Cell wall polysaccharides from in vitro propagated Cereus hildmannianus K. Schum. plants.

Cereus hildmannianus is a cactus exhibiting morphological and physiological adaptation of its cladodes which ensuring growth in climatic and soil conditions unfavourable for many plant species. Currently, limited water resources and increasing demand for renewable energy make cacti a biomass source for the production of biofuels. Somaclones regenerated from callus in vitro can be a source of new raw material in useful plants. The objective of this work was to determine if the regenerated plants showing two different morphologies present polysaccharide composition different from the wild plant. Somaclones aqueous extraction shows the absence of soluble polysaccharides as mucilage. The alkaline extraction of in vivo cultivated plant showed the presence of starch, type I arabinogalactan, and arabinoxylan and the somaclones showed type I arabinogalactan and arabinoxylan in both morphologies. Hemicelluloses found in the somaclones are not different from in vivo cultivated plants, but somaclones not almost biosynthesize mucilage and starch.

Changes in mercury distribution and its body burden in delphinids affected by a morbillivirus infection: Evidences of methylmercury intoxication in Guiana dolphin.

An unusual mortality event (UME) attributed to morbillivirus infection was identified in two Guiana dolphin populations from the Southeastern Brazilian coast. The aim of this study was to characterize total mercury (THg), methylmercury (MeHg) and selenium (Se) bioaccumulation and body burden in Guiana dolphins from Sepetiba Bay (RJ) collected before (n = 61) and during the UME (n = 20). Significantly lower Se concentrations were found in the livers of individuals collected during the UME (Mann-Whitney test; p = 0.03), probably due to impairment of the detoxification process in the liver. There were differences in THg and Se concentrations in the organs and tissues of individuals (Kruskal-Wallis test, p < 0.05), but not MeHg (Kruskal-Wallis test, p = 0.07). For THg, the liver showed the higher concentrations and differed among organs and tissues analyzed such as blubber (Tukey's test for unequal N; p = 0.003). For Se concentrations, the skin and kidney presented the higher concentrations and varied among other tissues/organs, like muscle (Tukey's test for unequal N; p = 0.02). Differences in body burdens were observed among specimens collected previously and during the UME probably due to the remobilization and transport of the muscle-stored MeHg to other tissues/organs. This abrupt input of MeHg into the bloodstream may cause serious health damage. Indeed, evidences of methylmercury intoxication was observed in Guiana dolphins in Sepetiba Bay. In conclusion, bioaccumulation patterns, the detoxification process and body burden were affected by morbillivirus.

The Allium cepa bioassay to evaluate landfarming soil, before and after the addition of rice hulls to accelerate organic pollutants biodegradation.

Landfarming is a soil bioremediation technology practiced by oil refineries in order to reduce or eliminate hydrocarbons from petroleum sludge. The goal of the current study was to use Allium cepa bioassay to assess landfarming and landfarming with rice hulls amendment before and after hydrocarbons biodegradation assay in the laboratory. Three cytogenetic endpoints were used: mitotic and chromosome abnormalities (MCA), micronucleus (MN) and nuclear buds (NB). Landfarming presented 13.5 g/kg of total petroleum hydrocarbons (TPHs) and caused strong clastogenic and mutagenic effects (p<0.05) in A. cepa. After 108 days of biodegradation, the landfarming reached the rate of 26.30 mmol of CO(2) released, the concentration of TPHs decreased by 27% and there was significant reduction in MCA, MN and NB. Landfarming treated with rice hulls had the highest release of CO(2), 110.9 mmol, associated with a remarkable reduction in TPHs concentration, 59%, and had the highest decrease in MCA, MN and NB (p>0.05). Our findings showed that the use of rice hulls accelerated the biodegradation efficacy of landfarming and reduced their clastogenicity, indicating that supplementary treatments are important to improve the efficiency of bioremediation processes.

Total-Hg and organic-Hg in Cephalopholis fulva (Linnaeus, 1758) from inshore and offshore waters of NE Brazil.

