ENZYMATIC ACTIVITY IN MICROSOMES, LIPID PEROXIDATION OF MICE HEPATOCYTES UNDER THE SODIUM FLUORIDE.

Fluorine is one of the most widespread and necessary microelements for the body of animals and humans, which is necessary in a clearly limited amount. Different concentrations of fluorine can affect the state of lipid peroxidation, as well as the functional state of the microsomes of liver hepatocytes. The studies were carried out on mature Wistar rats weighing 180-220 g. Animals were inoculated with an aqueous solution of sodium fluoride once a day for 60 days at doses of 1/10, 1/100 and 1/1000 DL50, which was 20 mg/kg, 2 mg/kg and 0,2 mg/kg body weight. Control rats received drinking water. Each group consisted of 10 animals, the studies of indicators were carried out on days 10, 20, 30, 50 and 60. The induction of free radical processes by sodium fluoride was confirmed using a chemiluminescent reaction of blood serum, the amount of diene conjugates in rat liver tissue homogenates was assessed spectrophotometrically, the content of TBA reactants in rat liver tissue homogenates was determined by the reaction of malondialdehyde and thiobarbituric acid (TBA). The level of chiff bases was determined with a spectrofluorometer, subcellular fractions of the liver were isolated by the method of differentiated centrifugation. An increase in the level of the indicator at a dose of 1/10 and 1/100 DL50 of the intensity of lemiluminescence on the 30th day and its decrease on the 60th day was established.Increase in the activity of NAD (P) H-cytochrome c reductase in the microsomal fraction of the liver at the beginning of the study and a gradual decrease on the 50th and 60th days when using both dosages. The same dynamics was observed for NADH-cytochrome c reductase. With respect to diene conjugates of TBA-reactants and chiff bases, a tendency to increase was observed at all periods of the experiment. Indicators of cytochrome P-450 cytochrome b5 were increased up to 30 days and gradually decreased by 60 days. Long-term administration of sodium fluoride can cause the formation of toxic products and a decrease in the activity of enzymes of the microsomal membrane of hepatocytes.

Evaluation of serum hepcidin and iron levels in patients with PCOS: a case-control study.

PURPOSE: Polycystic ovary syndrome (PCOS) is the most common cause of chronic anovulation with a prevalence of 5-10% in women of reproductive age. The etiology of this disease is not well known, and hepcidin is one of the factors affecting the pathogenesis of the disease. The aim of this study was to evaluate plasma levels of hepcidin in patients with PCOS and its correlation with serum iron level. METHODS: In this case-control study, plasma levels of hepcidin, IL-6, and ferritin using ELISA method and serum iron levels using a spectrophotometric method were tested on 56 women with PCOS (case group) and 41 healthy subjects (control group). The results were analyzed using t test, General Linear Model, Binary logistic regression, and linear regression tests. RESULTS: The mean hepcidin levels were 1.97 ± 0.53 and 2.40 ± 0.25 pg/ml in the case and control groups, respectively. The t-test results showed significant difference between the two groups (p = 0.0001). The mean serum iron levels were 72.89 ± 28.97 and 70.62 ± 31.18 g/dl in the case and control groups, respectively. The t test analysis indicated no significant difference between the two groups. The serum ferritin and iron levels had no significant relation with serum hepcidin level in two groups. CONCLUSION: Despite the differences in the serum levels of hepcidin between the two groups, no significant relation was observed between serum iron levels and hepcidin level in this group of patients. This implies the need for more comprehensive studies on gene expression in hepcidin and iron pathways using real-time and Western techniques to investigate more precisely serum hepcidin level and its relationship with the factors mentioned.

Gamma-secretase inhibitor does not modulate angiogenesis in colon adenocarcinoma in obese mice.

