preventing chemically induced URO accumulation and suggest that ascorbate might have a role in preventing URO accumulation in humans.

Effect of ascorbic acid on pituitary prolactin secretion in the non-ascorbate synthesizing osteogenic disorder shionogi (ODS) rat

We examined the secretion of prolactin (PRL) in the hereditary scurvy-prone Osteogenic Disorder Shionogi (ODS) rat to investigate the role of ascorbic acid (AsA) in pituitary lactotroph and hypothalamic function. Plasma PRL concentrations of conscious AsA-deficient or control ODS rats were measured before and after the administration of metoclopramide (MCP), 5- hydroxy-L-tryptophan (5-HTP) or saline. The basal plasma PRL levels did not differ between the two groups. However, the AsA-deficient rats exhibited significantly larger changes in plasma prolactin concentration in response to MCP or 5-HTP than the control ODS rats. Thus, AsA deficiency enhanced the stimulated, but not the basal, secretion of PRL in ODS rats. Our findings suggest that AsA may have an inhibitory effect on PRL secretion.

Increased CYP1A2 content and capacity to activate Glu-P-1 and Trp-P-2 in liver microsomes of scorbutic ODS rats.

Osteogenic Disorder Shionogi (ODS) rats, which cannot synthesize ascorbic acid due to a deficiency of L-gulonolactone oxidase, become scorbutic when not supplied with dietary ascorbic acid. We used the deficient rats to study the effects of ascorbic acid on the amount of cytochrome P450 enzymes in liver microsomes. The total amount of hepatic cytochrome P450 in ODS rats deprived of ascorbic acid was lower by approximately 40%, whereas ODS rats fed with ascorbic acid and the wild strain had the same level of total hepatic cytochrome P450. Western blot analysis for various forms of cytochrome P450 in liver microsomes indicated that the amount of CYP1A2 was significantly higher in ascorbic acid deficient rats. On the other hand, amounts of CYP2B2 and 3A were lower, and those of CYP2E1 and CYP2C6/11 were unaffected. In accordance with the higher amount of CYP1A2, Northern blot analysis showed increased expression of CYP1A2 mRNA. The capacity of microsomes to produce mutagens from 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole acetate (Glu-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) was higher in scorbutic ODS rats by the Ames test. These results indicate that the effects of ascorbic acid deficiency on the expression of cytochrome P450 in ODS rat livers are form-specific and that the increased CYP1A2 is associated with increased metabolic activation of promutagens in the scorbutic state.

A missense mutation of L-gulono-gamma-lactone oxidase causes the inability of scurvy-prone osteogenic disorder rats to synthesize L-ascorbic acid.

The osteogenic disorder Shionogi (ODS) rat is a mutant Wistar rat that is subject to scurvy, because it lacks L-gulono-gamma-lactone oxidase, a key enzyme in L-ascorbic acid biosynthesis. Sequencing of polymerase chain reaction-amplified cDNAs for mutant and normal rat L-gulono-gamma-lactone oxidases demonstrated that the mutant cDNA has a single base mutation from G to A at nucleotide 182, which mutation alters the 61st amino acid residue from Cys to Tyr. To test the effect of this mutation on the expression of L-gulono-gamma-lactone oxidase, we inserted a region of the cDNAs coding for normal and mutant L-gulono-gamma-lactone oxidases into an expression vector, pSVL, and transfected COS-1 cells with such vectors. The result indicated that the defined amino acid substitution does decrease both the amount of immunologically detectable protein and the level of enzyme activity to about one-tenth of their normal values, while it does not affect the amount of the mRNA produced in the transfected cells. This situation is similar to our previous observation that L-gulono-gamma-lactone oxidase is expressed in the liver of the ODS rat at a very low level irrespective of the presence of a normal amount of L-gulono-gamma-lactone oxidase-specific mRNA of a normal size (Nishikimi, M., Koshizaka, T., Kondo, K., and Yagi, K. (1989) Experientia (Basel) 45, 126-129). Thus it became clear that the Cys-->Tyr substitution is responsible for the L-gulono-gamma-lactone oxidase deficiency in the ODS rat.

Inhibition of Insulin Secretion and Increase of Plasma Non-Esterified Fatty Acids Induced by Biotin Deficiency in Osteogenic Disorder Shionogi Rats.

