Persistence of vertebral growth plate cartilage in aged cynomolgus monkeys.

Growth plates at each end of vertebral bodies play a pivotal role in longitudinal spinal growth. Epiphyseal closures are formed in adult humans. Although monkeys are frequently employed in bone and disc research, the age of epiphyseal closure has not been well documented. In this study, histological analyses of lumbar vertebral end plates and the surrounding tissue were performed in 11 normal cynomolgus monkeys aged approximately 9 to 15 years, and unclosed growth plate cartilage was detected in all the end plates. The data from this study constitute the first documentation of persistent vertebral growth plate cartilage in cynomolgus monkeys. The persistence of growth plate cartilage in cynomolgus monkeys approximately 15 years of age or younger, which differs from the complete epiphyseal closure exhibited in adult humans, may affect the biomechanical behavior of the spine. This is an important factor to consider in extrapolating the results of spine and intervertebral disc research using cynomolgus monkeys to adult humans.

Anti-angiotensin and hypoglycemic treatments suppress liver metastasis of colon cancer cells.

The effect of diabetic conditions on liver metastasis was examined using CT26 mouse colon cancer cells. CT26 cells produced angiotensin (A)-I and A-II from angiotensinogen; the production was abrogated by inhibitors of renin and chymase. Renin expression and A-II production increased with an increase in the concentration of glucose in the medium. In a streptozotocin-induced BALB/c mouse diabetes model that was fed a high-calorie diet, the blood sugar level increased and was associated with an increasing size and number of CT26 liver metastases. In this diabetic mouse model, liver metastasis of CT26 cells was suppressed by anti-angiotensin treatment with a chymase inhibitor, a renin inhibitor, and an A-II receptor blocker. Moreover, concurrent hypoglycemic and anti-angiotensin treatments showed a synergistic inhibitory effect on CT26 cell liver metastasis. These results suggest that angiotensin activation ability associated with diabetic conditions enhances liver metastasis of colon cancer. Therefore, treatment with anti-angiotensin and hypoglycemic agents might be relevant for baseline management of colon cancer patients with the diabetic condition for the prevention of liver metastasis. This scheme needs to be examined in a clinical setting.

A novel glucokinase activator TMG-123 causes long-lasting hypoglycemia and impairs spermatogenesis irreversibly in rats.

The importance of glucose is well known as an energy source in testes. In order to evaluate the effects of long-lasting hypoglycemia on testes, a novel glucokinase activator, TMG-123, was dosed to rats at 5, 20 and 100 mg/kg for 13 weeks. As a result, plasma glucose levels decreased for several hours with increasing doses over the dose range of 5 to 100 mg/kg. No toxicological findings attributable to the test article were observed in clinical observation, measurements of body weight and food consumption, necropsy, and organ weight measurement. Histopathology showed scattered degeneration of seminiferous tubules in testes, and exfoliation of germ cells related to the degeneration of seminiferous tubules was observed in the lumen of both epididymides in the same animals at the end of the dosing period. Similar histopathological findings were noted at the end of the recovery period. In addition, a fertility study was conducted at the same doses for 13 weeks for males and 5 weeks for females. Sperm analysis showed decreases in the sperm concentration and the motility index and an increase in the incidences of sperm malformations. However, there were no abnormalities in the copulation or fertility rate. These results suggest that long-lasting hypoglycemia in rats is harmful to spermatogenesis and the testicular damage does not recover.

Dietary linoleic acid and glucose enhances azoxymethane-induced colon cancer and metastases via the expression of high-mobility group box 1.

OBJECTIVE: High-mobility group box 1 (HMGB1) was closely associated with progression and metastasis of colorectal cancer. METHODS: We examined the significance of HMGB1 in causing colon carcinogenesis induced by azoxymethane (AOM) injection in Fischer 344 rats fed on a control diet (group C), a 15% linoleic acid (LA) diet (group L), a control diet with 10% glucose drink (group G), and a 15% LA diet with a 10% glucose drink (group L+G). RESULTS: Group L+G showed the highest body weight and calorie intake. Serum and mucosal HMGB1 levels were temporally increased in all groups, while the highest levels were observed in group L+G. Mucosal HMGB1 levels were correlated with cancer multiplicity and nodal metastases. In the AOM-injected rats fed the 15% LA diet with 10% glucose drink, administration of HMGB1 antibody suppressed serum HMGB1 concentration and cancer multiplicity. CONCLUSION: These data suggest that dietary LA and glucose provided the synergistic effect on AOM-induced rat colon cancer through HMGB1 induction.