To investigate whether source proximity or bioavailability is the major factor controlling both Hg concentration and Hg speciation in marine fishes, total- and organic-Hg content in muscle and liver tissues from different populations of Cephalopholis fulva (piraúna) from inshore and offshore waters of the Brazilian northeastern coast were analyzed. Average total-Hg in muscle (104 ng x g(-1) w.w.) and liver (2,865 ng x g(-1) w.w.) tissues, as well as organic-Hg concentrations in muscle (169 ng x g(-1) w.w.) and liver (1,038 ng x g(-1) w.w.), were much higher in the offshore population of C. fulva than in the inshore ones. In the inshore population total-Hg and organic-Hg average concentrations in muscle tissue were similar and reached only 49 ng x g(-1) w.w., while in liver they reached 412 ng x g(-1) w.w. for total-Hg and 180 ng x g(-1) w.w., for organic-Hg. Concentrations of both Hg species in the two populations were higher in liver than in muscle. The average percentage contribution of organic-Hg to the total Hg content was higher in muscle (98-100%) than in liver (42-53%), but similar between the two populations. Total-Hg and organic-Hg concentrations in muscle and liver from the offshore population showed significant (P < 0.05) positive correlation with fish length. However, in the inshore population only the total-Hg and organic-Hg in muscle tissues correlate significantly with fish size. Although the coastal environments are enriched in total Hg relative to open waters, the significant higher Hg concentrations in the offshore population of C. fulva and the significant correlation found between organic-Hg in liver with fish size suggest higher bioavailability of Hg in offshore waters relative to inshore ones.

Total-Hg and organic-Hg in Cephalopholis fulva (Linnaeus, 1758) from inshore and offshore waters of NE Brazil

To investigate whether source proximity or bioavailability is the major factor controlling both Hg concentration and Hg speciation in marine fishes, total- and organic-Hg content in muscle and liver tissues from different populations of Cephalopholis fulva (piraúna) from inshore and offshore waters of the Brazilian northeastern coast were analyzed. Average total-Hg in muscle (104 ng.g-1 w.w.) and liver (2,865 ng.g-1 w.w.) tissues, as well as organic-Hg concentrations in muscle (169 ng.g-1 w.w.) and liver (1,038 ng.g-1 w.w.), were much higher in the offshore population of C. fulva than in the inshore ones. In the inshore population total-Hg and organic-Hg average concentrations in muscle tissue were similar and reached only 49 ng.g-1 w.w., while in liver they reached 412 ng.g-1 w.w. for total-Hg and 180 ng.g-1 w.w., for organic-Hg. Concentrations of both Hg species in the two populations were higher in liver than in muscle. The average percentage contribution of organic-Hg to the total Hg content was higher in muscle (98-100 percent) than in liver (42-53 percent), but similar between the two populations. Total-Hg and organic-Hg concentrations in muscle and liver from the offshore population showed significant (P < 0.05) positive correlation with fish length. However, in the inshore population only the total-Hg and organic-Hg in muscle tissues correlate significantly with fish size. Although the coastal environments are enriched in total Hg relative to open waters, the significant higher Hg concentrations in the offshore population of C. fulva and the significant correlation found between organic-Hg in liver with fish size suggest higher bioavailability of Hg in offshore waters relative to inshore ones.

As concentrações de Hg-total e Hg-orgânico foram determinadas em diferentes populações de Cephalopholis fulva (piraúna) capturadas em águas costeiras e em bancos oceânicos do litoral nordeste do Brasil. A comparação entre as duas populações permitiu investigar o efeito da proximidade de fontes sobre as concentrações, e a especiação de Hg em músculo e fígado desta espécie. As concentrações médias de Hg-total em músculo (104 ng.g-1 w.w.) e fígado (2,865 ng.g-1 w.w.), assim como as concentrações de Hg-orgânico em músculo (169 ng.g-1 w.w.) e fígado (1,038 ng.g¹ w.w.) foram muito maiores na população capturada nos bancos oceânicos do que na população costeira. Nesta, as concentrações médias de Hg-total e Hg-orgânico na musculatura de C. fulva foram similares e baixas (49 ng.g-1 w.w.), enquanto que atingiram 412 ng.g-1 w.w. de Hg-total e 180 ng.g-1 w.w. de Hg-orgânico no fígado destes animais. As concentrações das duas espécies de Hg foram significativamente maiores no fígado do que na musculatura. A contribuição percentual média de Hg-orgânico para a concentração total de Hg nos peixes foi maior para músculo (98-100 por cento) que para fígado (42-53 por cento), mas semelhante entre as duas populações. As concentrações de Hg-total e Hg-orgânico na musculatura e no fígado de C. fulva mostraram-se positivamente correlacionadas com o tamanho do animal (P < 0,05). Entretanto, na população costeira somente as concentrações destas espécies de Hg na musculatura apresentaram correlações significativas com o tamanho do animal. Embora o ambiente costeiro seja relativamente enriquecido em Hg, em relação aos bancos oceânicos, as maiores concentrações de Hg foram verificadas na população oceânica de C. fulva. A correlação significativa entre Hg-orgânico no fígado e tamanho do animal no fígado sugerem uma maior biodisponibilidade do Hg em águas oceânicas quando comparada às águas costeiras.