We determined the changes in the sources of energy supply in biotin-deficient osteogenic disorder Shionogi rats. Plasma glucose level of biotin-deficient rats decreased gradually and steadily from the 59th day of the dietary regimen. Plasma insulin decreased sharply on day 59, although plasma glucose was still sufficient. Plasma insulin in response to an oral glucose load in the deficient rats was approximately one-third less in concentration than that of the control. The decrease in the insulin level may be induced by biotin deficiency in the early stage in the deficient rats. In the later stage of the deficiency period, when plasma glucose decreased to under 50% compared with the control level, the plasma non-esterified fatty acid level greatly increased. This suggests that the source of energy supply in the biotin-deficient rats changed from glucose to non-esterified fatty acid supplied by adipose tissue. The time of increase in the plasma non-esterified fatty acid level coincided with the onset of the clinical features of biotin deficiency in the rats.

Comparison of the anti-scorbutic activity of L-ascorbic acid and ester C® in the non-ascorbate synthesizing Osteogenic Disorder Shionogi (ODS) rat

The Osteogenic Disorder Shionogi (ODS) rat, Clea Inc., Tokyo, Japan lacks the ability to synthesize L-ascorbic acid (AA). As with man, monkey and the guinea pig, this rat lacks L-gulonolactone oxidase necessary for the synthesis of AA from glucose. This study shows this animal to be an alternative to the guinea pig in AA studies. The anti-scorbutic potency of Ester C@(EC), a calcium ascorbate and calcium threonate mixture, was compared with an AA dose of equal ascorbate activity equivalents (AAE) for anti-scorbutic activity in the ODS rat. The minimal anti-scorbutic dose of EC was determined to be 0.44 mg/kg/day (AAE), while an AA dose of 0.51 mg/kg/day (AAE) was not anti-scorbutic in a 24 day study. At 24 days EC rats gained 125% of initial body weight (BW) and the AA rats only 45% BW. Scorbutic signs at 24 days were scored on a 0(min) to 3(max) scale. The EC/AA ratio scores were: hemorrhage 0/1.4, behavior change 0/2.0, piloerection 0/2.2, mobility 0.4/2.2, dysbasia 0.6/2.8 and ataxia 0.4/1.0. Pearson's correlation coefficient for BW versus AAE was r=.34 for the AA group and r = .90 for the EC group. The morbidity index for EC was 0/5 and for the AA group 2/5. The AAE dose of AA which was 16% higher/day than the EC AAE dose was not anti-scorbutic, while the EC dose was anti-scorbutic. EC rats had 3.5X greater weight gain, a sensitive indicator of scurvy, than the AA rats. EC rats had 3–4 times less, if any, scorbutic signs than AA rats. The results clearly show that, based on ascorbate activity equivalents, EC has more available ascorbate activity/potency than AA. The mechanism of this increased potency is believed to be due to the facilitated transport of AAE into the cell by the threonate (a normal in vivo metabolite of AA) present in the EC product. In addition, previous studies have shown EC (AAE) to be higher in plasma and excreted less rapidly than the AAE derived from AA administered orally.

Cholesterol metabolism in inherently scorbutic rats (ODS rats).

We observed the cholesterol metabolism of a colony of Wistar rats with a hereditary defect in vitamin C synthesizing ability (the ODS (osteogenic disorder-Shionogi) rats) in six kinds of experiments. Female ODS rats aged 36 days had a low HDL (high-density lipoprotein)-cholesterol level in serum as compared with age-matched control rats in spite of the absence of scorbutic symptoms. Female ODS rats aged 63 days which revealed severe scorbutic symptoms had a very low HDL-cholesterol level (mean value; 17 mg/dl). And male ODS rats, whose lives had been prolonged by supplementing with L-ascorbic acid, also had lower serum HDL-cholesterol and had increased total cholesterol in serum and liver when the acid supplement dose was relatively insufficient. On the other hand, we examined HDL2- and HDL3-cholesterol levels in serum to determine the mechanism of low HDL-cholesterol. As a result, we observed a low HDL2-cholesterol level in ODS rats but normal HDL3-cholesterol level. But the authors observed no decrease of LCAT (lecithin: cholesterol acyltransferase) activity in serum of ODS rats. These results could be due to disturbance of lipid metabolism in a vitamin C-deficient condition, that is to say, there might be abnormalities of the cholesterol excretion pathway of bile acid from liver, and maturity of the HDL-cholesterol particle due to other factors except that of LCAT activity.