Linoleic-acid-induced growth suppression induces quiescent cancer cell nests in nude mice.

We examined the effect of linoleic acid (LA) on tumor formation. Cell growth was suppressed by LA in a dose-dependent manner in MKN28 and Colo320 cells. Continuous treatment with LA provided growth arrest in both cells at 5-7 weeks after the treatment. LA-pretreated MKN28 and Colo320 cells showed higher tumorigenicity (9/10 and 10/10, respectively) than nontreated cells (2/10 and 3/10, respectively; p < 0.01) in nude mice. In contrast, LA-pretreated MKN28 and Colo320 cells showed more suppressed tumor growth than nontreated cells (p < 0.01). LA-pretreated MKN28 and Colo320 cells with LA administration after the inoculation did not form macroscopic tumors. Histological examination revealed small cancer cell aggregations, which showed no proliferative activity. In LA-treated MKN28 and Colo320 cells, protein production of Bcl-2 was increased, whereas Bak, EGFR and VEGF levels were decreased. These findings suggest that LA might induce quiescence and subsequent dormancy in cancer cells.

A purified MAA-based ELISA is a useful tool for determining anti-MAA antibody titer with high sensitivity.

Atherosclerosis is widely accepted to be a chronic inflammatory disease, and the immunological response to the accumulation of LDL is believed to play a critical role in the development of this disease. 1,4-Dihydropyridine-type MAA-adducted LDL has been implicated in atherosclerosis. Here, we have demonstrated that pure MAA-modified residues can be chemically conjugated to large proteins without by-product contamination. Using this pure antigen, we established a purified MAA-ELISA, with which a marked increase in anti-MAA antibody titer was determined at a very early stage of atherosclerosis in 3-month ApoE-/- mice fed with a normal diet. Our methods of Nε-MAA-L-lysine purification and purified antigen-based ELISA will be easily applicable for biomarker-based detection of early stage atherosclerosis in patients, as well as for the development of an adduct-specific Liquid Chromatography/Mass Spectrometry-based quantification of physiological and pathological levels of MAA.

Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes.

Endogenous formaldehyde is abundantly present in our bodies, at around 100 µM under normal conditions. While such high steady state levels of formaldehyde may be derived by enzymatic reactions including oxidative demethylation/deamination and myeloperoxidation, it is unclear whether endogenous formaldehyde can initiate and/or promote diseases in humans. Here, we show that fluorescent malondialdehyde-formaldehyde (M2FA)-lysine adducts are immunogenic without adjuvants in mice. Natural antibody titers against M2FA are elevated in atherosclerosis-prone mice. Staining with an antibody against M2FA demonstrated that M2FA is present in plaque found on the aortic valve of ApoE -/- mice. To mimic inflammation during atherogenesis, human myeloperoxidase was incubated with glycine, H2O2, malondialdehyde, and a lysine analog in PBS at a physiological temperature, which resulted in M2FA generation. These results strongly suggest that the 1,4-dihydropyridine-type of lysine adducts observed in atherosclerosis lesions are likely produced by endogenous formaldehyde and malondialdehyde with lysine. These highly fluorescent M2FA adducts may play important roles in human inflammatory and degenerative diseases.

The effects of long-lasting hypoglycemia on male reproductive organs in rats.

Glucose has an important role in spermatogenesis. Nevertheless there are few reports in which the effects of long-lasting hypoglycemia on male reproductive organs have been evaluated. Therefore, insulin was administered subcutaneously at 100, 200, and 400 IU/kg to male rats twice a day for one month. This treatment regimen produced plasma glucose levels that rapidly decreased after treatment, with decreased glucose levels lasting for several hours after each administration on the first and final treatment days. During the treatment period, no abnormalities in clinical signs or body weight were observed. No statistically significant differences were noted in the weights of testes, epididymides, prostates and seminal vesicles, or pituitary glands. Histopathological examination revealed that the insulin-treated animals exhibited degeneration of seminiferous tubules in the testes and exfoliation of germ cells in the lumens of epididymides as a secondary change related to the testicular lesions. The incidences of the histopathological findings were found to be proportional to insulin dose. Sperm analysis of the group receiving the highest dosage indicated that the sperm concentration tended to decrease and the incidences of sperm malformations tended to increase. Our results suggest that long-lasting hypoglycemia affects male reproductive organs in rats.

