Ação genotóxica e antiproliferativa de Polygonum punctatum Elliott (Polygonaceae) sobre o ciclo celular de Allium cepa L / Genotoxic and antiproliferative action of Polygonum punctatum Elliott (Polygonaceae) on the cell cycle of Allium cepa L

RESUMO: A espécie Polygonum punctatum Elliott (Polygonaceae) é amplamente utilizada pela população como planta medicinal. O objetivo deste trabalho é o de avaliar o potencial genotóxico e mutagênico de P. punctatum utilizando raízes de bulbos e radículas em sementes germinadas de Allium cepa através do teste in vivo, e realizar comparações da extração do material vegetal por calor (infusões) e extração a frio (extrato). Para isso, foram preparadas dois tipos de soluções, infusões e extratos foliares de P. punctatum, em duas concentrações 0,4 g mL-1 e 2,4 g mL-1. A infusão foi preparada pela adição das folhas secas em água destilada fervente (100ºC), permanecendo por 10 minutos enquanto o extrato foi preparado através da maceração das folhas secas em água destilada fria. Para o teste em A. cepa foram utilizados, para cada tratamento, seis grupos de quatro bulbos e seis caixas gerbox com 50 sementes em cada caixa. Duas lâminas para cada tratamento foram obtidas através da técnica de esmagamento das raízes e coradas com orceína acética 2%. Foram contadas 2000 células por grupo de bulbos e 3000 células por grupo de sementes, observando-se a ocorrência de interrupções em metáfases, alterações cromossômicas estruturais, bem como a inibição ou aumento da divisão celular. Os valores do índice mitótico foram calculados e analisados estatisticamente pelo Teste χ2 (p≤0,05). Os resultados demonstraram que as infusões e os extratos de folhas apresentaram redução nos valores de índices mitóticos nas concentrações utilizadas em relação ao controle em água destilada. Foram identificadas alterações cromossômicas na divisão celular, tais como pontes anafásicas, em todas as concentrações de infusões e extratos indicando assim que P. punctatum possui atividade antiproliferativa e genotóxica.

ABSTRACT: The species Polygonum punctatum Elliott (Polygonaceae) is widely used by the Brazilian population as a medicinal plant. The aims of this study are to evaluate the genotoxic and mutagenic potential of P. punctatum, using its root bulbs and rootlets in germinated seeds of Allium cepa by in vivo testing, and to compare the extraction of plant material by heat (infusions) and cold (extract). Thus, two types of solutions - infusions and leaf extracts - of P. punctatum were prepared at the two concentrations of 0.4 g ml -1 and 2.4 g mL- 1. The infusion was prepared by addition of dry leaves in boiling distilled water (100ºC), remaining for 10 minutes, while the extract was prepared by maceration of dried leaves in cold distilled water. For the A. cepa, we used for each treatment six groups of six bulbs and six seedling boxes with 50 seeds each. Two slides for each treatment were obtained by the technique of crushing the roots, and they were stained with 2 % acetic orcein. For the analysis, 2000 cells per group of bulbs and 3000 cells per group of seeds were counted, and we noted the occurrence of interruptions in the metaphase, chromosomal aberrations, as well as inhibited or increased cell division. The values of the mitotic index were calculated and statistically analyzed by the χ2 test (p ≤ 0.05). The results showed that the infusions and extracts of leaves showed reduced values of mitotic indices in the concentrations used compared to the control in distilled water. Chromosomal alterations were identified in the cell division, in all concentrations of infusions and extracts, thus indicating that P. punctatum has an antiproliferative and genotoxic activity.

Treatment of hyperhomocysteinaemia in haemodialysis patients at high cardiovascular risk.