BACKGROUND: Notch is a signaling molecule which plays a role in angiogenesis and γ-secretase is required for processing of Notch. In this study, we investigated the effect of γ-secretase inhibitor (DAPT) on tumor angiogenesis in diet-induced obese mice. METHODS: 18 mice were divided into three groups; control, obese (diet-induced) and obese+DAPT. After 15 weeks, the obese mice were subjected for tumor induction of CT26 colon adenocarcinoma cells (5 x 105 cells). When the tumor size reached approximately 350 ± 50 mm3, half of the obese animals received DAPT (10mg/kg/day) subcutaneously. Blood samples were taken after 14 days and the tumors harvested for immunohistochemical staining and capillary density were reported as CD31 positive cells/mm2. RESULTS: The obese animals had higher serum leptin and NO concentrations, while, serum VEGF and VEGFR-1 concentrations were not different compare to control group. Administration of DAPT in obese mice significantly reduced serum VEGFR-1 and leptin concentrations and increased serum NO level (p < 0.05). Capillary density in the tumors of obese animals was not different compare to control groups. DAPT administration could not alter capillary density in the tumors. CONCLUSION: Administration of DAPT in obese mice altered serum angiogenic factors, however, it could not modulate tumor angiogenesis in diet-induced obese mice (Fig. 4, Ref. 26).

Systemic administration of ghrelin did not restore angiogenesis in hindlimb ischemia in control and diet-induced obese mice.

BACKGROUND: Ghrelin is a novel growth hormone releasing peptide that mainly regulates food intake and energy homeostasis, however, recently, it is indicated that it may be closely related with physiological and/or pathological angiogenesis. OBJECTIVES: The objective of the present study was to evaluate the effect of systemic ghrelin administration on angiogenesis in hindlimb ischemia in normal and diet-induced obese mice. METHODS: 24 male C57BL/6 mice were fed with high-fat diet (HFD) or standard for 14 weeks. Then, the mice underwent unilateral hindlimb ischemia. Next, each group was divided into the two subgroups: treatment with ghrelin (100 µg/kg, twice daily, Sc) or without treatment. After 10 days, the animals were sacrificed, blood samples were taken and the gastrocnemius muscles removed. RESULTS: There was no significant difference in capillary/fiber ratio in hind limb ischemia between obese and control groups. Administration of ghrelin reduced serum nitric oxide (NO) and leptin and increased vascular endothelial growth factor (VEGF) concentrations in obese mice, however, did not change the capillary/fiber ratio in ischemic legs. CONCLUSION: Systemic administration of ghrelin did not restore angiogenesis in hindlimb ischemia in control and diet-induced obese mice (Fig. 4, Ref. 35).

Pan-PPAR Agonist, Bezafibrate, Restores Angiogenesis in Hindlimb Ischemia in Normal and Diabetic Rats.

Introduction. The aim of this study was to investigate the effect of bezafibrate as a pan-PPAR agonist on angiogenesis and serum nitrite, the main metabolite of nitric oxide (NO), vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) concentrations in hindlimb ischemia model of normal and type I diabetic rats. Methods. 28 male Wistar rats were divided into control and diabetic groups. Then, all rats underwent unilateral hindlimb ischemia. After recovery, they were randomly assigned to one of the following experimental groups: (1) control; (2) control + bezafibrate (400 mg/kg/day); (3) diabetic; (4) diabetic + beztafibrate. After three weeks, blood samples were taken and capillary density was evaluated in the gasterocnemius muscle of ischemic limb. Results. Bezafibrate increased capillary density and capillary/fiber ratio in ischemic leg of diabetic and control rats (P < 0.05). Serum VEGF and VEGFR-2 concentrations did not alter after bezafibrate administration, however, serum nitrite concentration was significantly higher in bezafibrate-treated groups than non-treated groups (P < 0.05). Discussion. It seems that bezafibrate, as a pan PPAR agonist, restores angiogenesis in hindlimb ischemic diabetic animals and is useful for prevention and/or treatment of peripheral artery disease in diabetic subjects.

Role of peroxisome proliferator-activated receptor ß agonist on angiogenesis in hindlimb ischemic diabetic rats.