Topoisomerase I-DNA covalent complexes in human colorectal cancer xenografts with different p53 and microsatellite instability status: relation with their sensitivity to CTP-11.

The topoisomerase I poison CPT-11 has proved efficient for the treatment of untreated metastatic colorectal cancers (CRC) and those refractory to fluoropyrimidines. However, the interpatient variability is important. A previous in vitro study suggested that measurements of the level of topoisomerase I-DNA intermediates trapped by camptothecin may be useful to estimate the chemosensitivity of colon carcinoma cell lines. To verify this hypothesis, we developed an immuno-assay to detect covalent topoisomerase I-DNA complexes in a series of human colorectal cancers xenografted in nude mice. Six human CRCs were selected for their distinctive p53 and microsatellite instability (MSI) status. Tumour lysates, prepared from mice untreated or treated with CPT-11, were fractionated onto CsCl gradients to separate free and DNA-bound topoisomerase I by centrifugation. Interestingly, significant levels of DNA-topoisomerase I complexes were detected in the tumours most responsive to the treatment with CPT-11, irrespective of their MSI and p53 phenotypes. Our in vivo study fully agrees with the predictions from the in vitro data indicating that evaluation of topoisomerase I-DNA complexes would be useful to predict the response of CRC to a treatment with CPT-11.

Synergistic efficacy of 3n-butyrate and 5-fluorouracil in human colorectal cancer xenografts via modulation of DNA synthesis.

BACKGROUND & AIMS: Butyrate, produced in the colon lumen, maintains mucosal cell homeostasis. Poorly diffusible, its access is compromised in growing colon cancers and absent in distant metastases. Butyrate regulates DNA synthesis. We postulated that systemic administration of butyrate should reduce colon cancer growth and enhance 5-fluorouracil (5-FU) efficacy. METHODS: A stable derivative of butyrate (3n-But) was used. The antitumoral efficacy of 5-FU and 3n-But, alone or combined, was evaluated in human colorectal cancers (hCRCs) subcutaneously, orthotopically, or intrasplenically grafted into nude mice. Thymidylate synthase (TS) and thymidine kinase (TK) mRNA expression, proliferation, apoptosis, and cell cycle alterations were studied. RESULTS: In vivo, 5-FU alone inhibited growth of only 3 of the 12 hCRCs tested and 3n-But alone had no effect; the 5-FU/3n-But combination inhibited growth of all 16 hCRCs tested. The hCRCs differed in their p53 and microsatellite instability status. 5-FU/3n-But decreased TK and TS mRNA expression by 20- and 40-fold, respectively, and TS activity by 75%, stopped cell proliferation without affecting cell differentiation, and significantly enhanced apoptosis. 3n-But potentiated the efficacy of Tomudex and methotrexate, 2 TS inhibitors, but not that of oxaliplatin. In vitro, 5-FU/3n-But inhibited [3H]thymidine but not bromodeoxyuridine incorporation and induced apoptosis in hCRC cell lines. Cells treated with 5-FU/3n-But did not accumulate in G1 nor in S phase of the cell cycle, while 5-FU and 3n-But arrested the cycle in S and in G1 phase, respectively. 3n-But prevented the cell rescue from 5-FU-induced cytotoxicity by uridine or thymidine. CONCLUSIONS: 3n-But and TS inhibitors acted synergistically against colorectal cancers, independently of the genetic alterations of the hCRCs. The mechanism of action of 5-FU/3n-But could be enhanced reduction of TS and prevention of thymidine salvage in DNA synthesis.

Sensitivity to CPT-11 of xenografted human colorectal cancers as a function of microsatellite instability and p53 status.

Biological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into nude mice. CRC xenografts differed in their p53 status (wt or muf) and in their microsatellite instability phenotype (MSI+ when altered). Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+ and three were MSI-. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg(-1) of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI-. At 40 mg kg(-1) of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI-/Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/mutp53) were more sensitive than X17LoVo (MSI+/mutp53. HT 29 tumours (MSI-Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI-/wtp53) were sensitive, showing that wtp53 improves the drug-response in these MSI- tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+ being more sensitive than MSI(-)CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11.

Methionine depletion enhances the antitumoral efficacy of cytotoxic agents in drug-resistant human tumor xenografts.