OBJECTIVES: Cardiovascular disease (CVD) is the main cause of death among haemodialysis (HD) patients. Emerging cardiovascular risk factors such as oxidative stress and chronic inflammation are involved in these patients together with traditional risk factors. Here we investigate the effects of a short-term folate treatment on some markers of chronic inflammation in two groups of HD patients with and without vascular occlusive disease (VOD). PATIENTS AND METHODS: Homocysteine (HCy), C-reactive protein (CRP), Folate, fibrinogen and alpha1 acid glycoprotein (alpha1AGP) were dosed before and after a 3-month course of high-dose folate (25 mg intravenous calcium laevofolinate pentahydride once weekly) and again after a one-month washout in 15 HD patients with established VOD (group A) and in 15 comparable HD patients with no diagnosis of VOD (group B). RESULTS: Baseline HCy and CRP were significantly elevated in patients of both groups A and B compared to normal values. Folate treatment significantly reduced HCy in patients of both groups A and B and alpha1AGP only in patients of group A, while the other markers were not modified. After the one-month washout a significant raise of CRP could be observed in patients of group A; again, the other markers were not modified. CONCLUSIONS: Our results suggest that significant reduction of serum HCy can be achieved in both patients with or without VOD after administration of high-dose folic acid. Hence, folic acid supply is useful in the treatment of hyperhomocysteinemia in HD patients, although it is not sufficient to modify their chronic inflammatory status.

N-3 PUFAs reduce oxidative stress in ESRD patients on maintenance HD by inhibiting 5-lipoxygenase activity.

Reactive oxygen species formation and release of pro-inflammatory/pro-atherogenic cytokines, that is, interleukin 1-beta and tumor necrosis factor-alpha, need the activation of the arachidonic acid cascade via the enzyme 5-lipoxygenase (5-Lox). 5-Lox activity and expression are significantly increased in peripheral blood mononuclear cells (PBMCs) of end-stage renal disease (ESRD) patients on maintenance hemodialysis (HD). Diets enriched with n-3 polyunsaturated fatty acids (PUFAs) (omega-3) have been associated to a lower incidence of coronary heart disease (CHD) and a reduction in atherosclerotic lesions. Omega-3 may interfere with the arachidonic acid cascade by inhibiting 5-Lox. Lipid peroxidation, leukotriene B(4) (LTB(4)) production, 5-Lox activity and expression were investigated in PBMC isolated from ESRD patients under maintenance HD before and after a 3-month oral supplementation with omega-3 at a daily dose of 2700 mg of n-3 PUFAs at the average eicosapentaenoic acid/docosaesaenoic acid ratio of 1.2 and finally after a further 3-month washout with no omega-3 supplementation. PBMCs from non-uremic volunteers were also investigated for comparison to normal parameters. Administration of omega-3 reduced significantly lipid peroxidation (P < 0.0001), LTB(4) synthesis (P < 0.0001) and 5-Lox activity (P < 0.0001), with no effect on 5-Lox protein expression. After the 3-month washout, all parameters were comparable to those observed before treatment. Our results resemble those obtained after oral administration of vitamin E and are consistent with a reversible, dose-dependent inhibition of 5-Lox by omega-3. Upregulation of 5-Lox may also be related to the increased mitochondrial damage and apoptosis of PBMCs observed in ESRD patients compared to non-uremic controls. Omega-3 may thus protect PBMCs of ESRD patients against oxidative stress.

Peritonectomy and hyperthermic antiblastic perfusion in the treatment of peritoneal carcinomatosis.

AIMS: Some low-grade malignant tumours arising in the abdomen tend to remain loco-regionally confined to peritoneal surfaces, without systemic dissemination. In these cases complete surgical tumour cytoreduction followed by intra- or post-operative regional chemotherapy has curative potential. The aim of this study was to evaluate the outcome for patients treated in this way. METHODS: Peritonectomy was performed, involving the complete removal of all the visceral and parietal peritoneum involved by disease. After peritonectomy, hyperthermic antiblastic perfusion was carried out throughout the abdominopelvic cavity for 90 min, at a temperature of 41.5-42.5 degrees C, with mitomycin C (3.3 mg/m2/l) and cisplatin (25 mg/m2/l) (for appendicular or colorectal primaries), or cisplatin alone (for ovarian primaries). Alternatively, the immediate post-operative regional chemotherapy was performed with 5-fluorouracil (13.5 mg/kg) and Lederfolin (125 mg/m2) (for colonic or appendicular tumours) or cisplatin (25 mg/m2) (for ovarian tumours), each day for 5 days. RESULTS: Thirty-five patients affected by extensive peritoneal carcinomatosis were submitted to peritonectomy, with no residual macroscopic disease in all cases except three. Twenty-six patients were able to undergo the combined treatment involving loco-regional chemotherapy. Complications were observed in 54% of the patients and led to death in four of them. At a mean follow-up of 17 months overall 2-year survival was 55.2%, with a median survival of 26 months. CONCLUSIONS: After a learning curve of 18 months the feasibility of the integrated treatment increased to more than 90%, while mortality decreased dramatically. The curative potential of the combined therapeutic approach seems high in selected patients with peritoneal carcinomatosis not responding to systemic chemotherapy. Careful selection of patients can minimize the surgical risk, but the treatment should currently be reserved for clinical trials.