Effects of estrogens and metabolites on endometrial carcinogenesis in young adult mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine.

The present study assessed effects of estrogens and their steroid metabolites on the endometrial carcinogenesis in young adult mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). A total of 272 female CD-1 (ICR) mice were used and equally divided into 17 groups. Mice were implanted cholesterol pellets to the back subcutis at 9 weeks of age. Pellets contained nothing (control) or one of the experimental agents, three different estrogens and their 13 different steroid metabolites, at a concentration of 0.5% (w/w). At 10 weeks of age, mice were given a single intra-uterine administration of ENNG at a dose of 25 mg/kg body weight. When reaching the 30 weeks of age (20 weeks after the ENNG treatment), mice were sacrificed to assess the development of endometrial proliferative lesions. While endometrial proliferative lesions, including hyperplasias and adenocarcinomas, were observed in all groups, the incidences of hyperplasias in the groups treated with 2-hydroxyestriol, 2-methoxyestradiol, 2-methoxyestriol and 16-epiestriol were significantly higher than that in the control group. On the other hand, adenocarcinomas were significantly developed in the groups treated with estrone, estradiol, estriol, 16beta-hydroxyestrone, 16alpha-hydroxyestrone and 17-epiestriol. These results indicate that, on the endometrial carcinogenesis in mice initiated with ENNG, estrogens and their metabolites belonging to the 16alpha-hydroxylation pathway and the upstream of the 16beta-hydroxylation pathway exert both promoting and progressing effects, whereas, the estrogen metabolites belonging to the 2- and 4-hydroxylation pathways (catechol estrogens) and the downstream of the 16beta-hydroxylation pathway exert only promoting or no effects. It is thus suggested that a metabolic profile of estrogens may be crucial for the endometrial carcinogenesis and that the rate of the 16alpha-hydroxylation may be associated with the increased carcinogenic risks of estrogens on the endometrium.

Experimental chemonucleolysis with recombinant human matrix metalloproteinase 7 in human herniated discs and dogs.

BACKGROUND CONTEXT: Chemonucleolysis has been proposed as a less invasive technique than surgery for patients with lumbar disc herniation. Once chymopapain had been approved as a chemonucleolysis drug, it was withdrawn because of serious complications. A novel agent with fewer complications would be desirable. PURPOSE: The purpose of this study was to investigate the effects of recombinant human matrix metalloproteinase 7 (rhMMP-7) in experimental chemonucleolysis in vitro and in vivo and examine its effects on tissue damage. STUDY DESIGN: The study design is the experimental study using human herniated discs and enzyme substrates in vitro and dogs in vivo. METHODS: The effects of rhMMP-7 on the degradation of human herniated discs were examined by measuring the wet weight in vitro. The correlations between the decrease in wet weight by rhMMP-7 and the conditions associated with herniated discs were also analyzed. The effects of rhMMP-7 on the proteoglycan and water contents were respectively examined with alcian blue staining and T2-weighted magnetic resonance imaging at 7 days after intradiscal injection in dogs. The distribution of [125I]-labeled rhMMP-7 was investigated by autoradioluminography at 7 days after intradiscal injection in dogs. An epidural injection study with rhMMP-7 was performed to evaluate the effects on the tissue damage around the discs at 1 and 13 weeks after the treatment in dogs. The Type 1 and 2 collagen cleavage rates were measured and compared with those of aggrecan in vitro. RESULTS: Recombinant human matrix metalloproteinase 7 concentration dependently decreased the wet weight of herniated discs in vitro. The decrease in wet weight of the discs by rhMMP-7 did not significantly correlate with the conditions associated with herniated discs. Intradiscal injection of rhMMP-7 reduced the proteoglycan and water contents, with an increase in the serum keratan sulfate levels. Radioactivity of [125I]-labeled rhMMP-7 was detected in the nucleus pulposus and annulus fibrosus but not in the muscle. Epidural injection of rhMMP-7 had no effect on the injection site or the nerve tissues. The Type 1 and 2 collagen cleavage rates of rhMMP-7 were 1,000-fold weaker than those of aggrecan. CONCLUSIONS: This study demonstrated experimental chemonucleolysis with rhMMP-7 in vitro and in vivo. The effects of rhMMP-7 were not affected by the conditions associated with herniated discs. The epidural injection study together with the autoradioluminography and in vitro enzyme assay suggests that intradiscal injection of rhMMP-7 may not induce tissue damage around the discs because of its distribution and substrate selectivity. Recombinant human matrix metalloproteinase 7 may be a novel and promising chemonucleolysis agent.