INTRODUCTION: Studies indicated that PPARß agonists play a role in modulation of angiogenesis. In this study, we evaluated the effect of specific PPARß agonist, GW0742, on angiogenesis and serum vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and nitrite concentrations in hindlimb ischemia in normal and diabetic rats. METHODS: Hindlimb ischemic rats were divided into four groups: control, diabetic, control, and diabetic treated with GW0742 (n=7 each). Diabetes was induced by injection of streptozotocin (55mg/kg, ip). GW0742 was injected 1day after surgery (1mg/kg, sc). After 21days, blood samples were taken, and gastrocnemius muscles were harvested for immunohistochemistry. RESULTS: GW0742 significantly increased serum nitrite and VEGFR-2 concentrations and VEGF-to-VEGFR-2 ratio in control and diabetic rats. Capillary density was lower in diabetic animals compared to the control, and GW0742 significantly restored the capillary density in the control and diabetic hindlimb ischemic rats. CONCLUSION: PPARß agonists restore skeletal muscle angiogenesis and can be considered for prevention and/or treatment of peripheral vascular complications in diabetic subjects.

The effect of mebudipine on cardiac function and activity of the myocardial nitric oxide system in ischaemia-reperfusion injury in rats.

OBJECTIVES: Previous studies have suggested that failure of the synthesis of nitric oxide is involved in the pathophysiology of myocardial ischaemia-reperfusion injury. In this study, we investigated the effect of mebudipine, a new dihydropyridine calcium channel blocker, on cardiac function and activity of the myocardial nitric oxide system in ischaemia-reperfusion injury in isolated rat hearts. METHODS: Forty male Wistar rats (250-300 g) were divided into four groups (n = 10): sham, control, vehicle and drug groups. The animals were anesthetised with sodium pentobarbital (6 mg/kg intraperitoneal). The hearts were quickly removed, mounted on a Longendorff apparatus and perfused with Krebs-Henseleit solution under constant pressure at 37°C. After 20 min stabilisation period, the ischaemic groups received 30 min global ischaemia and 120 min reperfusion. For the drug and vehicle groups, before ischaemia the hearts were perfused with mebudipine (10(-3) µM) or ethanol-enriched solution (0.01%) for 25 min, respectively. Myocardial function, and creatine kinase, lactate dehydrogenase and total nitric oxide metabolite (nitrite and nitrate) levels were analysed. RESULTS: Cardiac functions had recovered significantly in the mebudipine group (p < 0.01). Furthermore, mebudipine remarkably reduced the levels of lactate dehydrogenase and creatine kinase in the coronary effluent and increased myocardial nitric oxide metabolite levels compared with the control group. CONCLUSION: Our results indicate that mebudipine reduced the intensity of myocardial ischaemia-reperfusion injury, and that activation of the myocardial nitric oxide system played an important role in this regard.

Efficacy of intra-hepatectomy 5-FU on recurrence and metastasis of human hepatocellular carcinoma in nude mice.

A novel intra-operative chemotherapy nude mouse model for human hepatocellular carcinoma (HCC) has been developed. Intra-peritoneal (i.p.) administration of 5-fluorouracil (5-FU) was begun 2 hr before hepatic resection of HCC and then continued post-operatively for 4 consecutive days. This regime, termed intra-hepatectomy chemotherapy (IHC), significantly prolonged animal survival compared with pre-operative 5-FU, neoadjuvant therapy, 5-FU post-operative adjuvant therapy, surgery alone, 5-FU without surgery, and the untreated control. The median survival of the intra-operative 5-FU-treated group was 127 days compared with 78 days for the neoadjuvantly-treated animals and 53 days for the control group (p<0.006). When all animals with neoadjuvant 5-FU treatment had died, 60% of the animals in the IHC group were still alive (p<0.011). Survival of all other treatment groups, including 5-FU without surgery, surgery alone, and adjuvant post-operative chemotherapy, was not significantly different from the untreated control group. Five animals in the IHC group were free of tumor when sacrificed at day 150 post-surgically. While 100% of animals in the control group had lymph nodes draining the liver involved with metastases, only 20% of animals in the IHC group had lymph node metastases. These data suggested that IHC therapy increased survival by preventing metastases of cancer cells not removed in the liver resection procedure. The results of this study indicate that IHC therapy for resection of HCC should be investigated clinically.

Antimetastatic intraoperative chemotherapy of human colon tumors in the livers of nude mice.