Treatment of peritoneal carcinomatosis with intent to cure.

BACKGROUND AND OBJECTIVES: Low-grade malignant tumors arise in the abdomen, do not infiltrate, and "redistribute" on the peritoneum with no extraregional spreading. In these cases, aggressive surgery combined with localized chemotherapy may provide cure. METHODS: After removing the tumor with the regional peritoneum en bloc, intraabdominal hyperthermic chemoperfusion was performed throughout the abdominopelvic cavity. Alternatively, early intraabdominal chemotherapy, starting on the first postoperative day, was administered for 5 days. RESULTS: Forty patients affected with extensive peritoneal carcinomatosis underwent peritonectomy, with no residual macroscopic disease except in four cases. Seventy-five percent of the patients underwent locoregional chemotherapy. Major complications were observed in 40% of the patients and led to death in five; there was a direct correlation to the duration of surgery (P = 0.03). At a mean follow-up of 20 months, the overall 2-year survival was 61.4%, with a median survival of 30 months. CONCLUSIONS: After a learning curve of 18 months, the feasibility of the integrated treatment increased to greater than 90%, and mortality dramatically decreased. The combined treatment resulted in a high survival rate in patients with extensive carcinomatosis who were no longer responsive to traditional therapies.

The integrated treatment of peritoneal carcinomatosis. A preliminary experience.

Some low-grade malignant tumors arising in the abdomen, lack of infiltrative attitude and "redistribute" on the peritoneum with no extraregional spreading. In this cases the complete tumor cytoreduction followed by intra- or postoperative regional chemotherapy has curative intent. Peritonectomy is the complete removal of all the parietal peritoneum and the visceral peritoneum involved by disease. After peritonectomy hyperthermic antiblastic perfusion is carried out throughout the abdomino-pelvic cavity for 60 minutes, at a temperature of 41.5 degrees C, with mitomycin C (3.3 mg/m2/Lt of perfusate) and cisplatin (25 mg/m2/Lt) (appendicular or colorectal primary), or cisplatin alone is (ovarian primary). Alternatively the immediate postoperative regional chemotherapy is performed with 5-fluorouracil (13.5 mg/Kg) and Lederfolin (125 mg/m2) (colic or appendicular tumor) or cisplatin (25 ng/m2) (ovarian tumor), each day for 5 days. Twenty patients affected by extensive peritoneal carcinomatosis (12 ovarian, 5 colonic, 1 appendicular, 1 mesothelial and 1 gastric primary) were submitted to peritonectomy with no residual macroscopic disease in all cases except three. Six patients were treated with intraoperative intra-abdominal hyperthermic antiblastic perfusion, while immediate postoperative intra-abdominal chemotherapy was given in 4 patients and systemic chemotherapy in other 5. Hospital mortality was 20%. At a mean follow-up of 11 months 14 patients are alive, 11 without disease and the median overall survival is 10.2 months. The curative potential of the combined therapeutic approach seems high in patients with peritoneal carcinomatosis from ovarian or colorectal primary not responding to systemic chemotherapy. Selection criteria of patients can strictly affect the surgical risk and the treatment has to be reserved for controlled clinical trials.

31Phosphorus magnetic resonance spectroscopy to evaluate medical therapy efficacy in peripheral arterial disease. A pilot study.

