Shuji Sassa

Effects of colloidal iron overload on renal and hepatic siderosis and the femur in male rats

Genetic hemochromatosis is an iron overload disorder, and osteopenic and osteoporotic. Femoral neck bone mineral density (BMD) appears to fall with rising hepatic iron concentrations. A critical role for iron in mediating tissue injury is played via hydroxyl radical formation in nephrotoxicity. We investigated the effects of a colloidal iron overload on renal function, organ siderosis, and femoral bone in male rats. Iron overload reduced body growth, and increased the weights of the liver and spleen. Marked deposition of iron was noted in liver and kidney. Activities of lactate dehydrogenase and alkaline phosphatase were decreased, and the concentrations of blood urea nitrogen and creatinine were increased with the reduction in plasma calcium and inorganic phosphorus levels, i.e. functions of the liver and kidney might be affected by reactive oxygen species such as the superoxide radical, H2O2, and the hydroxyl radical produced by overloaded iron. Damage to the proximal tubular epithelial cells of the kidney and a loss of connectivity of cancellous bone in the epiphysis and of trabecular bone in the metaphysis of the distal femur were observed in iron-overloaded rats with a reduction of femoral bone mineral density, i.e. reabsorption of calcium from the proximal tubular epithelial cells of the kidney might be affected and urinary discharge of calcium might be elevated. It was suggested that iron overload gave rise to osteoporosis combined with renal dysfunction and liver iron overload syndrome.

Chronic effect of hyperprolactinemia on blood glucose and lipid levels in mice.

We studied the chronic effects of hyperprolactinemia, induced by ectopic pituitary grafting, on blood glucose and lipid levels in adult male mice. For one year after pituitary grafting, we measured the blood levels of prolactin, growth hormone (GH), insulin, glucose and free fatty acid (FFA) at various intervals. The graft caused consistent hyperprolactinemia without changes in the serum GH levels. Hypoglycemia developed at 1 and 3 months after grafting but was not accompanied by any changes of the serum insulin levels. Thereafter, the blood glucose and serum insulin levels began to increase in the pituitary-grafted (PG) mice, and at 12 months after the operation, both levels became significantly higher in PG mice than controls. The serum FFA levels and the weight of epididymal fat bodies were significantly lower in PG mice than controls from 3-12 months after the grafting. Thus, hyperprolactinemia leads to persistent hypolipidemia and biphasic changes in the blood glucose level.

Suppression of the development of uterine adenomyosis by danazol treatment in mice

Inhibitory effects of danazol, an isoxazol derivative of synthetic steroid 17 alpha-ethinyl-testosterone, on the development of uterine adenomyosis, a pathological disorder of endometrial tissue defined as the presence of endometrial glands and stroma in the myometrium, were investigated in mice of SHN strain. Mice treated with 0.5 microgram danazol for 5 weeks during 4-9 weeks of age and killed at 21 weeks of age showed significantly lower incidence of the spontaneous development of adenomyosis than the age-matched intact control mice. The inhibitory effects of danazol were also evident in mice bearing pituitary isografts which were effective in inducing an early and a high incidence of adenomyosis. Furthermore, the treatment with danazol resulted in the decrease of serum levels of luteinizing hormone (LH) and prolactin (PRL) associated with hypofunction of ovaries and persistent diestrus. These results support the usefulness of danazol for the clinical treatment of gynecological disorders except for hypofunction of ovaries.

Effects of angiogenesis inhibitor TNP-470 on the development of uterine adenomyosis in mice

OBJECTIVE To investigate the effects of angiogenesis inhibitor TNP-470 on uterine microvessels in mice. Pituitary grafting frequently induced uterine adenomyosis. DESIGN In vivo experimental study. SETTING Department of Biological Sciences, University of Tokyo and Medical Research Institute, Tokyo Medical and Dental University. ANIMAL(S) SHN mice, which are known to develop uterine adenomyosis spontaneously, and also very soon after pituitary grafting. INTERVENTION(S) Immunohistochemical study on uterine blood vessels using an antibody to von Willebrand factor in pituitary gland-implanted mice with or without TNP-470. MAIN OUTCOME MEASURE(S) Reduced incidence of uterine adenomyosis. RESULT(S) Twelve of 15 mice developed uterine adenomyosis with dilated blood vessels, but none of the TNP-470-treated mice with shrunken microvessels. The number of bromodeoxyuridine immunoreactive cells and activities of thymidylate synthase and thymidine kinase in uterine tissues were markedly reduced in TNP-470-treated mice. CONCLUSION(S) TNP-470, a potent inhibitor of the development of vascular endothelium, reduced the development of endometrial blood vessels resulting in a lowered incidence of uterine adenomyosis induced by pituitary grafting in mice, and reduced the increase in S-phase cells and enzyme activity for pyrimidine nucleotide synthesis.