We have developed a new antimetastatic chemotherapeutic strategy for combination with hepatic resection of human colon cancers in a high-metastasis nude mouse model. The new procedure involves i.p. administration of 5-fluorouracil (5-FU) 2 h before hepatic resection of the human colon tumors, with therapy continued postoperatively for 4 consecutive days. We termed this strategy neo-neoadjuvant chemotherapy. The regime significantly prolonged animal survival compared with preoperative 5-FU neoadjuvant therapy, 5-FU postoperative adjuvant therapy, surgery alone, 5-FU without surgery, or the untreated control. The median survival of neo-neoadjuvant i.p. 5-FU-treated group was 81 days, compared with 27 days for the control group (P < 0.009). The median survival of animals in the neoadjuvant group was 37 days (P < 0.021 compared with the control group). There was also a significant difference between the median survival of neo-neoadjuvant, and the neoadjuvant group (P < 0.031). When all animals in the control group had died, 70% of animals with neo-neoadjuvant and 60% of animals with neoadjuvant 5-FU were still alive (P < 0.003 and P < 0.011, respectively). When all animals with neoadjuvant 5-FU treatment had died, 70% of animals with neo-neoadjuvant treatment were still alive (P < 0.003). Survival of all other treatment groups, including 5-FU without surgery, surgery alone, and adjuvant postoperative chemotherapy, was not significantly different from the untreated control group. Two animals in the neo-neoadjuvant group were free of tumors when sacrificed at days 154 and 165 post surgery. Whereas 100% of animals in the control, 90% in the 5-FU alone, 70% in the surgery alone, 60% in the 5-FU adjuvant, and 40% in the neoadjuvant groups had metastases in the lymph nodes draining the liver, only 10% of animals in the neo-neoadjuvant group had metastases. These data suggest that the neo-neoadjuvant therapy increased survival by preventing metastasis of cancer cells not removed in the liver resection procedure. The results of this study indicate that the neo-neoadjuvant treatment strategy for resection of colon cancer liver metastasis should be explored clinically.

An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.

Whether liver metastases from colon cancer are capable of metastasizing to other sites is an important question in surgical oncology. To answer this question, we have developed a highly metastatic orthotopic transplant model of a liver metastasis from a human colon cancer patient in nude mice that targets the liver and lymph nodes. The metastatic human tumor was transplanted in athymic nude mice by surgical orthotopic implantation (SOI) of a liver metastasis from a colon cancer patient. The human colon tumor was then subsequently implanted in the colon by SOI or, in an additional series of nude mice, in the liver by surgical hepatic implantation (SHI). The mice were then explored over time for lymph node involvement beginning 10 days after implantation. After SOI, 100% of the animals had liver metastasis within 10 days, and subsequently, 19 days after SOI, all lymph nodes draining the liver were involved with metastasis without any retroperitoneal or lung tissue involvement. After SHI, all sites of lymphatic drainage of the liver, including portal, celiac, and mediastinal lymph nodes, were massively involved by metastasis in 100% of the animals as early as 10 days after tumor implantation on the liver. The results of this study demonstrate that liver metastases from colon cancer are capable of remetastasizing to other sites. This study thus suggests that in colon cancer patients with liver metastasis, mediastinal, celiac, and portal lymph node metastases originate from the liver metastasis and not, as previously thought, from primary colon cancer.

A nude mouse model of massive liver and lymph node metastasis of human colon cancer.

Liver and lymph nodes metastasis are the main causes of treatment failure for advanced colon cancer. However, currently-available animal models of human colon cancer do not demonstrate sufficient metastasis to represent highly malignant colon cancer that extensively metastasizes to these sites. A liver metastasis from a patient with highly malignant, poorly differentiated adenocarcinoma of the colon was established in nude mice by surgical orthotopic implantation to the mouse colon. The human origin of the tumor growing in nude mice was confirmed by in situ hybridization of human DNA. After 20 passages from the first implantation, massive liver and lymph nodes metastasis, occurred in 100% of the transplanted animals. Lymph nodes metastasis were found at the sites of lymph node drainage of the liver: celiac, portal and mediastinal lymph nodes. However no mesenteric and retroperitoneal nodes or lung tissue metastases were observed. Our data suggest that the mediastinal, celiac and hepatic lymph nodes metastases are derived form the liver metastasis, confirming the concept of metastasis of metastases or "remetastasis" of colon cancer.