Efficacy of chemotherapy is limited in numerous tumors by specific cellular mechanisms that inactivate cytotoxic antitumoral drugs, such as ATP-dependent drug efflux and/or drug detoxification by glutathione. In reducing ATP pools and/or glutathione synthesis, it might be possible to enhance the efficacy of drugs affected by such resistance mechanisms. Reduction of the ATP pool and glutathione content is achievable in cancer cells by depleting the exogenous methionine (Met) supply and ethionine. Thus, the rationale for the present study was to use Met depletion to decrease the ATP and glutathione pools so as to sensitize tumors refractory to cytotoxic anticancer drugs. Met depletion was achieved by feeding mice a methionine-free diet supplemented with homocysteine. The effects of Met depletion combined with ethionine and/or chemotherapeutic agents were studied using human solid cancers xenografted into nude mice. TC71-MA (a colon cancer) SCLC6 (a small cell lung cancer), and SNB19 (a glioma) were found to be refractory to cisplatin, doxorubicin, and carmustine, respectively. These three drugs are used to treat such tumors and are dependent for their activity on the lack of cellular ATP- or glutathione-dependent mechanisms of resistance. TC71-MA, SCLC6, and SNB19 were Met dependent because their proliferation in vitro and growth in vivo were reduced by Met depletion. Cisplatin was inactive in the treatment of TC71-MA colon cancer, whereas a methionine-free diet, alone or in combination with ethionine, prolonged the survival of mice by 2-fold and 2.8-fold, respectively. When all three approaches were combined, survival was prolonged by 3.3-fold. Doxorubicin did not affect the growth of SCLC6, a MDR1-MRP-expressing tumor. A Met-deprived diet and ethionine slightly decreased SCLC6 growth and, in combination with doxorubicin, an inhibition of 51% was obtained, with survival prolonged by 1.7-fold. Combined treatment produced greater tumor growth inhibition (74%) in SCLC6-Dox, a SCLC6 tumor pretreated with doxorubicin. Growth of SNB19 glioma was not inhibited by carmustine, but when it was combined with Met depletion, survival duration was prolonged by 2-fold, with a growth inhibition of 80%. These results indicate the potential of Met depletion to enhance the antitumoral effects of chemotherapeutic agents on drug-refractory tumors.

Doppler echocardiographic indexes and 24-h ambulatory blood pressure data in sedentary middle-aged men presenting exaggerated blood pressure response during dynamical exercise test.

Previous studies have pointed out that exaggerated blood pressure (BP) response during physical exercise could be an early marker of essential hypertension. Apparently some of the exaggerated BP responders present changes in the heart geometry and function that are usually found in the early course of the hypertensive disease. To evaluate the association between exaggerated BP response and these changes, we submitted 20 normotensive men presenting elevated BP response during bicycle exercise (hyperreactive group, systolic BP > or = 220 mmHg at maximal workload) to 24-h ambulatory blood pressure monitoring (ABPM) and to two-dimensionally guided M-mode echocardiography and pulsed Doppler. The results from this group were contrasted with those of a comparable group, which otherwise, presented normal BP response during the same procedure (control group, systolic BP < or = 210 mmHg at maximal workload). The ABPM measurements were normal and analogous between the two groups: the mean 24-h systolic blood pressure (SBP) was respectively 126 +/- 6 mmHg and 129 +/- 5 mmHg, diastolic blood pressure (DBP) 82 +/- 4 mmHg in both groups, and heart rate (HR), respectively 76 +/- 9 and 74 +/- 7 bpm. The univariate correlation (R) between the maximal BP response during bicycle exercise and BP measurements in the ABPM were in general weak, and as a whole, the hyperreactive group presented the weakest correlation coefficients. M-mode echocardiographic data such as the left ventricular mass index (LVMI, 80 +/- 10 vs. 81 +/- 11 g/m2), posterior wall and interventricular septal thickness (PWT, 8.8 +/- 0.6 vs. 8.6 +/- 0.7 mm; IVST, 9.0 +/- 0.4 vs. 8.8 +/- 0.6) were also normal and comparable between the groups. LV systolic functional indexes such as fractional shortening (LVFS, 39 +/- 2.8 vs. 40 +/- 3.5%) and ejection fraction (LVEF, 70 +/- 3.5 vs. 71 +/- 3.7%) were also normal and similar. Doppler-derived LV diastolic functional indexes such as the peak velocity of early flow divided by the peak velocity of late flow (RE/A) and isovolumetric relaxation time (IVRT) were also equivalent (RE/A, both 1.3 +/- 0.2, IVRT 79 +/- 7 vs. 81 +/- 6 msec). These results support the concept that an exaggerated BP elevation during physical activity, when not accompanied of higher levels of BP during daily activities are not associated with changes in the heart geometry or in the ventricular function, and might represent an hemodynamical behavior of limited pathological and clinical importance. These conclusions must be taken cautiously since personal characteristics such as life style, family history of hypertension, gender, race and also the levels of BP chosen to delimit a normal and an exaggerated BP response might be important factors determining the consequences of the hyperreactive behavior.