Splenomegaly induced by recombinant human granulocyte-colony stimulating factor in rats

Spontaneous ruptures of the spleen have been observed in donors and patients with malignancy during mobilization of peripheral blood stem cells. Thus, we investigated the morphological and histological alteration of the spleen, and mRNA expression levels and activities of the DNA-synthesizing enzymes thymidylate synthase (TS) and thymidine kinase (TK) in the splenic cells, of rats treated with recombinant human granulocyte-colony stimulating factor (rhG-CSF). Daily injections of rhG-CSF for 5 or 7 days slightly enhanced the splenic weight. Single or 3-day treatment with rhG-CSF markedly enhanced the number of granulocytes in peripheral blood. Expression levels of TS and TK mRNA in the splenic cells were significantly increased 6 hours after rhG-CSF treatment. Early expression of TS and TK mRNA in the splenic cells may indicate a reseeding of hematopoietic cells from the bone marrow. Daily injections with rhG-CSF enhanced the TS and TK activities in the splenic cells. As continuous elevations of DNA-synthesizing enzyme activity and spleno-megaly are suggestive of a possible splenic rupture, the monitoring of peripheral granulocytes and splenic size is necessary during the rhG-CSF treatment.

Chemopreventive effect of a vitamin D3 analog, alfacalcidol, on colorectal carcinogenesis in mice with ulcerative colitis

An increased incidence of colorectal carcinoma is known to occur in patients with ulcerative colitis (UC), which displays a cycle of recurrence–remission in the colorectal mucosa. Repeated oral doses of 3% dextran sulfate sodium subsequent to a single intraperitoneal injection of azoxymethane induced a chronic UC resulting in an increased incidence of high-grade dysplasia and submucosal-invasive adenocarcinomas in the mouse colorectum. The active form of vitamin D3 is a calcium-regulating hormone that increases serum calcium levels and intestinal calcium absorption. It has been reported that there is an inverse correlation between serum levels of the active metabolite of vitamin D and colorectal carcinoma stage. The features of the colitis induced in this animal model are very similar to the UC in patients in terms of both clinical and histological characteristics. Treatment with a vitamin D3 analog, alfacalcidol, in mice prevented colitis and carcinogenesis; this is shown by inhibition of the decrease in colorectal length and inhibition of the increased incidence of colorectal dysplasia, with a reduction in the mRNA expression of the DNA-synthesizing enzyme, thymidine kinase, in colorectal tissues.

Down regulation by a low-zinc diet in gene expression of rat prostatic thymidylate synthase and thymidine kinase

BackgroundZinc has a wide spectrum of biological activities and its deficiency is related to various abnormalities of cell metabolism.MethodsWistar male rats, at age of 4 weeks, were fed a low-zinc diet for six weeks. The levels of bromodeoxyuridine incorporated into the prostatic DNA and the mRNA expression levels of prostate thymidylate synthase and thymidine kinase were examined.ResultThe low-zinc diet caused a marked reduction in the body growth and organ weights, resulted in a low hematopoiesis, hypo-albuminemia and hypocholesterolemia. Although there were few differences in plasma biochemical markers, plasma levels of luteinizing hormone and testosterone were reduced by the low-zinc diet. Bromodeoxyuridine-immunoreactive (S-phase) cells and mRNA expression levels of thymidylate synthase and thymidine kinase in the prostate cells were markedly affected by the low-zinc diet.ConclusionA low-zinc diet appears to reduce the body growth and organ weights including prostate, causing low plasma levels of luteinizing hormone and testosterone and reduction in prostate DNA replication in growing-rats.

Decrease in the number of sperm associated with decreased blood testosterone levels in male rats treated with extracts from seven plants consumed by natives of northern Thailand.

Natives of northern Thailand believe that certain plants preserved in alcohol provide sexual elevation although there is no scientific proof. To ascertain whether those plants have any biological effects on reproductive activity, we administered the extracts from seven kinds of plants to male rats. Treatments with these extracts resulted in a decrease in the number of sperm associated with decreased serum testosterone levels and an elevation of serum prolactin levels. The circulating levels of follicle stimulating hormone were only slightly affected by all extracts, although some extracts significantly decreased the luteinizing hormone levels. Thus, the extracts may contain a biologically active substance such as phytoestrogen and lead to a hormonal imbalance.

Effects of Conjugated Estrogens with or without Medroxyprogesterone Acetate on Mammary Carcinogenesis, Uterine Adenomyosis and Femur in Mice

Although combined hormone replacement therapy using estrogens and progestins reduces the risk of endometrial cancer in postmenopausal women, it has been unclear whether the combined therapy is associated with an alteration of the risk of mammary cancer. Virgin mice of the SHN strain, which has a high potential for the development of mammary cancer and uterine adenomyosis, were given diets containing conjugated estrogens with or without medroxyprogesterone acetate for 230 days. The combined administration of conjugated estrogens and medroxyprogesterone acetate completely suppressed the development of uterine adenomyosis with a decrease in uterine thymidylate synthetase activity, significantly enhanced the bone mineral density in the femur and slightly shortened the latent period of mammary carcinogenesis.

EARLY PROGRESSION FROM DIMETHYL SULFOXIDE-INDUCED G0/G1 ARREST IN L1210 CELLS

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G0/G1 arrest in mouse leukemia L1210 cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA‐synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36h. In conclusion, mouse leukemia L1210 cells, which were treated with 1.5% DMSO for 96h, tolerated the treatment and reversed the cell cycle arrest within 36h.