A highly metastatic Lewis lung carcinoma orthotopic green fluorescent protein model.

The Lewis lung carcinoma has been widely used for many important studies. However, the subcutaneous transplant or orthotopic cell-suspension injection models have not allowed the expression of its full metastatic potential. A powerful new highly metastatic model of the widely-used Lewis lung carcinoma is reported here using surgical orthotopic implantation (SOI) of tumor fragments and enhanced green fluorescent protein (GFP) transduction of the tumor cells. To achieve this goal, we first developed in vitro a stable high-expression GFP transductant of the Lewis lung carcinoma with the pLEIN retroviral expression vector containing the enhanced Aequorea victoria GFP gene. Stable high-level expression of GFP was found maintained in vivo in subcutaneously-growing Lewis lung tumors. The in vivo GFP-expressing tumors were harvested and implanted as tissue fragments by SOI in the right lung of additional nude mice. This model resulted in rapid orthotopic growth and extensive metastasis visualized by GFP-expression. 100% of the animals had metastases on the ipsilateral diaphragmatic surface, contralateral diaphragmatic surface, contralateral lung parenchima, and in mediastinal lymph nodes. Heart metastases were visualized in 40%, and brain metastases were visualized in 30% of the SOI animals. Mice developed signs of respiratory distress between 10-15 days post-tumor implantation and were sacrificed. The use of GFP-transduced Lewis lung carcinoma transplanted by SOI reveals for the first time the high malignancy of this tumor and provides an important useful model for metastasis, angiogenesis and therapeutic studies.

Antimetastatic efficacy of adjuvant gemcitabine in a pancreatic cancer orthotopic model.

Gemcitabine is a promising new agent that has been recently studied for palliation of advanced (stage IV) unresectable pancreatic cancer. We hypothesized that adjuvant gemcitabine would reduce recurrence and metastases following surgical resection of pancreatic cancer. To test this hypothesis, we evaluated gemcitabine on a green fluorescent protein (GFP) transductant of the human pancreatic cancer cell line BxPC-3 (BxPC-3-GFP) using surgical orthotopic implantation (SOI) in nude mice. GFP enabled high resolution fluorescent visualization of primary and metastatic growth. Five weeks after SOI, the mice were randomized into three groups: Group I received exploratory laparotomy only. Group II underwent surgical resection of the pancreatic tumor without further treatment. Group III underwent tumor resection followed by adjuvant treatment with gemcitabine, 100 mg/kg every three days for a total of four doses, starting two days after resection. The mice were sacrificed at thirteen weeks following implantation and the presence and location of recurrent tumor was recorded. Gemcitabine reduced the recurrence rate to 28.6% compared to 70.6% with resection only (P = 0.02) and reduced metastatic events 58% in the adjuvant group compared to resection only. This study, demonstrating that gemcitabine is effective as adjuvant chemotherapy post-pancreatectomy, suggests this new indication of the drug clinically.

Efficacy of recombinant methioninase in combination with cisplatin on human colon tumors in nude mice.