Methionine deprivation and methionine analogs inhibit cell proliferation and growth of human xenografted gliomas.

Growth of numerous malignant tumors depends on an exogenous methionine (MET) supply, while endogenously synthesized MET supports normal cell proliferation. Because an antitumor effect should be obtained by aggravating the altered MET metabolism in gliomas, MET dependency of human xenografted gliomas was evaluated and a therapeutic approach using MET deprivation or MET analogs to induce MET starvation was applied. In vitro proliferation inhibition of glioma cell lines by MET deprivation and two MET analogs, ethionine (ETH) and trifluoromethylhomocysteine (TFH), was measured. Proliferation of 7 human glioma cell lines tested was inhibited in MET-free medium, and was poorly or not reversed by homocysteine (HCY). ETH or TFH (concentration range: 0.005-2 mg/ml) inhibited proliferation of all cell lines tested. MET analog-induced inhibition was abolished by MET and enhanced by HCY. Cell-cycle alterations due to MET deprivation were optimally assessed after 30 h of culture and bromodeoxyuridine incorporation. In MET- medium, cells were arrested in the G1-phase. ETH induced a dramatic accumulation of cells in the G2-phase. ATP contents were reduced by MET analogs only in HCY+ medium, suggesting complementary effects of MET analogs and HCY. Human glioma bearing nude mice were fed an amino acid-substituted MET- HCY-supplemented diet (MET-HCY+) and/or treated with MET analogs, injected intraperitoneally daily. Using two human xenografted tumors derived from gliomas, antitumor effects were obtained by subjecting tumor-bearing nude mice to MET starvation. TG-1-MA was more sensitive to MET depletion (40% of growth inhibition, P < 0.10) than TG-8-OZ (no growth inhibition). Antitumor effects of a MET-HCY+ diet and 200 mg/kg of ETH were potentiated when co-administered to glioma-bearing mice (77% GI, P < 0.025 and 67%, P < 0.0057 to TG-1-MA and TG-8-OZ respectively). A dose-response effect with no toxicity was obtained when the ETH dose was increased 10 fold. Potentiation of the effects of ETH and a MET-free diet indicates that they probably act on the same pathway but not the same target. In conclusion, experimentally induced MET deprivation and MET-analog treatment retarded the growth of human gliomas. Combination of MET-analog therapy with MET substitution by HCY enhanced their respective effects.

Growth of methionine-dependent human prostate cancer (PC-3) is inhibited by ethionine combined with methionine starvation.

Methionine (MET) is required for cell metabolism. MET endogenously synthesized from homocysteine (HCY) supports the proliferation of normal cells, but not that of numerous malignant cells, as shown previously. MET starvation should have an anti-tumour effect, and its deleterious effects on the hosts might be prevented by HCY. Anti-tumour effects of MET starvation must be reinforced by ethionine (ETH), a MET analogue. MET dependency of PC-3, a human prostate cancer cell line, was studied in vitro. Proliferation of PC-3 cells, cultivated in MET-free medium, was 29% compared with growth in MET+HCY- medium. Addition of HCY to MET-free medium increased the proliferation rate to 56%. The concentration of ETH required to decrease the PC-3 cell proliferation rate to 50% (IC50) was 0.5 mg ml(-1) in MET-HCY- medium. ETH-induced inhibition was abolished by MET addition and was reinforced by HCY. PC-3 cell cycle was blocked in the S-G2-phase after 30 h culture in the absence of MET; this blockage was not reversed by addition of HCY. ETH at the IC50 in MET-HCY+ medium blocked DNA replication. Apoptotic cells appeared after 30 h incubation in MET-HCY+ medium only when ETH was added. ATP pools were decreased after 15 h of culture in MET-free medium. In vivo, MET starvation was obtained by feeding tumour-bearing mice a diet containing a synthetic amino acid mixture as the protein supply, in which HCY replaced MET. Given to nude mice bearing xenografted PC-3, from day 1 after grafting and for 3 weeks, this diet inhibited tumour growth (34% on day 20, P < 0.007); this effect was potentiated by ETH (200 mg kg(-1) day(-1) i.p.) (56% on day 20, P < 5 x 10(-5)). The differences between the effects of these two treatments were significant (P < 0.017) and optimal on day 20. These data showed that combination of ETH and HCY slowed the proliferation of prostate cancer cells in vitro and in vivo, decreased ATP synthesis and caused cell cycle arrest and apoptosis. Experimental therapy based on cancer cell MET metabolism deficiency could be efficient for treating advanced prostate cancers refractory to current therapies.