BACKGROUND: Human and animal studies have shown that propionyl-L-carnitine, increasing carnitine content, improves the energy metabolism of ischemic skeletal muscle. The aim of the study was to evaluate the accuracy of Doppler continuous wave, Treadmill test and 31Phosphorus magnetic resonance spectroscopy in determining the efficacy of propionyl-L-carnitine in patients with peripheral arterial disease. EXPERIMENTAL DESIGN: Prospective study. SETTING: University hospital. PATIENTS: Eighteen male patients with peripheral arterial disease (category 3) and 8 healthy volunteers form the basis of the study. Patients quit smoking, start physical training (2-3 Km walk per day) and were assigned to medical therapy consisting of propionyl-L-carnitine (8 patients) or placebo (10 patients). Patients were studied with Doppler continuous wave, Treadmill test and 31Phosphorus magnetic resonance spectroscopy at day 0 and at day 90. The following parameters were assessed by the principal component analysis: clinical (absolute claudication, ankle brachial index at rest and at 2, 5 and 10 minutes after completing Treadmill exercises) and biochemical (inorganic phosphorus/phosphocreatine ratio and pH profiles at 20% and 50% of the maximum load, the recovery half time of phosphocreatine, number of exercise steps and slope of linear relationship between muscle power and inorganic phosphorus/phosphocreatine ratio). RESULTS: Final evaluation showed a significant improvement of clinical and biochemical variables (p < 0.05 and p < 0.02 respectively). Breaking down the results on the basis of the two study arms, 31Phosphorus magnetic resonance spectroscopy showed a significant improvement of biochemical variables in the group of patients treated with propionyl-L-carnitine (p < 0.05) and was more sensitive in the evaluation of changes induced by 90-day treatment as compared with the other noninvasive examinations. CONCLUSIONS: 31P-MRS permits the evaluation of muscle metabolic effect induced by PLC after a 90-day-period in patients affected by category 3 of peripheral arterial disease and it is a more sensitive tool in the evaluation of the pharmacological effects of medical therapy.

[Preoperative blood storage and intraoperative blood recovery in elective treatment of abdominal aorta aneurysm]. / Predesposito e recupero intraoperatorio di sangue nel trattamento elettivo degli aneurismi dell'aorta addominale.

We evaluated the efficiency and costs-effectiveness of blood predonation and intraoperative salvage in elective abdominal aortic aneurysm surgery. Between January 1992 and January 1994, 66 patients (59 male and 7 female, aged 69.9 +/- 0.8 years) who underwent elective surgical repair of an AAA were selected for the study. Thirty-six (54.5%) patients (Group 1) intra- and/or postoperatively received homologous blood whereas 30 (45.5%) patients (Group 2) received autologous blood predonation and intraoperative blood aspiration and reinfusion. The two groups were similar for demographic data, aneurysmal diameter and associated diseases and/or risk factors (p = NS). Operative mortality was comparable between the two groups (p = NS). The mean intraoperative blood loss was 803.4 +/- 104.5 ml in group 1 and 812.8 +/- 44.8 ml in group 2 (p = NS). Group 2 patients received intra- or postoperatively a mean of 0.8 +/- 0.2 units of homologous blood (p < 0.001). Aneurysmal diameter did not influence the transfusion requirement between the two groups (p = NS). The cost per unit of homologous banked blood was significantly higher (p < 0.01). Cumulative costs of the procedures did not show statistical differences between the two groups (p = NS). Aortic surgery is the ideal target for predonation and intraoperative blood salvage.

Hyperthermic antiblastic perfusion for the treatment of soft tissue limb sarcoma.

Eighty patients with locally advanced, high grade soft tissue sarcoma of the extremities were studied prospectively in order to determine the efficacy of hyperthermic antiblastic perfusion (H.A.P.) as the first step of a combined multimodality therapy. All of the patients have been evaluated in terms of functional results, loco-regional control and survival according to the different treatment schedules adopted. The first clinical trial employed H.A.P., followed by surgery alone. Because the results obtained were unsatisfactory, the protocol was modified to include a continuous intra-arterial (i.a.) infusion of doxorubicin (dx) or radiotherapy before surgery. The best results have been obtained with the radiotherapy-including protocol showing a conservative surgery rate of 100% and a 94% rate of loco-regional control. The disease-free, distant disease-free and overall survival rates were 68%, 75% and 70%, respectively. The importance of the treatment protocol has been confirmed in a multivariate analysis which demonstrated that the treatment protocol adopted is one of the prognostic factors with an independent value (p = 0.06).