Advanced glycation end products (AGE) induce the receptor for AGE in the colonic mucosa of azoxymethane-injected Fischer 344 rats fed with a high-linoleic acid and high-glucose diet.

BACKGROUND: Advanced glycation end products (AGE) and the receptor for advanced glycation end products (RAGE) are closely associated with colorectal cancer progression. The association between RAGE and AGE in colon carcinogenesis needs to be clarified. METHODS: Levels of RAGE and AGE were examined in azoxymethane (AOM)-injected Fischer 344 rats fed a control diet (Group C), a 15 % linoleic acid (LA) diet (Group L), a control diet with 10 % glucose drink (Group G), and a 15 % LA diet with 10 % glucose drink (Group L + G). Group L + G showed the most pronounced increase of body weight, blood sugar, and serum insulin. RESULTS: The rats in Group L + G showed the most pronounced multiplicity of aberrant crypt foci (ACF) and carcinomas with increased mucosal RAGE and AGE. IEC6 rat intestinal epithelial cells treated with AGE showed increased RAGE expression, which was inhibited by treatment with metformin or losartan. In the AOM-injected rat colon cancer model, the levels of RAGE and AGE, and the multiplicity of ACF and carcinomas, in Group L + G rats were suppressed by treatment with metformin or losartan. CONCLUSIONS: These results suggest that AGE-RAGE induced by high-LA and high-glucose diets substantially enhances colon cancer development; thus, suppression of AGE-RAGE could be a potential target for colon cancer chemoprevention.

Diabetes-associated angiotensin activation enhances liver metastasis of colon cancer.

We examined the effects of hyperglycemic conditions on liver metastasis of colorectal cancer (CRC). Angiotensin (A)-II increased growth, invasion, and anti-apoptotic survival in HT29 and CT26 cells. In contrast, angiotensinogen (ATG) increased these features in HT29 cells but not in CT26 cells. HT29 cells expressed A-II type 1 receptor, chymase, and rennin, whereas CT26 cells did not express renin. Renin expression and ATG-induced cell growth, invasion, and survival induced and increased as glucose concentration increased in HT29 cells and also CT26 cells. An inhibitor of renin or chymase abrogated A-II production in HT29 cells. Reduction of hepatic ATG production by cholesterol-conjugated antisense S-oligodeoxynucleotide suppressed liver metastasis of HT29 cells. An examination of 121 CRC patients showed that diabetes in CRC cases was associated with higher blood HbA1c, higher renin and A-II concentrations in the primary tumors, and higher incidence of liver metastasis than in nondiabetic cases. These results suggest that diabetes-associated angiotensin activation enhances liver metastasis of CRC and may therefore provide a possible target for antimetastatic therapy in CRC.

Inhibitory effect of linoleic acid on transformation of IEC6 intestinal cells by in vitro azoxymethane treatment.

The effect of linoleic acid (LA) on growth and transformation of IEC6 intestinal cells was examined. IEC6 cells expressed mRNAs of 15-lipooxygenase (LOX15) and peroxisome proliferator-activated receptor (PPAR)gamma but not COX-2. Cell growth was suppressed by LA in a dose-dependent manner in IEC6 cells. Three-week treatment with LA provided IEC6 cells a quiescent state. LA-induced growth inhibition was abrogated by exposure to antisense S-oligodeoxynucleotides (S-ODNs) for LOX15 and/or PPARgamma. In an in vitro carcinogenesis model, IEC6 cells, which had confirmed CYP2E1 expression and activity, were continuously treated with AOM and/or LA for 40 weeks. DNA injury in AOM-treated cells was suppressed to the control level by concurrent LA treatment. Colony formation of AOM-treated cells in soft agar was suppressed by treatment with LA, which was reversed by exposure to antisense S-ODNs for LOX15 and/or PPARgamma. AOM-treated IEC6 cells formed s.c. tumors in 9 of 12 mice, whereas AOM+LA-treated cells formed no tumor. IEC6 cells showed no remarkable alteration of protein production by AOM treatment, whereas cells treated with AOM+LA showed decreased epidermal growth factor receptor (EGFR) and phospho-EGFR and increased BAX. These findings suggest that LA inhibited AOM-induced transformation of COX-2-negative IEC6 cells, which was possibly mediated with PPARgamma ligands generated by LOX15 from LA.

A case report of a choroid plexus carcinoma spontaneously occurring in the right lateral ventricle of a 14-week-old, female Donryu rat.