The present treatment of colon cancer is based on 5-fluorouracil (5-FU). Despite promising results of combining leucovorin or levamisole with 5-FU, the 5-year survival rate of patients with advanced colon cancer has not increased significantly. Colon tumors in vitro have been shown previously to have an elevated requirement for methionine, suggesting a new therapeutic target. In this study, targeting the methionine dependence of colon tumors is effected by recombinant methioninase (rMETase), alone and in combination with cisplatin (CDDP). In vitro results demonstrated that CDDP and rMETase act synergistically on the human colon cancer cell line SW 620, with a combination index (CI) of 0.45, as well as on the human colon cancer cell line Colo 205 with a CI of 0.7. Human colon cancer lines HCT 15, HT 29, Colo 205, and SW 620 growing in nude mice were treated with rMETase to determine an effective dose for depletion of tumor methionine. rMETase at 15 units/g/day for 5 days depleted tumor methionine in all four tumor types to approximately 30% of untreated control. rMETase alone arrested growth of HCT 15 and HT29 in nude mice for 1 week after treatment termination. Colo 205 and SW 620 were partially arrested by rMETase. However, CDDP in combination with rMETase resulted in tumor regression of Colo 205 and growth arrest of SW 620 in nude mice. The ratio of the treated:control group (T:C) tumor weights for Colo 205 was 8% when CDDP was given on day-5, followed by treatment on days 5-9 with rMETase. This treatment schedule resulted in two of the six animals having no detectable tumor when the experiment was terminated on day 16. SW620 was resistant to CDDP alone and only partially sensitive to rMETase alone. However, when SW 620 was treated with rMETase from days-5 to -9 and CDDP on day-5, tumor growth was arrested. The results demonstrate that rMETase used simultaneously in combination with CDDP had significant antitumor efficacy in colon cancer in vitro and in vivo. The data suggest a novel and promising therapeutic approach by targeting the elevated methionine dependence of colon cancer.

Minimal liver resection strongly stimulates the growth of human colon cancer in the liver of nude mice.

Partial hepatectomy has been widely employed in clinical practice as the therapy of choice for primary and metastatic liver tumors. However, the recurrence rate after the treatment remains high, which is most likely due to the growth of residual microscopic lesions. Previous studies in murine models demonstrated that a 70% hepatectomy significantly accelerated the growth of ectopically implanted tumors. In this study, we reported the effect of partial hepatectomy on the growth of two human colon cancers (Co-3 and AC3603) implanted in the liver of nude mice using the technique of surgical implantation of histologically intact tumor tissue. Our results showed a dramatic acceleration of tumor growth following 30% partial hepatectomy, which resembles clinical procedures. Tumor volumes were assessed with calipers on day-15 by abdominal palpation and on day-30 at autopsy by direct measurement. For both Co-3 and AC3603, tumor volumes in the hepatectomized animals were significantly larger than the control at the above two time points (P < 0.001). The results demonstrate the stimulating effect of partial hepatectomy directly on the tumor growth in the liver, in contrast to previous studies on ectopic tumors. Furthermore, since conservative partial hepatectomy (30%) is normally used in clinical practice for surgical treatment of liver metastasis, the animal models presented here should be useful for the clinical investigation of the high recurrence rate of liver metastasis following partial hepatectomy.

Factors predicting chronic hepatic encephalopathy after distal splenorenal shunt: a multivariate analysis of clinical and hemodynamic variables.

BACKGROUND: This study was aimed at evaluating several factors that promote chronic hepatic encephalopathy by multivariate analysis of data for patients with cirrhosis with good or moderate liver function submitted to distal splenorenal shunts. METHODS: The study group comprised 131 patients: 55 had alcoholic and 76 nonalcoholic cirrhosis. Seventy patients were in Child's class A and 61 in class B. Cerebral function was assessed by a complete neurologic examination. Angiography with venous phase was performed before and within 1 month after the shunt operation. In 84 cases the original Warren technique was used and in 20 cases a Britton's modified procedure was used. Twenty-seven patients had distal splenorenal shunts with a splenopancreatic disconnection. Statistical analysis was performed by two multivariate analyses based on stepwise selection. RESULTS: Thirty-nine patients died during a follow-up period of 51 +/- 32 months. Chronic encephalopathy occurred in 18 patients (14%). According to the multivariate analysis of the preoperative prognostic factors, only age (p = 0.0001) and albumin values (p = 0.0002) were independent predictive risk factors for chronic encephalopathy. In the multivariate analysis concerning the hemodynamic consequences of the selective shunts, independent risk factors promoting chronic encephalopathy were postoperative portal perfusion (p = 0.0001), postshunt portal pressure (p = 0.001), and surgical disconnection (p = 0.0064). CONCLUSIONS: Our study has shown that chronic encephalopathy after selective shunt surgery is promoted by both clinical and hemodynamic factors. A better selection of the candidates for shunt surgery and prevention of the development of portal malcirculation by accurate surgical disconnection should further decrease the risk of chronic encephalopathy.