We encountered a brain tumor arising in the right lateral ventricle of a 14-week-old, female Donryu rat and investigated its histological and immunohistochemical characteristics. Macroscopically, the tumor appeared as a grayish mass with a size of 10 mm in diameter, present in front of the right hemicerebrum and well circumscribed on the cut surface. Histological examination revealed the tumor to be a hypercellular mass occupying the front part of the right lateral ventricle and expanding into the area in front of the hemicerebrum, continuing to the ependymal area at its edge. The tumor was constituted by columnar- or pleomorphic-shaped, highly atypical cells of epithelial origin surrounding fibrovascular cores as single or multiple cell layers. Growth was papillary with high proliferating activity. Immunohistochemically, the tumor cells proved positive for cytokeratin but negative for vimentin, S100 protein or glial fibrillary acidic protein, a profile characteristic for the epithelial cells of the choroid plexus, whereas the ependymal cells were found to be positive for all 4 items. In conclusion, the present tumor was diagnosed as a rat choroid plexus carcinoma, only the third such case to be reported in the world literature, with particular features.

Maternal exposure to low doses of bisphenol a has no effects on development of female reproductive tract and uterine carcinogenesis in Donryu rats.

Effects of maternal exposure to low doses of bisphenol A (BPA), including those comparable with human exposure levels, on growth and development of the female reproductive system and uterine carcinogenesis in Donryu rats were investigated. Dams were administered BPA (0, 0.006 and 6 mg/kg/day) daily by gavage from gestation day 2 up to the day before weaning (postnatal day 21 at offspring). The serum levels of BPA were significantly elevated in the dams receiving 6 mg/kg/day, however, BPA levels in the milk of dams, and those in the serum and liver of offspring were similar between control and treated groups. The treatment did not exert any influences on uterine development including weight, gland genesis and estrogen receptor alpha expression, vaginal opening and gonadotropin secretion in the female offspring up to puberty. After maturation, no effects were evident with regard to estrous cyclicity in female offspring treated with BPA. In addition, the treatment had no effects on age-related morphological changes of the reproductive and endocrine organs and uterine carcinogenesis until 15 months of age. The results demonstrate that maternal exposure to BPA at levels comparable to human exposure did not have any effects on the female reproductive system of offspring in rats. In addition, BPA was also found in the serum, milk and liver of control dams and pups, and low levels of BPA were detected in drinking water and pellet diet. The present study showed that the experimental animals were also exposed to environmental BPA in the animal room.

Promotion, but not progression, effects of tamoxifen on uterine carcinogenesis in mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine.

Effects of tamoxifen (TAM) on development of uterine endometrial carcinogenesis were studied in intact and ovariectomized (OVX) mice initiated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). In experiment I, animals were implanted with cholesterol (ChL, controls) or TAM (5% w/w) and/or 17beta-oestradiol (E(2), 0.5% w/w) pellets s.c. from 9 to 25 weeks of age, until the termination of the experiment, and all received a single intra-uterine administration of ENNG (12.5 mg/kg) at 10 weeks of age. They were divided into four groups: ENNG + ChL (control), ENNG + TAM, ENNG + E(2) and ENNG + TAM + E(2). Endometrial proliferative lesions (hyperplasias and/or carcinomas) were observed in all groups, the incidences in the TAM- and/or E(2)-treated groups being two times higher than in the ChL-treated control animals. High induction (11/20, 55%) of adenocarcinomas was observed in the E(2) group but this was significantly decreased in combination with TAM (2/20, 10%), no carcinomas being found in the TAM group. In experiment II, animals pre-treated with TAM (10 weeks) and receiving E(2) post-treated (4 weeks) developed adenocarcinomas, although no cancers were observed in mice treated by ChL instead of TAM. In animals pre-treated with TAM and post-treated with ChL or TAM, no adenocarcinomas were also developed. In OVX mice (experiment III), proliferative lesions were observed in the TAM- and/or E(2)-treated groups, at incidences significantly higher than in ChL-treated animals, in which these lesions were completely absent. However, no adenocarcinomas were found, only slight hyperplasias being observed in the TAM group, although the incidence of adenocarcinoma was highest in the E(2) alone group, and significantly decreased in combination with TAM, as in experiment I. These results indicate that TAM may itself exert promotion effects, while exhibiting an anti-progression influence on uterine carcinogenesis in adult mice initiated by ENNG and receiving E(2).